CN105683362A - Suspension and clustering of human pluripotent stem cells for differentiation into pancreatic endocrine cells - Google Patents

Abstract

The present invention provides methods of preparing aggregated pluripotent stem cell clusters for differentiation. Specifically, the invention discloses methods of differentiating pluripotent cells into beta cell, cardiac cell and neuronal cell lineages using suspension clustering. The methods involve preparing the aggregated cell clusters followed by differentiation of these clusters.

Description

Be used for the suspension of the human pluripotent stem cells that is divided into pancreatic endocrine cell and troop

The cross reference of related application

The application is the part continuation application of U. S. application 13/998,974 (being filed on December 30th, 2013), its requirementU.S. Provisional Application 61/747,799 (being filed on December 31st, 2012) and U.S. Provisional Application 61/962,158 (are filed inOn November 1st, 2013) priority, these two applications are all incorporated herein by reference in full.

Technical field

The invention belongs to Cell Differentiation field, comprise that preparing embryonic stem cell and other keeps the many of the cell cluster assembledEnergy property is to be divided into the pluripotent cell of entoderm CFU-GM, pancreatic endocrine cell, mesoblastema or ectoderm cell. ?An aspect, the invention discloses and generate trooping and holding it in suspension culture to be divided into of multipotential stem cellThe method of pancreas entoderm, pancreatic endocrine progenitor cells and single hormone pancreatic endocrine cell.

Background technology

The progress of cell replacement therapy and the shortage of portable pancreas islet for IDDM have made people notingPower concentrates on to be developed on the source that is suitable for the insulin secretory cell (being β cell) of transplanting. One method is by multipotential stem cellSuch as embryonic stem cell systematic function β cell.

In vertebrate embryonic development, pluripotent cell can produce and comprise three embryos in the process that is called primitive gut formationThe cell mass of layer (ectoderm, mesoderm and entoderm). Tissue (such as thyroid gland, thymus gland, pancreas, intestines and liver) will be from interior embryoLayer was grown via the interstage. Interstage in this process is the formation of definitive entoderm.

Till primitive gut forms, entoderm is divided into the anterior-posterior territory that can identify by the expression of a group factor, described inThe factor uniquely mark endoblastic before, in and back zone. For example, HHEX and SOX2 identify endoblastic proparea, and CDX1, CDX2Identify endoblastic back zone with CDX4.

The migration of entoderm tissue makes entoderm approach from different mesoderm tissue tights, and this contributes to dividing of intestinal tubeDistrict. This completes by multiple EF such as FGF, Wnt, TGF-β, retinoic acid (RA) and BMP part and antagonist thereof. ExampleAs, it is reported, FGF4 and BMP promote the expression of the CDX2 in the rear gut entoderm of supposition and check the table of front gene HH EX and SOX2Reach (2000Development, 127:1563 – 1567). WNT signal transduction also shown and the multiple operation of FGF signal transduction, withPromote hindgut grow and suppress anterior intestine destiny (2007Development, 134:2207 – 2217). Finally, secreted by mesenchymaRetinoic acid regulation and control anterior intestine-hindgut borders (2002CurrBiol, 12:1215-1220).

The expression of idiosyncratic transcription factor can be used for the identity of specified tissue. Change into entodermal canal at definitive entodermProcess in, intestinal tube becomes and divides extensive territory into, described extensive territory can by restriction gene expression pattern on molecular levelObserve. For example, in intestinal tube, the pancreas territory of subregion shows the high expression of PDX1 and the extremely low expression of CDX2 and SOX2.PDX1, NKX6.1, PTF1A and NKX2.2 highly express in pancreatic tissue; And CDX2 highly expresses in intestinal tissue.

Definitive entoderm is divided into pancreas entoderm and causes forming pancreas. Dorsal part and veutro pancreas territory produce on anterior intestineSkin. Anterior intestine also produces esophagus, tracheae, lung, thyroid gland, stomach, liver, pancreas and biliary system.

The endoblastic cellular expression pancreas-duodenum of pancreas hox genes PDX1. In the time not there is not PDX1, pancreas formsAfter ventral pancreatic bud and dorsal pancreatic bud, no longer grow. Thereby PDX1 expresses the committed step indicating in the generation of pancreas organ. RipePancreas comprise the exocrine tissue and the endocrine tissue that are produced by the endoblastic differentiation of pancreas.

The people such as D ' Amour have described under the existence of high concentration activin and low serum, and what human embryo stem cell was originated determinesGeneration (NatureBiotechnol2005, the 23:1534-1541 of the endoblastic enrichment culture thing of shape; U.S. Patent number 7,704,738). These cell transplantations are caused being divided at the subrenal capsule of mouse and there is the more ripe of entoderm tissue signatureCell (United States Patent (USP) 7,704,738). Adding after FGF10 and retinoic acid, the definitive entoderm in human embryo stem cell source is thinBorn of the same parents can also be divided into PDX1 positive cell (U.S. Patent Application Publication 2005/0266554A1). These diabetes existFollow-up transplanting in the fat pad of immunodeficient mouse caused forming function pancreatic endocrine cell after 3-4 month maturity period(United States Patent (USP) 7,993,920 and United States Patent (USP) 7,534,608).

People's reports such as Fisk are for being produced the system (United States Patent (USP) 7,033,831) of islet cells by human embryo stem cell.Micromolecular inhibitor is also for inducing pancreatic endocrine progenitor cells. For example, the little molecule of TGF-beta receptor and bmp receptor presses downPreparation (Development2011,138:861-871; Diabetes2011,60:239-247) for significantly increasing pancreasThe number of endocrine cell. In addition, activation of small molecule agent is also for generating definitive entoderm cell or diabetes(CurrOpinCellBiol2009,21:727-732；NatureChemBiol2009,5:258-265)。

The scheme that is used for cultivating CFU-GM (such as multipotential stem cell) in improvement has obtained huge progress. PCT announcesWO2007/026353 (people such as Amit) discloses in two-dimentional culture systems human embryo stem cell has been remained on to undifferentiated stateUnder. The people such as Ludwig disclose for cultivate people in matrix at 2006 (NatureBiotechnology, 24:185-7)The TeSR1 defined medium of embryonic stem cell. U.S. Patent Application Publication 2007/0155013 people such as () Akaike is openUse the adherent carrier to multipotential stem cell in suspension, the grow method of multipotential stem cell and U.S. Patent application public affairsCloth 2009/0029462 people such as () Beardsley discloses use microcarrier or cell, and to be encapsulated in the multipotency that increases in suspension dry thinBorn of the same parents' method. PCT announces WO2008/015682 people such as () Amit and discloses under the condition of culture that there is no substrate adhesivenessThe method of amplification and maintenance human embryo stem cell in suspension culture.

U.S. Patent Application Publication 2008/0159994 (people such as Mantalaris) disclose in dimensional culture system and trainedSupport the method that is encapsulated in the human embryo stem cell in alginate beads.

Although there are these progress, but still need to be in dimensional culture system successfully cultivate can be divided in functional and divideSecrete the method for the multipotential stem cell of cell.

Brief description of the drawings

In the time reading by reference to the accompanying drawings foregoing invention content and following detailed description of the invention, can carry out betterUnderstand. For the purpose of illustrating the invention, accompanying drawing has been shown embodiments of the invention. But should be appreciated that the present invention does not limit toIn the accurate arrangement, example and the instrument that illustrate.

Fig. 1 a illustrates and then after promoting (the left figure) and in non-adherent static culture thing 24 according to embodiment 1The warp of dry (" hES ") the clone H1 of the people embryo of (right figure) after hour The micrograph of the cell of processing. Promote (a left sideFigure) cell is afterwards similar to the individual layer fragment that average fragment diameter is about 20-30 micron, and each fragment is by cell mass groupBecome. In non-adherent static culture thing, after 24 hours, cell is presumed to the columnar structure of trooping.

Fig. 1 b illustrates and is containing 25mL according to embodiment 1In the 125mL rolling bottle of culture medium, cultivate after 4 daysThe warp of human embryonic stem cell H1CD9, SSEA4, CXCR4, TRA-1-60 and the TRA-1-81 of the cell of processingFlow cytometry result. For versatility mark (CD9, SSEA4, TRA-1-60 and TRA-1-81), cell shows heightExpress, and for differentiation mark CXCR4, almost do not express.

Fig. 1 c is illustrated in the warp of the 1st stage human embryonic stem cell H1 after differentiation 72 hours and 96 hours while finishingThe micrograph of the cell of processing. In Fig. 1 c, can see loose thin after 72 hours under 4 x magnificationsBorn of the same parents' aggregation (left figure), the loose cell aggregation (middle graph) after 96 hours under 4 x magnifications, and put at 10 timesLoose cell aggregation (right figure) after 96 hours under large rate.

Human embryo stem cell when the 1st stage differentiation that Fig. 1 d is illustrated in label CD9, CD184 (CXCR4) and CD99 finishesIt is the warp of H1The flow cytometry result (referring to embodiment 1) of the cell of processing. As shown in Figure 1 d, versatility markThe expression of thing CD9 is almost eliminated, and the differentiation mark CXCR4 (CD184) of definitive entoderm and the expression of CD99 are quite high.

Fig. 1 e illustrates compared with undifferentiated H1 (WA01) hES cell, the selected gene being associated with versatility withAnd with the warp of the human embryonic stem cell H1 in the time that the 1st stage finishedThe definitive entoderm of the cell of processing is associatedThe result (referring to embodiment 1) of quantitative RT-polymerase chain reaction (qRT-PCR) of expression of gene. With undifferentiatedWA01hES cell is compared, and cell when the 1st stage finished demonstrates versatility gene (CD9, NANOG and POU5F1/OCT4)Express significantly and reduce, and the gene being associated with definitive entoderm (CXCR4, CERBERUS (CER1), GSC, FOXA2, GATA4,GATA6, MNX1 and SOX17) expression roll up.

Fig. 1 f illustrates along with cell further breaks up towards pancreas entoderm from definitive entoderm, human embryonic stem cell H1WarpThe micrograph (referring to embodiment 1) of the cell of processing. The clear metamorphosis of cell and cell cluster can quiltBe considered as from the 1st day the 2nd stage (top left figure) to the 3rd day the 2nd stage (top right figure) to the 4th day the 3rd stage (bottom Zuo Tu)And the atomization in the 4th stage the 1st day (lower right figure).

Fig. 2 a and is transferring to and basis after being illustrated in and cultivating 2 days in stirring suspension culture after EDTA processesHuman embryonic stem cell H1 before the differentiation culture of the versatility of example 2 mark associated with differentiation phase through EDTA placeThe flow cytometry data of the cell of reason. For versatility mark (CD9, SSEA4, TRA-1-60 and TRA-1-81), dataDemonstrate high expressed, and for differentiation mark (CXCR4), almost do not express.

Fig. 2 b illustrates the 3rd day the 1st stage cell of growing in rolling bottle according to being divided into of example 2 and at rolling bottle or coneThe 2nd day the 2nd stage of growing in shape bottle, the warp of the human embryonic stem cell H1 of the 4th the 1st day stage and the 4th stage the 3rd day cellThe micrograph of the cell that EDTA processes. The culture of suspension differentiation forms substantially equal even homogeneities in sphere aggregatesCell mass.

The 1st stage that Fig. 2 c is illustrated in multipotential cell surface marker and entoderm noble cells surface marker finishesTime the flow cytometry data of cell of processing through EDTA of human embryonic stem cell H1. In Fig. 2 c, can see multipotencyThe expression of property mark CD9 is almost eliminated, and the expression of the differentiation mark CXCR4 (CD184) of definitive entoderm is quite high.

Fig. 2 d be illustrated in the 1st stage while finishing compared with undifferentiated H1 (WA01) hES cell, selected and versatilityThe gene being associated and the gene being associated with the definitive entoderm of cell of processing through EDTA of human embryonic stem cell H1The qRT-PCR result (referring to example 2) of expression. Fig. 2 d illustrates the table of versatility gene (CD9, Nanog and POU5F1/OCT4)Reach minimizing, and the gene being associated with definitive entoderm (CXCR4, CERBERUS (" CER1 "), FOXA2, GATA4, GATA6,MNX1 and SOX17) expression roll up.

Fig. 2 e illustrates according to the instruction of example 2 and is divided in pancreas by being suspended in rolling bottle or conical flask from the 1st stageThe mark of the differentiation of the cell of processing through EDTA of the human embryonic stem cell H1 of germinal layer cell (NKX6.1, CDX2, SOX2 andChromograin) flow cytometry data. These flow cytometry data illustrate high-caliber NKX6.1 (functional β cell instituteThe transcription factor needing), and high-caliber endocrine pancreas mark, such as synaptophysin (data are not shown) with have two kinds and hangThe chromograin of floating form.

Fig. 2 f illustrates according to the selected of example 2 with further from the 1st stage by being suspended in rolling bottle or conical flaskBe divided into the gene of the differentiation phase association of the cell of processing through EDTA of pancreas endoderm cell's human embryonic stem cell H1The qRT-PCR result of expressing. Expression in these data and WA01hES cell is compared. RT-PCR result illustrates before pancreasThe high level expression of body gene.

Fig. 3 a is illustrated in useThe human embryonic stem cell H1's having promoted from static culture thing after processing is thinBorn of the same parents' micrograph. As shown in Figure 3 a, cell removes from surface as little aggregation.

Fig. 3 b is illustrated in useAfter processing, promote from static culture thing, and then in suspension cultureThe cell of the human embryonic stem cell H1 increasing three days differ micrograph. In Fig. 3 b, can see substantially spherical uniformlyThe formation that cell mass is trooped.

Fig. 3 c is illustrated in useAfter processing, promote from static culture thing, then in suspension culture, increaseThree days, and then useThe micrograph of cell cluster of human embryonic stem cell H1 of continuous passage dissociates.

The different phase that Fig. 4 a is illustrated in differentiation is used the people embryo of the clone H1 of directed differentiation scheme suspension cultivation to doThe micrograph of cell. In Fig. 4 a, can see the micrograph at the cell of each differential period.

The different phase (starting differentiation latter several hours) that Fig. 4 b is illustrated in differentiation is used the suspension of directed differentiation scheme to cultivateThe flow cytometry result of the differentiation mark (CXCR4, CD56 and PDX1) of the human embryo stem cell of clone H1. On the 4th rankWhen the atomization of the 4th day of section finishes, express for PDX1, the cell of high percentage is positive.

Fig. 4 c illustrates to have transplanted and is encapsulated in TheraCyte^TMThe non-fasting blood of the SCID-Bg mouse of the noble cells in deviceSugar level.

Before Fig. 5 a is illustrated in the differentiation culture of transferring to the mark associated with versatility and differentiation phase, people embryo is dryThe flow cytometry data of the cell of processing through EDTA of clone H1. As shown in Figure 5 a, observe the versatility mark of high expressedWill thing CD9, SSEA4, TRA-1-60 and TRA-1-80.

Fig. 5 b sets for three kinds of different feedings during being illustrated in for the 1st stage, CXCR4/CD184 and CD99 (differentiation markThing) and cell phase difference image and the flow cytometry data of CD9 (versatility mark). Test condition is as follows: (A) drawingSend out differentiation and change culture medium in latter 24 hours, and located not change culture medium at 48 hours; (B) causing differentiation replacing training in latter 24 hoursSupport base and located to add grape sugar-pill at 48 hours; And (C) whole the 1st stage without glucose change culture medium andCause differentiation and add GDF8 ball in latter 24 hours, then within latter 48 hours, locate to add grape sugar-pill in initiation.

Fig. 5 c illustrates the phase difference image of the noble cells that shows pancreas entoderm form, and described noble cells is fixed in formationDuring shape entoderm, use with lower feeding and set differentiation: (A) within latter 24 hours, change culture medium in initiation differentiation, and located at 48 hoursDo not change culture medium; (B) causing latter 24 hours replacing culture mediums of differentiation and locating to add grape sugar-pill at 48 hours; And(C) change culture medium in whole the 1st stage without glucose and causing latter 24 hours interpolation GDF8 balls of differentiation, then drawingAfter sending out, within 48 hours, locate to add grape sugar-pill.

Fig. 5 d is illustrated in the selection marker thing (NKX6.1 and having a liking for of the 4th stage pancreas gene expression of noble cells while finishingChromium corpuscular protein) and select the flow cytometry result of non-pancreas gene (CDX2 and SOX2), described noble cells form fixedDuring shape entoderm, use with lower feeding and set differentiation: (A) within latter 24 hours, change culture medium in initiation differentiation, and located at 48 hoursDo not change culture medium; (B) causing latter 24 hours replacing culture mediums of differentiation and locating to add grape sugar-pill at 48 hours; And(C) change culture medium in whole the 1st stage without glucose and causing latter 24 hours interpolation GDF8 balls of differentiation, then drawingAfter sending out, within 48 hours, locate to add grape sugar-pill.

Fig. 5 e is illustrated in the 4th stage selection pancreas of noble cells and the qRT-PCR of non-pancreas gene expression knot while finishingReally, the use during forming definitive entoderm of described noble cells is set differentiation with lower feeding: (A) causing latter 24 hours of differentiationChange culture medium, and located not change culture medium at 48 hours; (B) causing latter 24 hours replacing culture mediums and little 48 of differentiationTime place adds grape sugar-pill; And (C) 24 little after changing culture medium in whole the 1st stage without glucose and causing differentiationShi Tianjia GDF8 ball, then locates to add grape sugar-pill for latter 48 hours in initiation. These data are shown as with undifferentiated H1 (WA01)The multiple that hES cell is compared is expressed poor (baseline is expressed as 1).

Fig. 5 f illustrates that C-peptide (changes culture medium in latter 24 hours causing differentiation, and at 48 hours having implanted according to condition APlace does not change culture medium) expression in the SCID-Bg mouse of the cell of differentiation. Each SCID-Bg mouse is all planted at subrenal capsule5,000,000 cells are entered. As shown in Fig. 5 f, implant latter 12 weeks, can detect that people c-peptide level is higher than 1ng/mL, and16 weeks time, c-peptide level is average 2.5ng/mL.

Fig. 5 g be illustrated in and use (for example, implanting) according to condition A (within latter 24 hours, change culture medium causing differentiation, andWithin 48 hours, locate not change culture medium) differentiation cell before and afterwards selected SCID-Bg mouse is carried out to glucose processingEffect. As shown in Fig. 5 g, glucose is processed the average 0.93ng/mL of people c-peptide from empty stomach state in induction circulation and is significantly increasedBe added to the 2.39ng/mL in feed state.

Fig. 5 h is illustrated in to use according to condition A and (is causing latter 24 hours replacing culture mediums of differentiation, and locating not at 48 hoursChange culture medium) use Streptozotocin (STZ) (, the sugar of Streptozotocin induction on the SCID-Bg mouse of cell of differentiationUrine is sick) effect. From Fig. 5 h, obviously, the animal with the graft of the effective tissue of functional GSIS (, has been usedThose animals of cell) keep euglycemia level, unlike the untreated control that develops into obvious diabetes.

Fig. 6 a is illustrated in differentiation and exists beforeThe cell of the human embryonic stem cell H1 growing on microcarrier beadMicrograph.

Each stage that Fig. 6 b is illustrated in differentiation is inThe human embryonic stem cell of growing on microcarrier beadThe micrograph of the cell of H1.

Fig. 6 c illustrates cell count (cell/cm²) as the human embryonic stem cell of growing and breaking up on the followingThe function of the differentiation number of days of the cell of H1: the plate (WTN3A/AA plate) in the culture medium that contains activin A (AA) and WNT3A, containsThere is the microcarrier (WTN3A/AA microcarrier) in the culture medium of activin A and WNT3A, in the culture medium that contains MCX and GDF8Plate (MCX/GDF8 plate) and include the microcarrier (MCX/GDF8 microcarrier) in the culture medium of MCX and GDF8.

Fig. 6 d illustrates the human embryonic stem cell H1 of cell count (cell/ml) as growth on the following and differentiationThe function of differentiation number of days of cell: the plate (WTN3A/AA plate) in the culture medium that contains activin A and WNT3A, contain activationMicrocarrier (WTN3A/AA microcarrier) in the culture medium of element A and WNT3A, the plate (MCX/ in the culture medium that contains MCX and GDF8GDF8 plate) and the culture medium that contains MCX and GDF8 in microcarrier (MCX/GDF8 microcarrier).

Fig. 6 e is illustrated in of the cell of growing on microcarrier culture or plane culture under the existence of the followingThe flow cytometry result of one differential period: (a) WNT3A and AA, or (2) MCX and GDF8, as CXCR4/CD184 (Y-axis) andThe dot chart of the cellular expression of CD9 (X-axis).

Fig. 6 f is illustrated in of the cell of growing on microcarrier culture or plane culture under the existence of the followingThe flow cytometry result of one differential period: (a) WNT3A and AA, or (2) MCX and GDF8, as every kind of mark (CXCR4 andCD9) total expression.

Fig. 6 g illustrates the expression of the differentiation phase of cell of selected and human embryonic stem cell H1 associated geneQRT-PCR result, described cell passes through on plane culture or in suspension culture under the existence of the followingOn microcarrier bead, growth is broken up: (a) WNT3A and AA, or (2) MCX and GDF8.

Fig. 7 illustrates for according to the cell of the scheme differentiation of example 7, in bioreactor from the 1st day the 1st stage to theThe cell count that each stage of the 3rd day 4 stages differentiation is located. Millions of cell/ml of cytometer illustrates, as passed through based on figureThe cell instrument of pictureMeasure.

During Fig. 8 is illustrated in the differentiation scheme of embodiment 7, average daily bioreactor culture base pH level is as time (differentiationNumber of days) function. PH level is passed through NOVAFLEX(NovaBiomedicalCorporation,Waltham, MA) determine.

Fig. 9 be illustrated in during the differentiation scheme of example 7 average daily bioreactor culture base lactate levels as the time (pointChange number of days) function. Lactate levels is passed through NOVAFLEX(NovaBiomedicalCorporation,Waltham, MA) determine.

During Figure 10 is illustrated in the differentiation scheme of example 7, average daily bioreactor culture base glucose level is as the timeThe number of days of (differentiation number of days). Glucose level passes through NOVAFLEX(NovaBiomedicalCorporation, Waltham, MA) determine.

Figure 11 illustrates for the versatility array that comprises the Select gene being associated with versatility, according to the scheme of example 7The not differentiation gene of the 1st day the 0th stage (, twenty four hours after the inoculation) cell of differentiation is expressed (as true by qRT-PCRFixed).

Figure 12 illustrates for the definitive entoderm that comprises the Select gene being associated with definitive entoderm (" DE ") array,The 1st day 0 stage not differentiation gene of (, twenty four hours after inoculation) cell is expressed (as determined by qRT-PCR) (referring to realityExample 7).

Figure 13 illustrates for the versatility array that comprises the Select gene being associated with versatility, and the 0th the 3rd day stage (,After inoculation 72 hours) the not differentiation gene of cell expresses (as determined by qRT-PCR) (referring to example 7).

Figure 14 illustrates for the DE array that comprises the Select gene being associated with DE, (, the inoculation rear seven of the 0th the 3rd day stage12 hours) the not differentiation gene of cell expresses (as determined by qRT-PCR) (referring to example 7).

Figure 15 illustrates CD9, the CD184/ to not breaking up the 3rd day the 0th stage (, latter 72 hours of inoculation) cellCXCR4, SSEA4, TRA-1-60 and TRA-1-81 carry out the result (referring to embodiment 7) of fluorescence-activated cell sorting (FACS).Result is also shown in Table 8.

Figure 16 illustrates according to the 1st day the 0th stage of the scheme differentiation of example 7 (, twenty four hours after inoculation) and the 0th rankThe not differentiation gene of the Select gene of the 3rd day (, latter 72 hours of inoculation) cell of section is expressed (as true by qRT-PCRFixed). Specifically, Figure 16 is illustrated in before directed differentiation during the 0th phase process, the gene of GATA4, GSC, MIXL1 and TExpressing appropriateness increases, and the expression of GATA2 increases >=100 times.

Figure 17 illustrates for the DE array that comprises the Select gene being associated with DE, according to the 0th of the scheme differentiation of example 7 theThe not differentiation of the 1st day stage (, twenty four hours after inoculation) and the 3rd day the 0th stage (, latter 72 hours of inoculation) cellGene expression (as determined by qRT-PCR). Specifically, Figure 17 is illustrated in before directed differentiation during the 0th phase process,The expression of CER1, FGF17 and FGF4 increases >=100 times.

Figure 18 and Figure 19 illustrate according to the gene expression of the 1st stage the 1st day cell of the scheme differentiation of example 7. Figure 18 showsGo out for the versatility array that comprises the Select gene being associated with versatility, the gene expression of the 1st stage the 1st day cell (asDetermine by qRT-PCR). Figure 19 illustrates for the DE array that comprises the Select gene being associated with DE, and the 1st the 1st day stage is thinBorn of the same parents' gene expression (as determined by qRT-PCR). Figure 18 and Figure 19 illustrate the remarkable change of gene expression pattern, such asThe expression of FOXA2 increases approximately 700 times, and the expression of CER1, EOMES, FGF17, FGF4, GATA4, GATA6, GSC, MIXL1 and TIncrease by 1000 times.

Figure 20 and Figure 21 illustrate according to the gene expression of the 1st stage the 3rd day cell of the scheme differentiation of example 7. Figure 20 showsGo out for the versatility array that comprises the Select gene being associated with versatility, the gene expression of the 1st stage the 3rd day cell (asDetermine by qRT-PCR). Figure 21 illustrates for the DE array that comprises the Select gene being associated with DE, and the 1st the 3rd day stage is thinBorn of the same parents' gene expression (as determined by qRT-PCR).

Figure 22 illustrate to according to CD9, the CD184 of the 1st stage the 3rd day cell of the scheme differentiation of example 7 (also referred to asCXCR4) and CD99 carry out the result of FACS. Observe the negative pluripotent cell group (figure of CD9 expression/CXCR4 while initiation from differentiation15) the homogeneity CXCR4 while end to the 1st stage expresses cell mass (98.3% cell is the CXCR4 positive, ± 1.9SD) (Figure 22)Approximate change completely.

Figure 23 illustrates for the DE array that comprises the Select gene being associated with DE, according to the 1st of the scheme differentiation of example 7 theThe 3rd day stage; The 2nd the 1st day stage; And the gene expression (as determined by qRT-PCR) of the 2nd stage the 3rd day cell. Figure 23While being illustrated in the 1st day the 2nd stage and the 3rd day, HNF4 α and GATA6 expression increase, and the 3rd day of the 1st stage with Gao ShuiFlat gene (CXCR4, EOMES, FGF17, FGF4, MNX1, PRDM1, SOX17 and VWF) of expressing shows in the time that the 2nd stage finishedGo out to express and reduce.

Figure 24 illustrates according to the 1st day the 2nd stage cell of the scheme differentiation of example 7 and the anterior intestine of the 2nd stage the 3rd day cellThe gene expression (as determined by qRT-PCR) of Gene A FP, PDX1 and PROX1. As shown in figure 24, the expression of these genes increasesAdd.

Figure 25 illustrates the 3rd stage the 3rd of the growth in the 3rd stage culture medium (table 7) to breaking up according to the scheme of example 7PDX1, FOXA2, chromograin, NKX2.2 and the SOX2 of it cell carries out the result of FACS. As shown in figure 25, cellular expressionWith as express by PDX1 and FOXA2 mark (90.9% ± 11.9SDPDX1 that measured entoderm pancreas pedigree is consistentThe positive and 99.2% ± 0.6SDFOXA2 positive).

Figure 26 illustrates for the 4th stage array that comprises the Select gene being associated with the 4th stage, according to the side of example 7The 1st day the 3rd stage of case differentiation and the gene expression (as determined by qRT-PCR) of the 3rd stage the 3rd day cell. Figure 26 illustratesThese cells show increase level the gene of conventionally expressing in pancreas (ARX, GAST, GCG, INS, ISL1, NEUROD1,NGN3, NKX2.2, NKX6.1, PAX4, PAX6, PTF1A and SST) host.

Figure 27 illustrates NKX6.1, the chromograin of the 4th stage the 3rd day cell to breaking up according to the scheme of example 7(CHGA), CDX2, SOX2, NKX2.2, PDX1, FOXA2 and NEUROD carry out the result of FACS. As shown in figure 27, in the 4th stageThe 3rd day time, cell keeps high-caliber PDX1 and FOXA2 express and further develop and pancreatic endocrine cell (28.1%± 12.5SD chromograin the positive) with the consistent table of mixture of pancreatic progenitor cell (58.3% ± 9.7SDNKX6.1 positive)Expression patterns.

Figure 28 illustrates for the 4th stage array that comprises the Select gene being associated with the 4th stage, according to the side of example 7The 3rd day the 3rd stage of case differentiation; The gene expression of the 4th the 1st day stage and the 4th stage the 3rd day cell is (as passed through qRT-PCRDetermine). Figure 28 illustrate the gene of conventionally expressing in pancreas (ARX, GAST, GCG, IAPP, INS, ISL1, MAFB, NEUROD1,NGN3, NKX2.2, NKX6.1, PAX4, PAX6, PTF1A, and SST) expression increase.

Figure 29 illustrates NKX6.1, the chromograin of the 4th stage the 3rd day cell to breaking up according to the scheme of example 7(CHGA), CDX2, SOX2, NKX2.2, PDX1, FOXA2 and NEUROD carry out the average result of FACS. Specifically, Figure 29 showsGo out the mean F ACS expression pattern of criticizing the pancreas precursor of inoculation material generation with 3L scale from difference.

Figure 30 illustrates NKX6.1, the chromograin of the 4th stage the 3rd day cell to breaking up according to the scheme of example 7(CHGA), CDX2, SOX2, NKX2.2, PDX1, FOXA2 and NEUROD carry out the average result of FACS. Thin the 3rd day the 4th stageBefore born of the same parents' differentiation, cell increases to form ISM, then the 0th stage be in customization inside culture medium " IH3 " orEssential8^TMIn any one in grow, the two is all supplemented with 0.5%BSA. The cell of growing in IH3 culture medium is" IH3-P auxocyte ", and at Essential8^TMThe cell of middle growth is " EZ8 auxocyte ". In different culture media, growCell between do not observe the significant difference of expression pattern.

Figure 31 illustrate the 4th stage the 3rd day cell to growing in the 0th stage with different pH levels before NKX6.1,The average result that chromograin (CHGA), CDX2, SOX2, NKX2.2, PDX1, FOXA2 and NEUROD carry out FACS is (referring to realityExample 7). In the 3rd day the 4th stage cell distribution, do not observe remarkable change.

Figure 32 compared NKX6.1 to following cell, chromograin (CHGA), CDX2, SOX2, NKX2.2, PDX1,FOXA2 and NEUROD carry out the result of FACS: the 3rd day the 4th stage cell of not processing with defoamer C, and with defoamer C breastThe 3rd day the 4th stage cell (referring to example 7) that liquid (94ppm) is processed. Observe defoamer C emulsion (Sigma catalog number (Cat.No.)A8011) can not affect the distribution of the 4th stage the 3rd day cell.

Figure 33 to Figure 35 illustrates according to the gene expression of the Select gene of the cell of the scheme differentiation of example 8 (as passed throughQRT-PCR determines). Figure 33 is illustrated in differentiation and starts twenty four hours before, and the gene expression of the Select gene of cell is (as passed throughQRT-PCR determines) (referring to example 8). As shown in figure 33, from the table of the cell maintainer gene versatility feature of bioreactorReach (POU5F1, NANOG, SOX2 and ZFP42) and demonstrate the minimum induction to Gene Differentiation feature or do not induce (AFPAnd FOXA2: express and increase < 50 times; FOXD3, GATA2, GATA4, GSC, HAND2, MIXL1 and T: express and increase < 10 times). Figure34 are illustrated in differentiation starts twenty four hours afterwards, the gene expression (as determined by qRT-PCR) of the Select gene of cell. Figure35 are illustrated in differentiation starts 72 hours afterwards, the gene expression (as determined by qRT-PCR) of the Select gene of cell.

Figure 36 (a) illustrates according to the scheme of example 8 from the differentiation of the 2nd 3 stages of stage to the and the 4th stage to Figure 36 (e)The gene expression (as determined by qRT-PCR) of the Select gene of cell. Specifically, these illustrate cell in the 2nd stageThe 1st day; The 2nd the 2nd day stage; The 2nd the 3rd day stage; The gene expression in the 3rd the 3rd day stage and the 1st day the 4th stage, specifically getsCertainly in gene. Figure 36 (a) illustrates the gene expression of AFP, ATOH1 and CDX2. Figure 36 (b) illustrate GAST, HAND1, HHEX andThe gene expression of HNF4a. Figure 36 (c) illustrates the gene expression of NKX2.2, NKX6.1, OSR1 and PDX1. Figure 36 (d) illustratesThe gene expression of PROX1, PFT1a, SOX17 and SOX2. Figure 36 (e) illustrates the gene expression of SOX9. These data be shown as with notThe difference (baseline is expressed as 1) of the expression that H1 (WA01) the hES cell of differentiation is compared.

Figure 37 illustrate according to the gene expression of the Select gene of the 4th stage the 3rd day cell of the scheme differentiation of example 8 (asDetermine by qRT-PCR). As shown in figure 37, in the time that the differentiation in the 3rd day the 3rd stage finishes, it is thin that cell has been divided into pancreas ancestralBorn of the same parents, compared with undifferentiated H1 human embryo stem cell, this pancreatic progenitor cell is characterised in that: the PDX1 (> 1 of high expression level ×10⁶Doubly induction) and ARX, the GCG of 1000 times of other pancreas genes (>, GAST, INS, ISL, NEUROD1, NGN3, NKX2.2,The induction of NKX6.1, PAX4, PTF1a and SST) and OCT4/POU5F1 express approach total losses.

Figure 38 illustrates according to cell count every day between the scheme idiophase of example 8. Specifically, Figure 38 illustrates that cell is closeDegree is as the function of processing number of days. Figure 38 is illustrated in pH6.8 and two reactors operation of carrying out for 7.2 times (PRD1205 andThe cell count of differentiation scheme PRD1207). The cell count of cell drift is also shown as a comparison.

Figure 39 (a) illustrates according to the scheme differentiation of example 8 and is implanted to the 4th rank in SCID-Bg mouse to Figure 39 (d)The in vivo bioactivity of the 3rd day cell of section. Cell passes through TheraCyte^TMInstall in the subcutaneous implantation of subrenal capsule, or ultralowAdhere in culture dish and implant after incubation. Blood sugar level and the C-peptide level of every surrounding monitoring mouse after graft is implanted.Figure 39 (a) is illustrated in TheraCyte^TMIn device, implant 5 × 10⁶Individual or 10 × 10⁶C-peptide after individual the 3rd day the 4th stage cellLevel is as the function of time. Figure 39 (b) is illustrated in TheraCyte^TMIn device, implant 5 × 10⁶Individual or 10 × 10⁶Individual the 4th rankThe non-fasting blood glucose level of animal after the 3rd day cell of section. Process mouse in Figure 39 (b) to eliminate with STZ before implantingHost's Instreptozotocin Induced. Figure 39 (c) is illustrated in TheraCyte^TMBefore implanting in device frozen the 3rd day the 4th stage cell itThe C-peptide level of rear generation is as the function of time (a few weeks longer after implanting). Figure 39 (d) has compared the kidney transplant by following cellThe C-peptide level of the mouse of thing processing: never frozen/fresh the 3rd day the 4th stage cell, or after thawing (D0) immediatelyImplantation or frozen the 3rd day the 4th stage cell that 1 day (D1) implants after thawing.

Figure 40 A to Figure 40 D illustrates according to CXCR4, the CD99 of the scheme differentiation cell of three days of example 9 and the FACS of CD9Figure, described cell was processed with the following the 1st day the 1st stage: MCX compound and GDF-8 (Figure 40 A); Only MCX (figure40B); WNT3A and activin A (Figure 40 C); And WNT3A (Figure 40 D) only. These figure instruction, in suspension culture, notExist in TGF-'beta ' family member's situation and to add 3 μ MMCX at the first day of differentiation and can generate and be equivalent at 3 μ M for first dayMCX adds that 100ng/mlGDF-8 or 20ng/mlWNT-3a add the water that 100ng/ml activin A obtains while processing cellFlat definitive entoderm.

Figure 41 A to Figure 41 D illustrates according to CXCR4, the CD99 of the scheme differentiation cell of three days of example 10 and the FACS of CD9Figure, described cell is processed at the MCX of the 1st day the 1st stage by various amounts. Specifically, used the following the 1st day the 1st stageProcess cell: 4 μ MMCX (Figure 41 A); 3 μ MMCX (Figure 41 B); 2 μ MMCX (Figure 41 C); And 1.5 μ MMCX (Figure 41 D).

Figure 42 A and Figure 42 B illustrate according to CXCR4, the CD99 of the scheme differentiation cell of three days of example 11 and the FACS of CD9Figure. Specifically, these illustrate the effect of culture medium replacement frequency in suspension culture. Figure 42 A illustrates according to example 10Scheme differentiation CXCR4, the CD99 of cell of three days and the FACS of CD9 figure, wherein carry out culture medium completely more in the 1st stageChange. Figure 42 B illustrates according to scheme differentiation CXCR4, the CD99 of cell of three days of example 10 and the FACS of CD9 figure, wherein the 3rdIt does not carry out culture medium replacing. Data show, in suspension culture systems, in the training that culture medium is changed of accepting for the 3rd day of differentiationSupport thing (Figure 42 A) produce definitive endoderm, its have can with do not accept at the 3rd day culture medium change culture (Figure 42 B) phaseThe efficiency of ratio.

Figure 43 A and Figure 43 B illustrate according to CXCR4, the CD99 of the scheme differentiation cell of three days of example 12 and the FACS of CD9Figure. Particularly, these illustrate the Glutamax in suspension culture^TMEffect. Be supplemented with 1X in the 1st stageGlutamax^TM(Figure 43 A) or containing Glutamax^TMOr any glutamine (0MGlutamax^TM) in the culture medium of (Figure 43 B)Cultured cell. Data show, in suspension culture systems, add Glutamax^TMSeem not affect the effect that produces definitive entodermRate.

The sodium acid carbonate that Figure 44 A to Figure 44 D illustrates various amounts is on according to the impact of the cell of the scheme differentiation of example 13. Figure44A and Figure 44 B illustrate according to scheme differentiation CXCR4, the CD99 of cell of three days of example 13 and the FACS of CD9 figure, Qi ZhongThe 1st stage was added 3.64g/l (Figure 44 A) or 2.49g/l (Figure 44 B). Figure 44 C and Figure 44 D illustrate according to the scheme of example 13 and divideChange three days cell differ micrograph, wherein add 3.64g/l (Figure 44 C) or 2.49g/l (Figure 44 D) in the 1st stage.

Cell count every day that Figure 45 illustrates cell density is as according to the differentiation of the cell of the scheme differentiation of example 14Function. Use the cell instrument based on imageObtain cell count.

Figure 46 be illustrated in during the differentiation scheme of embodiment 14 average daily bioreactor culture base pH level as the time (pointChange number of days) function. PH level is passed through NOVAFLEX(NovaBiomedicalCorporation,Waltham, MA) determine.

During Figure 47 is illustrated in the differentiation scheme of example 14, average daily bioreactor culture base glucose level is as the timeThe function of (differentiation number of days). Glucose level passes through NOVAFLEX(NovaBiomedicalCorporation, Waltham, MA) determine.

During Figure 48 is illustrated in the differentiation scheme of example 14, average daily bioreactor culture base lactate levels is as the timeThe function of (differentiation number of days). Lactate levels is passed through NOVAFLEX(NovaBiomedicalCorporation, Waltham, MA) determine.

Figure 49 illustrates for the versatility array that comprises the Select gene being associated with versatility, according to the scheme of example 14The 1st day to the 3rd day the 0th stage and the 1st day the 1st stage to the gene expression of the 3rd day cell of differentiation (are determined by qRT-PCRFor the multiple compared with neoblast is expressed). Figure 50 illustrates for the DE array that comprises the Select gene being associated with DE, rootThe the 1st to the 3rd day the 0th stage of the scheme of example 14 differentiation factually; The 1st the 1st day stage is to the 3rd day and the 1st day the 2nd stage toThe gene expression (multiple being defined as compared with neoblast by qRT-PCR is expressed) of 3 days cells.

Figure 51 illustrates the mark being associated with versatility to the 0th phase cell before breaking up according to the scheme of example 14Thing (CD184/CXCR4, SSEA4, TRA-1-60 and TRA-1-81) carries out the result of FACS. Specifically, Figure 51 illustrates with manyThe high expressed of the mark that energy property is associated.

Figure 52 illustrates the definitive entoderm mark of the cell breaking up in the time that the 1st stage finished according to the scheme of example 14The FACS figure of CXCR4, CD99 and CD9.

Figure 53 illustrated according to the 1st day the 2nd stage of the scheme differentiation of example 14; The 2nd the 2nd day stage and the 2nd stage the 3rdThe gene expression of GAPDH, AFP, HHEX, HNF4 α, PDX1 and the PROX1 of it cell (is defined as and is not broken up carefully by qRT-PCRThe multiple that born of the same parents compare is expressed). Figure 53 illustrates that the expression of anterior intestine gene (AFP, HHEX, PDX1 and PROX1) increases.

Figure 54 illustrates that the 1st day to the 3rd day and the 3rd stage, the 1st day extremely according to the 2nd stage of the scheme differentiation of example 14GAPDH, AFP, CDX2, GAST, HNF4A, NKX2-2, the OSR1 of the 3rd day cell, the gene expression of PDX1 and PFT1A is (by qRT-The multiple that PCR is defined as compared with neoblast is expressed). As shown in Figure 54, the expression of PDX1 finished from the 3rd day the 2nd stageTime exceed contrast 12,000 times in the time that the 3rd day the 3rd stage finished, exceed contrast 739,000 times increased by 60 times.

Figure 55 illustrates according to the 1st day to the 3rd day the 3rd stage of the scheme differentiation of example 14 and the 1st day the 4th stage to theThe gene expression (multiple being defined as compared with neoblast by qRT-PCR is expressed) of some gene of 3 days cells. ParticularlySay, the top graph of Figure 55 illustrates GAPDH, AFP, ALB, ARX, CDX2, CHGA, GAST, GCG, IAAP, INS, ISL1 and MAFBGene expression. The bottom diagram of Figure 55 illustrate MAFB, MUCS, NEUROD1, NEUROG3, NKX2-2, NKX6-1, PAX4, PDX1,The gene expression of POUSF1, PTF1A, SST and ZlC1.

Figure 56 shows according to the terminal stage micrograph of the cell of the scheme differentiation of example 14. In Figure 56, can seeArrive the representative micrograph (4X) of the cell cluster in the time that the differentiation in the 0th stage and the 1st 4 stages of stage to the finishes.

Figure 57 to Figure 80 is illustrated in following Gene Differentiation 0 hour, 6 hours, 24 hours, 30 hours, 48 hours and 72 hours(be defined as and do not break up carefully by qRT-PCR according to the gene expression of the cell of the various embodiment differentiation of the scheme of example 15 afterwardsThe multiple that born of the same parents compare is expressed): AFP (Figure 57), CD99 (Figure 58), CD9 (Figure 59), CDH1 (Figure 60), CDH2 (Figure 61), CDX2(Figure 62), CER1 (Figure 63), CXCR4 (Figure 64), FGF17 (Figure 65), FGF4 (Figure 66), FOXA (Figure 67), GADPH (Figure 68),GATA4 (Figure 69), GATA6 (Figure 70), GSC (Figure 71), KIT (Figure 72), MIXL1 (Figure 73), MNX1 (Figure 74), NANOG (figure75), OTX2 (Figure 76), POUF5F1 (Figure 77), SOX17 (Figure 78), SOX7 (Figure 79) and T (Figure 80).

Figure 81 illustrates according to the various embodiments differentiation of the scheme of embodiment 15 6 hours, 24 hours, 30 hours, 48 littleTime and 72 hours after G0/G1 cell cycle of cell in cell percentage. Specifically, Figure 81 is illustrated in of differentiationOne day result of trooping with a kind of processing in following six kinds of conditions: (1) is pure, and (2) 3 μ MMCX add 100ng/mlGDF-8 (catalog number (Cat.No.) 120-00, Peprotech), (3) are 3 μ MMCX only, (4) only 100ng/mlGDF-8, (5) 20ng/mlWNT-3A (catalog number (Cat.No.) 1324-WN-002, R&DSystems, MN) adds 100ng/ml activin A (catalog number (Cat.No.) 338-AC, R&DSystems, MN) or (6) only 20ng/mlWNT-3A.

Figure 82 illustrates that EDU processes the effect of the cell cluster to breaking up according to the scheme of example 15. A left side of Figure 82 illustratesBreak up 0 hour, 6 hours, 24 hours, 30 hours, 48 hours and 72 hours according to the various embodiments of the scheme of embodiment 15Cell percentage in the G2/M cell cycle of cell afterwards. Specifically, a left side illustrates below the first day of differentiation is usedThe result of trooping of a kind of processing in six kinds of conditions: (1) is pure, (2) 3 μ MMCX add 100ng/mlGDF-8 (catalog number (Cat.No.)120-00, Peprotech), (3) are 3 μ MMCX only, (4) only 100ng/mlGDF-8, (5) 20ng/mlWNT-3A (catalog number (Cat.No.)1324-WN-002, R&DSystems, MN) add 100ng/ml activin A (catalog number (Cat.No.) 338-AC, R&DSystems, MN) or(6) 20ng/mlWNT-3A only. In one group of data, these are trooped and also process with EDU. The right side of Figure 82 illustrates according to example 15The various embodiment of scheme break up 0 hour, 6 hours, 24 hours, 30 hours, 48 hours and be for 72 hours the cell of the EDU positivePercentage.

Figure 83 is illustrated in the general work parameter using in the scheme of example 15.

Figure 84 illustrate break up 6 hours, 24 hours, 30 hours, 48 hours according to the various embodiment of the scheme of example 15 andThe amount of the EDU combination of the cell after 72 hours. Specifically, the first day that Figure 84 is illustrated in differentiation is with in following six kinds of conditionsA kind of result of EDU incubation cell cluster of processing: (1) is pure, and (2) 3 μ MMCX add 100ng/mlGDF-8 (catalog number (Cat.No.)120-00, Peprotech), (3) are 3 μ MMCX only, (4) only 100ng/mlGDF-8, (5) 20ng/mlWNT-3A (catalog number (Cat.No.)1324-WN-002, R&DSystems, MN) add 100ng/ml activin A (catalog number (Cat.No.) 338-AC, R&DSystems, MN) or(6) 20ng/mlWNT-3A only.

Figure 85 illustrates according to the various embodiments differentiation of the scheme of embodiment 15 6 hours, 24 hours, 30 hours, 48 littleTime and 72 hours after G0/G1 cell cycle of cell in cell percentage. Specifically, Figure 85 is illustrated in of differentiationOne day result of trooping with a kind of processing in following six kinds of conditions: (1) is pure, and (2) 3 μ MMCX add 100ng/mlGDF-8 (catalog number (Cat.No.) 120-00, Peprotech), (3) are 3 μ MMCX only, (4) only 100ng/mlGDF-8, (5) 20ng/mlWNT-3A (catalog number (Cat.No.) 1324-WN-002, R&DSystems, MN) adds 100ng/ml activin A (catalog number (Cat.No.) 338-AC, R&DSystems, MN) or (6) only 20ng/mlWNT-3A.

Figure 86 illustrates according to the various embodiments differentiation of the scheme of embodiment 15 6 hours, 24 hours, 30 hours, 48 littleTime and 72 hours after S phase cell cycle of cell in cell percentage. Specifically, Figure 86 is illustrated in first of differentiationIt is by the result of trooping of a kind of processing in following six kinds of conditions: (1) is pure, and (2) 3 μ MMCX add 100ng/mlGDF-8(catalog number (Cat.No.) 120-00, Peprotech), (3) are 3 μ MMCX only, (4) only 100ng/mlGDF-8, (5) 20ng/mlWNT-3A(catalog number (Cat.No.) 1324-WN-002, R&DSystems, MN) adds 100ng/ml activin A (catalog number (Cat.No.) 338-AC, R&DSystems, MN) or (6) only 20ng/mlWNT-3A.

Figure 87 illustrated according to the various embodiments differentiation a few hours, 6 hours, 24 hours of the scheme of embodiment 15,30 littleTime, cell after 48 hours and 72 hours the S phase cell cycle in cell percentage. Specifically, Figure 87 is illustrated in pointThe result of trooping of a kind of processing in following six kinds of conditions for the first day of changing: (1) is pure, and (2) 3 μ MMCX add 100ng/MlGDF-8 (catalog number (Cat.No.) 120-00, Peprotech), (3) are 3 μ MMCX only, (4) only 100ng/mlGDF-8, (5) 20ng/mlWNT-3A (catalog number (Cat.No.) 1324-WN-002, R&DSystems, MN) adds 100ng/ml activin A (catalog number (Cat.No.) 338-AC, R&DSystems, MN) or (6) only 20ng/mlWNT-3A.

Figure 88 A to Figure 88 E is illustrated in and breaks up basis after 0 hour, 6 hours, 24 hours, 30 hours, 48 hours and 72 hoursThe gene expression of the cell of the various embodiment differentiation of the scheme of example 15 (is defined as compared with neoblast by qRT-PCRMultiple express). Figure 88 A illustrates that the gene expression of CD99, CD9, CDH1 and CDH2 (is defined as and is not broken up carefully by qRT-PCRThe multiple that born of the same parents compare is expressed). Figure 88 A illustrates that the gene expression of CXD2, CER1, CXCR4 and FGF17 (is defined as by qRT-PCRMultiple compared with neoblast is expressed). Figure 88 C illustrates that the gene expression of FGF4, FOXA, GATA4 and GATA6 is (by qRT-The multiple that PCR is defined as compared with neoblast is expressed). Figure 88 D illustrates the gene expression of GSC, KIT, MIXL1 and MNX1(multiple being defined as compared with neoblast by qRT-PCR is expressed). Figure 88 E illustrate NANOG, OTX2, POUF5F1 andThe gene expression (multiple being defined as compared with neoblast by qRT-PCR is expressed) of SOX17. Figure 88 F illustrates SOX7 and T'sGene expression (multiple being defined as compared with neoblast by qRT-PCR is expressed). The master data of Figure 88 A to Figure 88 F is shownIn Figure 58 to Figure 67 and Figure 69 to Figure 80.

Figure 89 illustrates the gene expression of the pluripotent cell of cultivating in differentiation of germinal layers culture medium outside according to the scheme of example 16Pattern (as determined by qRT-PCR). As shown in Figure 89, cell breaks up towards nerve cell pedigree. Specifically, a left side of Figure 89Illustrate the gene expression pattern of the induced multi-potent stem cells system being produced by umbilical cord tissue cell (UTC). The right side of Figure 89 illustratesThe gene expression pattern of the WB0106 subclone of H1hES clone.

Figure 90 illustrates the gene expression of the pluripotent cell of cultivating in mesoderm differential medium according to the scheme of example 16Pattern (as determined by qRT-PCR). As shown in Figure 90, cell breaks up towards heart cell pedigree. Specifically, a left side of Figure 90Illustrate the gene expression pattern of the induced multi-potent stem cells system being produced by umbilical cord tissue cell (UTC). The right side of Figure 90 illustratesThe gene expression pattern of the WB0106 subclone of H1hES clone.

Figure 91 illustrates the gene expression of the pluripotent cell of cultivating in differentiation of germinal layers culture medium outside according to the scheme of example 16Pattern (as determined by qRT-PCR). As shown in Figure 91, cell breaks up towards nerve cell pedigree. Specifically, a left side of Figure 91Illustrate the gene expression pattern of the induced multi-potent stem cells system being produced by umbilical cord tissue cell (UTC). The right side of Figure 91 illustratesThe gene expression pattern of the WB0106 subclone of H1hES clone.

Figure 92 illustrate according to the scheme of example 16 cultivate in differentiation of germinal layers culture medium outside the pluripotent cell of three days PAX6,The gene expression pattern (as determined by FACS) of SOX2 and POU5F1/OCT4. Specifically, a left side of Figure 92 illustrates by umbilical cordPAX6, the SOX2 of the induced multi-potent stem cells system that histocyte (UTC) produces and the expression pattern of POU5F1/OCT4. Figure 92'sThe right side illustrates PAX6, the SOX2 of WB0106 subclone of H1hES clone and the protein expression mode of POU5F1/OCT4.

Figure 93 illustrates the gene expression of the pluripotent cell of cultivating in mesoderm differential medium according to the scheme of example 16Pattern (as determined by qRT-PCR). As shown in Figure 93, cell breaks up towards heart cell pedigree. Specifically, a left side of Figure 93Illustrate the gene expression pattern of the induced multi-potent stem cells system being produced by umbilical cord tissue cell (UTC). The right side of Figure 93 illustratesThe gene expression pattern of the WB0106 subclone of H1hES clone.

Figure 94 illustrates the micrograph of the cell breaking up in mesoderm differential medium according to the scheme of example 16. As Figure 94Shown in, cell breaks up towards heart cell pedigree. Specifically, a left side of Figure 94 illustrates the 3rd day, the 5th day and the in differentiationThe micrograph of the cell of the WB0106 subclone of the H1hES clone of 10 days. The right side of Figure 94 illustrates after differentiation 10 days, byThe micrograph of the induced multi-potent stem cells system that umbilical cord tissue cell (UTCiPSC) produces.

Figure 95 illustrates the micrograph of the cell breaking up in differentiation of germinal layers culture medium outside according to the scheme of example 16. As Figure 95Shown in, cell breaks up towards nerve cell pedigree. Specifically, a left side of Figure 95 illustrates the 3rd day, the 5th day and the in differentiationThe micrograph of the cell of the WB0106 subclone of the H1hES clone of 10 days. The right side of Figure 95 illustrates after differentiation 10 days, byThe micrograph of the induced multi-potent stem cells system that umbilical cord tissue cell (UTCiPCS) produces.

Detailed description of the invention

The application relates to and prepares embryonic stem cell and other pluripotent cells, and this pluripotent cell keeps assembling the many of cell clusterEnergy property is to be divided into entoderm CFU-GM, pancreatic endocrine cell, mesoblastema or ectoderm cell. For with not limitedThe clear explanation disclosure of mode of system, is divided into the specific embodiment of the present invention following description or illustrates some spy of the present inventionLevy, the trifle of embodiment or application.

Definition

Stem cell is by its neoblast that not only self but also the ability of breaking up define in unicellular level.Stem cell can produce daughter cell, comprises self CFU-GM, non-renewal CFU-GM and terminally differentiated cells. The spy of stem cellLevy and be also that it is divided into the function from the various kinds of cell pedigree of multiple germinal layers (entoderm, mesoderm and ectoderm) in vitroThe ability of cell. Stem cell also produces the tissue of multiple germinal layer afterwards in transplanting, and after being expelled in blastocyst, facilitates basicUpper to most of (if not all) tissue.

Stem cell is according to its potentiality of development classification. " cell cultivation " or " cultivation " generally refer to from live organism and obtainAnd the cell of growth (" cultivation " or " by cultivating ") under controlled condition. Primitive cell culture is in first cultivation of going down to posterityCell, tissue or organ that front cultivation directly obtains from organism. When being conducive to the one or two of Growth of Cells and/or divisionUnder person's condition, when cell is placed in growth medium, they increase and produce larger cell mass in cultivation. When cell existsWhile amplification in cultivation, sometimes measure cell proliferation rate by the double required time quantum of cell number (being called the doubling time).

As used herein, " amplification " is the process that increases the number of multipotential stem cell by cultivation, such as increase at least about5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、60％、75％、90％、100％、200％、500%, 1000% or more, and level in these percentages. Should be appreciated that can obtain from single pluripotent stem cell manyThe number of energy stem cell depends on the multiplication capacity of multipotential stem cell. The multiplication capacity of multipotential stem cell can by cell doublyThe increasing time (that is, cell experiences the required time of mitosis in cultivation), and multipotential stem cell can remain on and not break up shapeCycle (it equals number of passages and is multiplied by the number of days between at every turn going down to posterity) in state calculates.

Differentiation is such process, and by this process, the cell of unspecialized (" nondirectional ") or few specialization can obtainObtain the feature of specialized cell (such as nerve cell or myocyte). The cell of differentiation or the cell of induction are to compose at cellIn system, occupy the more cell of (" orientation ") position of specialization. Term " directed " in the time being applied to atomization, refer in differentiationIn approach, proceed to so a kind of cell of degree: under home, it can continue to be divided into specific cell typeOr cell type subgroup, and under home, can not be divided into another cell type or be returned to the cell that differentiation degree is lowerType. " dedifferente " phalangeal cell and be returned to the process of specialization (or directed) status that degree is lower in the middle of the pedigree of cell. Herein" cell lineage " used limits the heredity of cell, and what cell it can produce from which cell and it. Cell lineage willCelluar localization is in the hereditary scheme of growing and break up. Pedigree Specific marker refers to the phenotype of the cell of paid close attention to pedigree brightReally relevant feature, and can be used to assess not committed cell to the differentiation of paid close attention to pedigree.

Nucleic acid or peptide molecule that " mark " used herein is differential expression in paid close attention to cell. In this linguistic contextIn, differential expression means the level rising of positive mark compared with neoblast and the level of negative mark declines.Compared with other cells, can detection level higher or lower fully in paid close attention to cell of mark nucleic acid or polypeptide, makeCan use in several different methods known in the art any by paid close attention to cell and other Identification cell lines with make a distinction.

As used herein, when special sign thing detected in cell time, cell " being positive for special sign thing "Or " positive ". Similarly, when special sign thing fully do not detected in cell time, cell " for special sign thing isNegative " or " negative ". Specifically, the positive detecting with FACS is greater than 2% conventionally, and the moon detecting with FACSProperty threshold value is less than 1% conventionally. The positive detecting with PCR is less than 34 circulations (Cts) conventionally; And the feminine gender detecting with PCRConventionally be greater than 34.5 circulations.

As used herein, " cell density " and " inoculum density " is used interchangeably herein, and refers to per unit areaSolid or the quantity of the cell inoculated of semi-solid smooth or bending culture medium.

As used herein, " suspension culture " refers to and is suspended in culture medium instead of adherent lip-deep cell, the list of arrivingIndividual cell or the culture of trooping.

As used herein, " serum-free " refers to not containing human or animal's serum. Therefore, serum free medium do not comprise serum orThe part of serum.

In the trial copying in functional pancreatic endocrine cell multipotential stem cell being broken up in cell culture,Atomization is regarded as being undertaken by multiple successive stages conventionally. As used herein, each stage is by incubation time, and this paperThe reagent of listing in the example comprising limits.

As used herein, " definitive entoderm " refers to have the spy of the cell producing from epiblast in primitive gut forming processProperty also forms the cell of intestines and stomach and derivative thereof. At least one in the following mark of definitive entoderm cellular expression: FOXA2(also referred to as HNF 3-β (HNF3 β)), GATA4, GATA6, MNX1, SOX17, CXCR4, Cerberus, OTX2,Brachyury, goosecoid, C-Kit, CD99 and MIXL1. The characteristic mark of definitive entoderm cell comprise CXCR4,FOXA2 and SOX17. Therefore, definitive entoderm cell can characterize by it in the expression to CXCR4, FOXA2 and SOX17. ThisOutward, depend on and allow cell to remain on the duration in the 1st stage, can be observed the growth of HNF4 α.

As used herein, " pancreatic endocrine cell " refers to express the cell of at least one following hormone: insulin, pancreasGlucagons, growth hormone release inhibiting hormone, ghrelin and pancreatic polypeptide. Except these hormones, pancreatic endocrine cell characteristicMark also comprises following one or more marks: NGN3, NeuroD1, ISL1, PDX1, NKX6.1, PAX4, ARX,NKX2.2 and PAX6. Express the pancreatic endocrine cell of the characteristic mark of β cell can be by it to insulin and at leastA kind of expression of following transcription factor characterizes: PDX1, NKX2.2, NKX6.1, NeuroD1, ISL1, HNF3 β, MAFA, PAX4And PAX6.

That be used interchangeably in this article is " d1 ", " d1 " and " the 1st day "; " d2 ", " d2 " and " the 2nd day "; " d3 ", " d3 " and " the 3rd day " etc. The combination of these digital alphabets refers to progressively breaks up the different phase in scheme process in present patent applicationThe concrete number of days of middle incubation.

" glucose " and " D-Glucose " is used interchangeably in this article, and refers to dextrose, conventionally sends out at occurring in natureExisting sugar.

That be used interchangeably in this article is " NeuroD " and " NeuroD1 ", and it is identified in pancreatic endocrine CFU-GMProtein and the encoding gene thereof of expressing.

" LDN " and " LDN-193189 " refers to ((6-(4-(2-(piperidin-1-yl) ethyoxyl) phenyl)-3-(pyridine-4-Base) pyrazolo [1,5-a] pyrimidine, hydrochloride; DM-3189)), one can trade mark STEMOLECULETM purchased from Stemgent,Inc., Cambridge, MA, the bmp receptor inhibitor of USA.

Separation, amplification and the cultivation of multipotential stem cell

Multipotential stem cell can express the TRA-1-60 of one or more appointments and TRA-1-81 antibody (people such as Thomson,1998, Science282:1145-1147). Multipotential stem cell differentiation in vitro causes TRA-1-60 and TRA-1-81 to expressForfeiture. Undifferentiated multipotential stem cell has alkaline phosphatase activities conventionally, and this activity can be by using 4% paraformaldehydeFixed cell, then usesRed develops the color to detect as substrate, as manufacturer (VectorLaboratories,Inc., Burlingame, CA) as described in. Undifferentiated multipotential stem cell is also expressed OCT4 and TERT conventionally, and this passes throughRT-PCR detects.

Another desirable phenotype of multipotential stem cell of propagation is that to be divided into all three germinal layers be entoderm, mesoderm and outerThe potential of the cell of germinal layer tissue. Can for example confirm in the following way the versatility of stem cell: cell is injected into heavilyIn disease combined immunodeficiency (" SCID ") mouse, use 4% the fixing teratoma forming of paraformaldehyde, then it is carried out to groupKnit to learn and detect, find out the evidence from the cell type of these three germinal layers. Select as another kind, can be by producing embryoidAnd versatility is determined in the existence of assessing in embryoid the mark relevant to three germinal layers.

Propagation multipotential stem cell be available standards G banding technique carry out karyotyping and with disclosed corresponding primate thingThe caryogram of planting is compared. Wish to obtain and there is the cell of " normal karyotype " (it means cell is euploid), wherein everyoneChromosome all exists and changes without obvious. Pluripotent cell can be used multiple feeder layer or pass through to use matrix in cultureThe container of protein coating easily increases. Alternatively, for example can use the culture medium definite with compositionCulture mediumThe definite surface of chemical composition of (StemCellTechnologies, Vancouver, Canada) combination is carried out conventional thinBorn of the same parents' amplification.

Cultivate in suspension culture according to the method for some embodiments of the present invention and pass through to allow cell survival and increasingGrow, but the cell density of restriction differentiation is inoculated multipotential stem cell and realizes in culture vessel. Conventionally, use and maintain cell and do not divideThe inoculum density of changing. Although should be appreciated that the single cell suspension that can inoculate stem cell, little cell cluster may be favourable.

For the multipotential stem cell of enough and sustainable supply nutriment and growth factor is provided in the time suspending cultivation, can be everyOr it is at predetermined time table, replaces or supplementing culture medium such as every 1-5 days. Large multipotential stem cell is trooped and can be caused cell to divideChange, therefore can take measures the multipotential stem cell aggregation of avoiding large. According to embodiments more of the present invention, formMultipotential stem cell is trooped and for example within every 2 days to 7 days, is dissociated, and individual cells or cellule agglomerate are assigned to other cultivation and holdIn device, (that is, go down to posterity), or remain in identical culture vessel and with replacement medium or other medium treatment.

Comprise by the dry cell mass of most energy of the multipotential stem cell pellet of centrifugal generation and can stand enzymic digestion and/or machineryOne or both in dissociating. The enzymic digestion of multipotential stem cell agglomerate can be by making agglomerate stand enzyme, such as IV Collagenase Type, pointLoose enzyme orProcessing carry out. The mechanical dissociation of large multipotential stem cell agglomerate can be used and be designed so that agglomerate is brokenThe device that is cleaved into preliminary dimension carries out. In addition or as another kind of select, mechanical dissociation can be used pin or pipette handMoving carrying out.

Being used for can according to the culture vessel of the method cultivation multipotential stem cell of some embodiments of the present invention at suspensionFor example, for any tissue culture vessel (, having the purity level that is suitable for cultivating multipotential stem cell), this culture vessel has and is designedBecome to make the multipotential stem cell cultivated therein can not be adherent or be attached to this lip-deep inner surface (for example,, with tissue trainingSupport the container of thing processing, attached or adherent to surface for preventing). Preferably, can to expand culture in order obtaining, to use and controlCulture systems (being preferably computer-controlled culture systems) realizes the cultivation according to some embodiments of the present invention, in controlIn culture systems processed, use suitable device monitoring and control culture parameters automatically, such as temperature, stirring, pH and oxygen. OnceDetermine required culture parameters, system can be set to automatically adjust as required culture parameters to strengthen multipotential stem cell amplificationAnd differentiation.

Can under dynamic condition, (, stand under the condition of persistent movement, for example, to stir in the time suspending cultivation at multipotential stem cellMix suspension culture systems) or at the lower multipotential stem cell of cultivating of non-dynamic condition (, static culture), retain its propagation energy simultaneouslyPower, versatility ability and the karyotype stability in repeatedly going down to posterity.

For the non-dynamic cultivation of multipotential stem cell, multipotential stem cell can be cultivated in following: culture dish, T-burnBottle,(CorningIncorporated,Corning,NY)、(CorningIncorporated, Corning, NY) or coating or uncoated cell factory (NUNC^TMCellFactory^TMSystemsThermoFisherScientific, Inc., Pittsburgh, PA). For the dynamic cultivation of multipotential stem cell, can closeIn suitable container (using plastics vibrator or agitator tank container such as rolling bottle or conical flask, stainless steel, glass or single), cultivateMultipotential stem cell. Culture vessel can be connected to control module, thereby presents control culture systems. Can stir continuously or off and on trainingSupport container (for example, rolling bottle or conical flask). Preferably, fully stir culture vessel to keep multipotential stem cell to suspend.

Multipotential stem cell can provide enough nutriments and environmental stimulus to promote any culture medium of growth and amplificationMiddle cultivation. Suitable culture medium comprises E8^TM, IH3 andOrCan regularly replace culture medium to upgradeNutriment supply also removes cell by-products. According to some embodiments of the present invention, culture medium is changed every day.

The source of multipotential stem cell

Any multipotential stem cell all can be used in method of the present invention. The exemplary types bag of spendable multipotential stem cellDraw together the pluripotent cell system of the foundation that derives from the tissue forming after gestation, be included in the embryo that any time of pregnancy duration obtainsFront tissue (for example blastocyst), embryonic tissue or fetal tissue, the described time is conventionally but not necessarily before approximately 10 to 12 weeks gestation.Non-limitative example be set up human embryo stem cell (hESC) be or human embryo stem cell, for example human embryonic stem cell H1,H7 and H9 (WiCellResearchInstitute, Madison, WI, USA). Suitable is to take from do not exist and raise in additionSupport the multipotential stem cell group's who cultivates in the situation of cell cell.

Suitable is in addition induced multi-potent cell (IPS) or the pluripotent cell that reprograms, and it can use multiple multipotency phaseBeing forced to express of the transcription factor of closing and derive from into human body cell, described transcription factor such as OCT4, NANOG, Sox2, KLF4And ZFP42 (AnnuRevGenomicsHumGenet2011,12:165-185). The people who uses in the method for the inventionEmbryonic stem cell also can be as (United States Patent (USP) 5,843,780 of being prepared of being described by people such as Thomson; Science, 1998,282:1145-1147；CurrTopDevBiol1998,38:133-165；ProcNatlAcadSciU.S.A.1995,92:7844-7848). Same suitable is the human embryonic stem cell of sudden change, such as for example BG01v(BresaGen, Athens, Ga.), or come from adult somatic cell, such as for example people such as Takahashi, CellDisclosed cell in 131:1-12 (2007). Being applicable to multipotential stem cell of the present invention can be according to describing in Publication about DocumentMethod obtains: the people such as Li (CellStemCell4:16-19,2009); The people such as Maherali (CellStemCell1:55-70,2007); The people such as Stadtfeld (CellStemCell2:230-240); People (the Nature such as NakagawaBiotechnology26:101-106,2008); The people such as Takahashi (Cell131:861-872,2007); And the U.S.Public announcement of a patent application 2011-0104805. Other sources of multipotential stem cell comprise pluripotent cell (IPS, Cell, 126 of induction(4): 663-676). Other sources of the cell that is suitable for using in the method for the invention comprise human umbilical tissue derived cell,People's amniotic fluid-derived cell, people's placenta derived cell, and people's parthenote. In one embodiment, umbilical cord tissue sourceCell can use United States Patent (USP) 7,510,873 method obtains, because this patent relates to separation and the sign of cell, itsDisclosure is incorporated herein by reference in full. In another embodiment, it is special that placenta tissue derived cell can use the U.S.The method of profit Shen Qing Publication 2005/0058631 obtains, because this patent relates to separation and the sign of cell, its disclosureBe incorporated herein by reference in full. In another embodiment, the cell of amniotic fluid-derived can use U.S. Patent application public affairsThe method of cloth number 2007/0122903 obtains, because this patent relates to separation and the sign of cell, its disclosure in full withWay of reference is incorporated to herein.

The feature of multipotential stem cell is well known to those skilled in the art, and other features of multipotential stem cell need to be continuedContinuous qualification. Multipotential stem cell mark for example comprises that one or more (for example 1,2,3,4,5,6,7,8,9,10,11,12,13,14 kinds or all) expression of following mark: ABCG2, cripto, FOXD3, CONNEXIN43, CONNEXIN45, OCT4,SOX2, NANOG, hTERT, UTF1, ZFP42, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81. In one embodiment,The multipotential stem cell that is suitable for using is in the method for the invention expressed the one in CD9, SSEA4, TRA-1-60 and TRA-1-81Or multiple (for example, 1,2,3 kind or all), but lack differentiation CXCR4 as arrived by Flow cytometry (also referred to asThe expression of label CD184). In another embodiment, be suitable for the multipotential stem cell table that uses in the method for the inventionReach one or more (for example, 1,2 kind or all) in CD9, NANOG and the POU5F1/OCT4 being detected by RT-PCR.

Exemplary multipotential stem cell comprises human embryonic stem cell H9 (NIH code: WA09), human embryonic stem cell H1(NIH code: WA01), human embryonic stem cell H7 (NIH code: WA07), and human embryonic stem cell SA002(Cellartis, Sweden). In one embodiment, multipotential stem cell is human embryo stem cell, for example H1hES cell. SeparatelySelect in embodiment, use the multipotential stem cell of non-embryonic origin.

Be divided into the cell of expressing pancreas entoderm pedigree characteristic label by multipotential stem cell

The amplification of multipotential stem cell

In some of following embodiment, the present invention relates to separate and cultivate stem cell, specifically, relate to cultivate dry thinBorn of the same parents troop, and it keeps the versatility of dynamic suspension culture systems. Pluripotent cell is trooped and can be broken up to produce functional β cell.

For the multipotential stem cell of method of the present invention before required terminal differentiation preferably at dynamic suspensionIn culture, increase. Advantageously, have been found that multipotential stem cell can be used as cell cluster in suspension in suitable cultivationIn base, cultivate and amplification, and do not lose versatility. This cultivation can be carried out in dynamic suspension culture systems, wherein cell or thinBorn of the same parents troop and keep mobile to prevent versatility loss fully. Available dynamic suspension culture systems comprise be equipped with for stirCultivate the system of device of content, such as by stirring, shake, recycle or gas sparging being stirred by culture mediumMoving. As long as keep enough cell clusters to move to promote increase and prevent too early differentiation, this stirring just can be intermittence orContinuous. Preferably, stir and comprise such as carrying out continuous stirring by the impeller rotating with special speed. Impeller can have circleShape bottom or flat bottom. The stir speed (S.S.) of impeller should make to troop and remains in suspension and sedimentation is minimized. In addition, leafThe angle of impeller blade can be adjusted to contribute to cell and troop move upward to avoid sedimentation. In addition impeller-type, angle,Become to make cell and troop to be arranged in the material that is rendered as even colloidal suspension liquid with the speed of rotation is can be whole coordinated.

Can by the stem cell of static culture is transferred to suitable dynamic cultivation system (such as disposable plastic, can weighUse again plastics, stainless steel or glass container, for example rolling bottle or conical flask) realize that suspension that multipotential stem cell troops is cultivated andAmplification. For example, the stem cell of cultivating in the adherent static environment (, plate or culture dish surface) can be first-selected by with chelating agent orEnzyme is processed and is removed from surface. Suitable enzyme includes but not limited to, type i collagen enzyme,(SigmaAldrichLLC, St.Louis, MO) or with trade nameWhat (SigmaAldrichLLC, St.Louis, MO) sold can businessThe commercially available formula of purchasing.

That the cell separation solution that comprises Collagenase and proteolytic enzyme (divides from shellfishFrom), and do not comprise mammal derivative products or bacterial derivation product. Therefore, in one embodiment, enzyme is collagen eggWhite enzyme and/or proteolytic enzyme, or the cell separation solution that comprises Collagenase and proteolytic enzyme. Suitable chelating agentInclude but not limited to ethylenediamine tetra-acetic acid (EDTA). In certain embodiments, cultivate with enzyme or chelating agent incubation multipotential stem cellThing, preferably until colony edge starts curling and lifting, but before bacterium colony separates completely from culture surface. A realityExecute in example, at room temperature incubation cell culture. In one embodiment, higher than 20 DEG C, higher than 25 DEG C, higher than 30 DEG C or highAt the temperature of 35 DEG C, for example, between approximately 20 DEG C and approximately 40 DEG C, between approximately 25 DEG C and approximately 40 DEG C, between approximately 30 DEG C and approximately 40 DEG CTemperature under, for example incubation cell at approximately 37 DEG C. In one embodiment, by cell incubation at least about 1 minute, at least about 5Minute, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, for example between approximately 1 minute and approximately 30 minutes, approximately 5Minute and approximately 30 minutes between, between approximately 10 minutes and approximately 25 minutes, between approximately 15 minutes and approximately 25 minutes, for example approximately 20 pointsClock. In one embodiment, the method relates to the step that removes enzyme or chelating agent after processing from cell culture. At oneIn embodiment, after removing enzyme or chelating agent, cell culture is washed once or twice or more times. At an embodimentIn, with suitable culture medium, such as(StemCellTechnologies, Vancouver, BC, Canada) washesWash cell culture. In one embodiment, provide Rho-inhibitors of kinases (for example, Y-27632, Axxora catalog number (Cat.No.)ALX-270-333, SanDiego, CA). The concentration of Rho-inhibitors of kinases can be approximately 1 to approximately 100 μ M, approximately 1 to 90 μ M, approximately 1To approximately 80 μ M, approximately 1 to approximately 70 μ M, approximately 1 to approximately 60 μ M, approximately 1 to approximately 50 μ M, approximately 1 to approximately 40 μ M, approximately 1 to approximately 30 μ M, approximately 1 to approximately20 μ M, approximately 1 to approximately 15 μ M, approximately 1 to approximately 10 μ M or approximately 10 μ M. In one embodiment, add at least 1 μ M, at least 5 μ M or extremelyThe Rho-inhibitors of kinases of few 10 μ M. Available scraper or rubber policeman promote cell from the surface of static culture system. Can useCulture medium and cell are transferred to dynamic cultivation system by glass pipet or other suitable devices. In a preferred embodiment,Change the culture medium in dynamic cultivation system every day.

In one embodiment, the invention provides in three-dimensional suspending culture and to cultivate and the side of amplification multipotential stem cellMethod. Specifically, the method provides gathering cell cluster by forming these multipotential stem cells multipotency of cultivating and increaseStem cell. Cell cluster can for example, for example, by using enzyme (, neutral proteinase, dispase) or chelating agent place before cultured cellReason multipotential stem cell is trooped and forms. Preferably cultured cell in stirring or shaking suspension culture systems. An enforcementIn example, the present invention also provides by this type of cluster of multipotential stem cell and forms and express the thin of pancreas entoderm pedigree characteristic markBorn of the same parents.

Preferably, cell cluster is the multipotential stem cell of assembling. The stem cell assembling is expressed one or more versatility marksNote thing, for example, in label CD9, SSEA4, TRA-1-60 and TRA-1-81 one or more (for example, 1,2,3 orAll), but lack the expression to one or more differentiation marker things, for example, lack the expression to CXCR4.

In one embodiment, the stem cell of gathering is expressed versatility label CD9, SSEA4, TRA-1-60 and TRA-1-81, but lack the expression to differentiation marker thing CXCR4.

An embodiment cultivates the method for cell cluster by multipotential stem cell in suspension culture. Cell cluster isThe gathering multipotential stem cell of cultivating in dynamic agitation or shake suspension culture systems. Can be by enzyme (such as neutral proteinase, for exampleDispase) as cell elevator, cell cluster is transferred to and stirred or shake suspension culture systems from plane adhere-wall culture thing.Exemplary suitable enzyme include but not limited to IV Collagenase Type, dispase orCell is stirring or shake suspensionIn culture systems (stirring suspension culture systems specifically), keep versatility.

Another embodiment of the invention is, in suspension culture, multipotential stem cell is cultivated to the side for cell clusterMethod, wherein cell cluster for example, for using chelating agent (EDTA) to shift from plane adhere-wall culture thing and stirring or shake suspensionThe gathering multipotential stem cell of cultivating in culture systems. Cell cluster is stirring or (the stirring specifically, of shake suspension culture systems(dynamically stirring) suspension culture systems) the middle versatility that keeps.

Another embodiment of the invention is, in suspension culture, multipotential stem cell is cultivated to the side for cell clusterMethod, wherein cell cluster is for usingEnzyme shifts and is stirring or shake suspension cultivation from plane adhere-wall culture thingThe gathering multipotential stem cell of cultivating in system. Cell cluster keeps versatility in dynamically stirring suspension culture systems.

Cell cluster of the present invention can be divided into mesoblastema such as heart cell, and ectoderm cell is thin such as nerveBorn of the same parents, single hormone positive cell or pancreas endoderm cell. The method also can comprise differentiation, for example, pancreas endoderm cell is broken upBecome diabetes and pancreatic hormone to express cell. In another embodiment, diabetes turns by expressing β cellRecord factor PDX1 and NKX6.1 characterize.

In one embodiment, differentiation step in suspension culture systems at least 12 hours, at least 24 hours, at least 36 littleTime, at least 48 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 144 hours, at least 168 hours, at leastAfter 196 hours or more hours, preferably carried out after approximately hour to 72 at approximately 48 hours. Can use nutrient media componentsProgressively develop such as breaking up described in example (for example,, referring to Table A and table 1a and table 1c).

In preferred embodiments, three-dimensional cell is trooped and is produced by following steps: multipotential stem cell is pasted in planeIn wall culture, grow; Multipotential stem cell is increased into gathering cell cluster; And use enzyme or chelating agent by multipotential stem cell groupCollection is transferred to dynamic suspension culture from plane adhere-wall culture thing.

Preferred embodiment is increased and break up many in dynamically stirring suspension culture systems by following steps in additionThe method of energy stem cell: multipotential stem cell is grown in plane adhere-wall culture thing; Multipotential stem cell is increased into gathering cellTroop; And use enzyme or chelating agent that multipotential stem cell is trooped and transferred to dynamic suspension culture from plane adhere-wall culture thing; AndAnd in dynamically stirring suspension culture systems, break up pluripotent cell and troop to produce diabetes group.

Another embodiment comprises moving of the differentiated stem cells prepared by the amplification multipotential stem cell suspension of troopingPlant the cellular products of source of human stem cell, described amplification multipotential stem cell is trooped and is divided into diabetes. More specifically, can moveThe product of planting source of human stem cell produces by following steps: multipotential stem cell is grown in plane adhere-wall culture thing; To be manyCan become to assemble cell cluster by expansion of stem cells; And use enzyme or chelating agent that multipotential stem cell is trooped from plane adhere-wall culture thingTransfer to dynamic suspension culture; And in dynamically stirring suspension culture systems, breaking up pluripotent cell troops. Portable is dry thinThe cellular products in born of the same parents source is preferably used for treating diabetes.

In another embodiment, the method comprises to be transplanted in diabetic animal body, taking in vivo further maturation asFunctional pancreatic endocrine cell.

Another embodiment is the method that increases and break up multipotential stem cell in suspension culture systems, and the method comprisesFollowing steps: multipotential stem cell is grown in plane adhere-wall culture thing; Use enzyme to remove multipotency from plane adhere-wall culture thing dryCell; By on the adherent multipotential stem cell microcarrier in static culture thing; In dynamically stirring suspension culture systems, amplification is manyEnergy cell; And in dynamically stirring suspension culture systems, break up pluripotent cell to produce diabetes group.

Microcarrier can be any form for attached cell as known in the art, and specifically, microcarrier can be micro-Pearl. Microcarrier can be made up of the material in the material of natural origin or synthetic source. The example of microcarrier comprises based on collagenMicrocarrier, microcarrier based on glucan or based on cellulosic microcarrier. For example, microcarrier bead can be and has the table of being attached toFace is to provide the MPS pearl of cation trimethyl ammonium on positive charge surface to microcarrier. Bead diameter can be at approximately 90 μ mTo approximately 200 μ m or approximately 100 μ m to approximately 190 μ m or approximately 110 μ m to the diameter of approximately 180 μ m or approximately 125 μ m to 175 μ mIn scope. Microcarrier bead also can be the thin layer of the denatured collagen that is chemically coupled to cross-link dextran matrix. Microcarrier beadCan be glass, pottery, polymer (such as polystyrene) or metal. In addition, microcarrier can be uncoated, or such as useSilicon or such as the protein coating of collagen. In yet another aspect, microcarrier can be by the combination that strengthens cell and microcarrierAnd strengthen cell from the compound composition of microcarrier disengaging or scribble described compound, described compound includes but not limited to transparentMatter acid sodium, poly-(be total to-succinic acid of single stearoyl glyceride), poly--PLG, fibronectin, layer adhesion eggElastin laminin, lysine, n-N-isopropylacrylamide, vitronectin and collagen in vain. Example also comprises having micro-electric currentMicrocarrier, such as thering is the relevant zinc of electric power of the low-level biology of generation and the microcarrier of copper particle galvanic couple; Or paramagnetic micro-Carrier, such as paramagnetism calcium alginate microcarrier.

In certain embodiments, the progressively differentiation that pancreas endoderm cell group troops by pluripotent cell obtains. At someIn embodiment, pluripotent cell is people's embryonic pleuripotent stem cell. In one aspect of the invention, express definitive entoderm pedigree featureThe cell of property mark is former precursor. On the other hand, express the cell of definitive entoderm pedigree characteristic markIt is mesendoderm cell.

In certain embodiments, the present invention relates to progressively break up the method for pluripotent cell, the method is included in dynamic suspensionIn culture, cultivate 3-5 phase cell. In certain embodiments, produced pancreas entoderm group being transplanted to diabetes movesIn object, taking further ripe as functional pancreatic endocrine cell in vivo. The present invention also provides for of the present inventionThe system using in method or kit.

The present invention also provides by the obtainable cell of method of the present invention or cell mass. The present invention also provides by thisCell or cell mass that the method for invention obtains.

The invention provides methods for the treatment of. Specifically, the invention provides one and be used for the treatment of and suffer from diabetes, or have and send outThe patient's of exhibition diabetes risk method.

The present invention also provides and can be obtained or be obtained for treatment by method of the present invention by method of the present inventionThe cell of method or cell mass. Specifically, the invention provides and can obtain or pass through of the present invention by method of the present inventionMethod obtains for treatment suffers from diabetes, or has cell or the cell mass of the patient's of development diabetes risk method. SugarUrine disease can be type 1 diabetes or diabetes B.

In one embodiment, methods for the treatment of comprises and will be obtained and maybe can be passed through side of the present invention by method of the present inventionIn the cell patients with implantation body that method obtains.

In one embodiment, methods for the treatment of comprises and makes multipotential stem cell be divided in vitro for the 1st the 2nd stage the 3rd of stageThe 4th stage of stage or the 5th phase cell, for example as described herein, and the cell of differentiation is implanted in patient body.

In one embodiment, before the method is also included in the step that makes multipotential stem cell differentiation, for example, as this paper instituteThe step of the cultivation multipotential stem cell of stating.

In one embodiment, the method also comprises the step of implantation step noble cells in vivo afterwards.

In one embodiment, patient is mammal, preferably the mankind.

In one embodiment, these cells can be implanted as the cell disperseing, maybe can be made these cells form and troop,These cell clusters can be infused in vena portae hepatica. Select as another kind, what cell can be arranged on to biocompatibility canIn degradation polymer type holder, in the non-degradability device of porous, maybe can encapsulate to protect it to avoid host immune shouldThe destruction of answering. Cell can be implanted in the appropriate location in acceptor. Implantation site comprises liver for example, natural pancreas, kidneySpace, intestines, stomach or subcutaneous pocket under space, nethike embrane, peritonaeum, serous coat under coating.

Implant further differentiation, survival rate or activity in vivo of cell in order to strengthen, can be before dosed cells, with itSimultaneously or use afterwards the extra factor, for example growth factor, antioxidant or antiinflammatory. These factors can be by endogenous cellSecretion original position are exposed to used cell. Can use by the endogenous growth factor of using known in the art and external sourceAny combination of growth factor is induced implantation Cell Differentiation.

Cell concentration used in transplanting depends on many factors, comprises patient's situation and the response to this therapy, andCan be determined by those skilled in the art.

In one embodiment, methods for the treatment of be also included in transplant before by described Cell binding in three dimensional support. ?Before being transplanted in patient body, this cells in vitro can be remained on this holder. Select as another kind, can be thin by comprising thisBorn of the same parents' holder is directly implanted in patient body and without carrying out other in vitro culture. Can optionally at least one be conducive to plantEnter the survival of cell and the medicament of function mixes in this holder.

In certain embodiments of the present invention, one or more in every of the following stated can be used for method of the present inventionIn.

Table A

The publication of quoting in the whole text in the document is incorporated herein by reference in full at this. The present invention is by following exampleFurther illustrate, but be not limited.

Example

Can be further described the present invention by following limiting examples。

Example 1

The suspension of the human embryo stem cell of the clone H1 carrying out with dispase/neutral proteinase and trooping the 41st generation peopleThe cell (WA01 cell, WiCell, MadisonWI) of embryonic stem cell line H1 PBS (catalog number (Cat.No.) 14190, Invitrogen)Wash once, and with 1mg/mL's(neutral proteinase, SigmaAldrichCoLLC, catalog number (Cat.No.) D4818,St.Louis, MO) solution in DMEM/F12 (Invitrogen catalog number (Cat.No.) 11330, GrandIsland, NY) processes.By cell incubation 15-25 minute at 37 DEG C, until colony edge start curling and promote, but at bacterium colony from culture surfaceBefore departing from completely. Then removeAnd with contain 10 μ MY-27632 (Axxora catalog number (Cat.No.) ALX-270-333,SanDiego, CA)(StemCellTechnologies, Vancouver, BC, Canada) culture medium willCulture dish washed twice. Then will contain 10 μ MY-27632'sCulture medium is with 5mL/60cm²Add cultivation toWare, and mention cell from surface with scraper or rubber policeman. Then use glass pipet that culture medium and cell are transferred to50mL conical tube, and will troop centrifugal 3 minutes with 90g (rcf).

After centrifugal, sucking-off culture medium, by cell lightly Eddy diffusion in 12mLIn culture medium andWherein grind simply, described inCulture medium contains the every 225-240cm of 10 μ MY-27632²Cultivate general layoutThing (being equivalent to a T225 flask or four 10cm culture dishes, about 9,000 ten thousand cells). Then cell suspending liquid is shifted(1mL/ hole) to contain 2mL/ hole fresh have 10 μ MY-27632'sCultivate in ultralow combination 6 holes of culture mediumWare (UltraLowBindingCulture6welldishes) (CorningIncorporated, catalog number (Cat.No.) 3471,Corning, NY) in. Cell promotes in the mode that is similar to individual layer fragment, and the average diameter that wherein promotes fragment is about 20-30(a), the each free cell mass of described lifting fragment forms Fig. 1 micron. By these individual layer fragments incubation 2 hours in suspension(incubative time can in the scope of 0.5-4 hour), now observes the aggregation of fragment. Then use 10ml glass pipet pairThis aggregation grinds simply, and in low board, is incubated overnight that (aggregation also can be cultivated and mould at untreated cellIncubation in the plastics of material and the processing of normal structure culture) (aggregation can directly enter into suspension).

Be incubated overnight (18 hours to 24 hours) afterwards, cell and culture medium are directly transferred to and comprised 25mLThe 125mL rolling bottle (Corning, the catalogue that stir with 50rpm (can in the scope of 30-80+rpm) of culture mediumNumber 4500-125, CorningNY) in, making final volume is about 75mL. In 4 days, change culture medium every day. After 4 days, trainingSupport in thing and measure versatility, and for versatility mark (CD9, SSEA4, TRA-1-60 and TRA-1-81), fluidic cellArt result demonstrates high expressed, and for differentiation mark (CXCR4), does not almost express. Referring to Fig. 1 b. These tables of dataBright, H1hES cell can be used as cell cluster utilization as cell elevatorBecome from plane adhere-wall culture thing formMerit is transferred to suspension culture, and keeps stirring the versatility in (dynamically) suspension culture systems. This example also can haveIn the shake suspension system of plate and conical flask, carry out, instead of carry out in stirring suspension system, there is comparable results.

In suspension culture, after 4 days, (differentiation also can start for 24-120 hour after forming aggregation, preferably existsStart to cultivate 2-3 days before differentiation), pluripotent cell aggregation breaks up to induce carefully under the progressively development of nutrient media componentsBorn of the same parents form pancreas destiny. Circulator turns on stirring for the speed differentiation aggregation with 65rpm. Culture medium and component are shown in tableIn 1a.

The 1st stage, while end, was carried out flow cytometry and PCR to sample. The 1st stage is while end, the cultivation of the differentiation that suspendsThing in loose aggregates, form evenly and the cell mass of homogeneity (c), wherein the expression of versatility mark (CD9) almost for Fig. 1Eliminate, and definitive entoderm differentiation mark is quite high, for CXCR4 (CD184), is 97.2% positive, and for CD99Be that (Fig. 1 d) for 97.3% positive.

These results are relevant to qRT-PCR result, and it shows compared with undifferentiated WA01hES cell, multipotency geneThe expression of (CD9, NANOG and POU5F1/OCT4) significantly reduces, and the gene being associated with definitive entoderm (CXCR4,CERBERUS, GSC, FOXA2, GATA4, GATA6, MNX1 and SOX17) expression roll up that (Fig. 1 e).

Then definitive entoderm troops by removing TGF-'beta ' family member GDF8, and adds FGF7 to culture mediumFurther towards former anterior intestine differentiation. After cultivating three days with FGF7, troop by all-trans retinoic acid being added to following trainingSupport any one in base and be divided into pancreas PDX1 and express destiny: the lipid that is rich in that comprises high glucose (25mM) and low concentrationThe culture medium of bovine serum albumin(BSA) (Albumax (LifeTechnologiesCorporation, Carlsbad, CA)), or bagContaining the not culture medium of the bovine serum albumin(BSA) of fatty acids of relatively low concentration of glucose (8mM) and 2%. Add these cultivations toDetailed composition as listed in base is in table 1a. In the time that differentiation finishes, analytic sample is to express the mark of diabetes. ObserveTo be clustered in following condition any one under break up: LG+2%FAF-BSA (A) or high glucose+0.1%(B), as express the required transcription factor of high-caliber NKX6.1, functional β cell and high-caliber in pointSecrete measured (the table 1b) of flow cytometry of pancreas label such as synaptophysin and chromograin. These results and RT-PCRResult is consistent, and it demonstrates high-caliber multiple pancreas precursor-genes of being expressed by condition A and B in sample, and (data are notIllustrate).

Along with cell cluster is by from definitive entoderm (DE), (Fig. 1 c) is divided into former anterior intestine and proceeds to pancreas entoderm(Fig. 1 is f) upper, and its representative configuration demonstrates the visible metamorphosis of cell and cell cluster. Conventionally, multipotency is trooped by differingMicroscopy and seem intensive and dim, then along with cell develops into the former anterior intestine in the 2nd stage and make outward appearance become loose.After all-trans retinoic acid is processed, the reversion of this form, and troop and utilize smooth cluster boundaries and again become more intensiveAnd uniformly.

In the 5th stage culture medium that comprises ALK5 inhibitor, protect in addition by the cell in the 4th stage according to condition B differentiationHold 5 days (referring to table 1c). This extra maturation is cultivated the remarkable increase that causes following endocrine marker expression: INS, GCG,SST, PPY and PCSK1. Then according to the research approach of IACUC approval, cell cluster is implanted to the kidney peplos of SCID-Bg mouseIn, the every 2-4 week of subsequently this mouse being carried out 20 weeks is by a definite date measured fasted/fed c-peptide. After implanting 4 weeks, little in fasting 20Time and subsequently glucose stimulate after, c-peptide do not detected. During by the 6th week, 2 in 5 mouse demonstrate people c-peptide areCertain positive (0.087&0.137ng/mL), and during by the 10th week, in 5 mouse 5 to the c-peptide (0.085-that is positive0.291ng/mL). In the time of the 16th week, after fasting 20 hours and glucose stimulation, all 4 (4/4) mouse are to c-peptide tableReach be all positive (0.377 – 3.627ng/mL).

These results show, can form pluripotent cell aggregation, and then in suspension culture, differentiation is thin to produce by βThe diabetes group that the expression of born of the same parents' transcription factor (such as PDX1 and NKX6.1) characterizes. In addition, implantation and allowThe ripe noble cells expression of insulin in response to the glucose load of proper level on physiology of trooping in vivo.

Table 1a: differentiation scheme

As used in the present embodiment and whole description, TppB (also referred to as TpB) is PKC activator, and it has chemistryName: (2S, 5S)-(E, E)-8-(5-(4-(trifluoromethyl) phenyl)-2,4-pentadiene acylamino-) benzo lactams and No. CAS:497259-23-1。

Table 1b: the flow cytometry result of selected differentiation mark

Table 1c: differentiation scheme

Example 2

The suspension of the human embryo stem cell of the clone H1 carrying out with EDTA and trooping

By PBS (catalogue for the cell of the 41st generation human embryonic stem cell H1 (WA01 cell, WiCell, MadisonWI)Numbers 14190, Invitrogen) washing once, and promote/go down to posterity agent EDTA (Lonza, catalog number (Cat.No.) 17-7-11E) with non-enzyme cellProcess. By at room temperature incubation 8 minutes of cell. Then remove EDTA, after 1 minute or 2 minutes, (EDTA exposes altogether 9Minute to 10 minutes), with comprising 10 μ MY-27632'sCulture medium (Axxora catalog number (Cat.No.) ALX-270-333,SanDiego, CA) rinse plate, and use glass pipet by the cell harvesting shifting out in 50ml conical tube. With comprising 10 μ MY-27632'sCulture medium rinses plate again, and merges the cell shifting out. It should be noted that at room temperature and exposeAfter EDTA9-10 minute, some cells still stay onboard, and the cell promoting is not exclusively depolymerized to single-cell suspensionLiquid. On the contrary, cell removes from surface as little aggregation. Then use glass pipet that culture medium and cell are transferred to 50mlIn conical tube, and carry out cell count (-ChemoMetecA/S, catalog number (Cat.No.) YC-T100,Denmark). Add as required and extra comprise 10 μ MY-27632'sCulture medium so that cell concentration be1.0 hundred ten thousand cell/ml to 1.5 1,000,000 cells/ml.

If be centrifuged into pellet and pass through pipette Eddy diffusion, trooping is that being also dissociated into of loose gathering is slenderBorn of the same parents, so do not carry out centrifugal to cell. On the contrary, make culture medium in pipe and cell gently turn until form unit for uniform suspension.If need, can also extend EDTA treatment cycle, and make cell near single-cell suspension liquid. Then will be thin with glass pipetBorn of the same parents' suspension is at the CO of 37 DEG C of humidity 5%₂In incubator, transferring to two with 3ml/ hole does not train with 6 holes of tissue culture processingSupport in ware (BectonDickinson, catalog number (Cat.No.) Falcon351146, FranklinLakes, NJ). Will be thin in suspensionBorn of the same parents' incubation 2 hours, now observes aggregation. Then grind aggregation to destroy greatly by move gently liquid with glass pipetAggregation also forms homogeneity, the suspension of trooping uniformly, then leaves standstill and is incubated overnight.

Then after 18-24 hour, make cell and culture medium in 50mL conical tube, rotate 3 points of sedimentations with 90g (rcf)Clock. Abandon the medium supernatant of using, cell aggregation is suspended in freshIn, and by suspension 37In the CO2 incubator of DEG C humidity 5%, transfer to the rolling bottle (CorningIncorporated, the catalog number (Cat.No.) that stir with 55rpm4500-125, CorningNY) in. In 2 days every day change culture medium continue. Before being transformed into differentiation culture, stirringIn suspension culture, after 2 days, determine versatility. The flow cytometry of CD9, SSEA4, TRA-1-60, TRA-1-81 and CXCR4Result illustrates with scatter diagram form in Fig. 2 a. For versatility mark (CD9, SSEA4, TRA-1-60 and TRA-1-81),These data demonstrate high expressed, and for differentiation mark (CXCR4), demonstrate low expression or do not express. These resultsShow, H1hES cell can use non-enzyme method for improving to transfer to suspension culture from plane adhere-wall culture thing form, and keepsDynamically stir the versatility in suspension culture systems.

In suspension culture, after 2 days, pluripotent cell aggregation breaks up under the progressively development of nutrient media components,Thereby inducing cell forms pancreas destiny. Circulator is stirred and remained under the speed of 55rpm. Culture medium and component are shown in table 2aIn.

The 1st stage, while end, was carried out flow cytometry and PCR to sample. The 1st stage is while end, the cultivation of the differentiation that suspendsThing in loose aggregates, form evenly and the cell mass of homogeneity (b), wherein the expression of versatility mark (CD9) almost for Fig. 2Eliminate, and definitive entoderm differentiation mark CXCR4 (CD184) is quite high, is 95.9% ± 1.8sd (figure on three rolling bottles2c). These results are relevant to qRT-PCR result, and it shows compared with undifferentiated WA01hES cell, multipotency gene (CD9,NANOG and POU5F1/OCT4) expression significantly reduce, and the gene being associated with definitive entoderm (CXCR4, CERBERUS,GSC, FOXA2, GATA4, GATA6, MNX1 and SOX17) expression roll up that (Fig. 2 d).

Then by the merging of trooping from the definitive entoderm of rolling bottle, and (shake is stirred to be distributed to another rolling bottle or conical flaskMoving system) in any one, and directed with by removing GDF8, and add FGF7 to culture medium and enter towards former anterior intestineOne step differentiation. After cultivating three days with FGF7, troop and comprise relatively low glucose by all-trans retinoic acid is added toConcentration (8mM) and 2% not the bovine serum albumin(BSA) of fatty acids culture medium and be divided into pancreas PDX1 express destiny. Add toDetailed composition as listed in these culture mediums is in table 2a. In the time that differentiation finishes, analytic sample is to express the mark of diabetesWill thing. Use flow cytometry, observe high-caliber NKX6.1 (required the turning of functional β cell with two kinds of form of suspensionThe record factor), and high-caliber endocrine pancreas mark, (show 2b and Fig. 2 e) such as synaptophysin and chromograin. TheseResult is consistent with RT-PCR result, and it is illustrated in express in the sample producing with rolling bottle form or conical flask form closely similarHigh-caliber multiple pancreas precursor-genes (Fig. 2 f).

These results show, can form pluripotent cell aggregation, then in suspension culture with multiple suspension cultureForm (comprising stirring system or shake suspension system) differentiation is to produce by β cell transcription factor (such as PDX1 and NKX6.1)The diabetes group that characterizes of expression.

Table 2a: nutrient media components and differentiation scheme

Table 2b: the flow cytometry result of selected differentiation mark

Example 3

The suspension of the human embryo stem cell of clone H1 is trooped and is suspended and go down to posterity continuously

To be coated withThe tissue culture processing of (CorningIncorporated, CorningNY)Polystyrene on the cell (WA01 cell, WiCell, MadisonWI) of the 40th generation human embryonic stem cell H1 that grows usePBS (catalog number (Cat.No.) 14190, Invitrogen) washed twice is also used(a part of PBS to of half intensity solutionPointSigma-Aldrich, catalog number (Cat.No.) A-6964, St.Louis, MO) process. By at room temperature incubation of cell3¹/₂Minute.That the cell separation solution that comprises Collagenase and proteolytic enzyme (divides from shellfishFrom), and do not contain mammal derivative products or bacterial derivation product. Then removeAfter 3 minutes (Expose 61/2 minute altogether) afterwards, with comprising 10 μ MY-27632'sCulture medium rinses plate, andUse glass pipet by the cell harvesting shifting out in 50ml conical tube. With comprising 10 μ MY-27632'sTrainingFoster base rinses plate again, and merges the cell shifting out. Be exposed toAfterwards, some cells still stay onboard,And the cell promoting is not exclusively depolymerized to single-cell suspension liquid. On the contrary, cell removes from surface as little aggregation that (Fig. 3 a).Then use glass pipet that culture medium and cell are transferred in 50ml conical tube, and carry out cell count. As requiredAdd and extra comprise 10 μ MY-27632'sCulture medium is so that cell concentration is 1.0 hundred ten thousand cell/ml to 1.51000000 cells/ml.

If be centrifuged into pellet and pass through pipette Eddy diffusion, trooping is that being also dissociated into of loose gathering is slenderBorn of the same parents, so do not carry out centrifugal to cell. On the contrary, make culture medium in pipe and cell gently turn until form unit for uniform suspension.Then with glass pipet by cell suspending liquid the CO 37 DEG C of humidity 5%₂In incubator with 3ml/ hole transfer to two ultralowIn 6 hole culture dishes. In suspension, by cell incubation 90 minutes, now observe aggregation. Then aggregation is carried outGrind simply, and it is directly transferred to and comprises 25mlThe 125ml rolling bottle stirring with 55rpm of culture mediumIn (total final volume is about 75mL). In 3 days every day change culture medium continue, and in the 3rd day definite culture multipotencyProperty. Troop differ micro-image be illustrated in static suspension culture form after 90 minutes and in culture amplification super(Fig. 3 b) for the uniform-spherical group who troops who spends three days. In suspension culture three days finish time, by mark CD9, SSEA4,The flow cytometry result of TRA-1-60, TRA-1-81 and CXCR4 is measured the versatility of cell. For versatility mark(CD9, SSEA4, TRA-1-60, TRA-1-81), cell keeps high expressed, and for differentiation mark CXCR4, does not almost showReach (table 3). These data show, H1hES cell can use enzyme method for improving (such as) from plane adhere-wall cultureThing form is transferred to suspension culture, and will keep dynamically stirring the versatility in suspension culture systems.

Then useIn 20 extra generations of dissociating, make the multipotency continuous passage of trooping. In every generation, by 5000Ten thousand cell gravitational settlings in 50ml conical tube 2 minutes, use PBS washed twice, and are addingTwo points afterwardsWhen clock and four minutes, under the turn gently of pipe, in 37 DEG C of water-baths, useHalf intensity solution-treated. At incubation sixAfter minute, willSucking-off from pipe and interference cell granulating not. And then by cell incubation 3 minutes (Expose 9 minutes altogether). Then with comprising 10 μ MY-27632'sCulture medium cleaning hose, uses glassGlass pipette grind twice, and make suspension cell pass 70 microns of cell filter screens (BDFalcon, catalog number (Cat.No.) 352350,FranklinLakes, NJ). With comprising 10 μ MY-27632'sCulture medium carries out twice extra rushing to pipeWash, and through cell filter screen.

Make culture medium in pipe and cell gently turn until form unit for uniform suspension. Then with glass pipet by cellSuspension is at the CO of 37 DEG C of humidity 5%₂In incubator, transfer in ultralow combination 6 hole culture dishes with 3ml/ hole, and at suspensionMiddle incubation 2 hours (test 0-28 hour), the glass rolling bottle that the final volume of now aggregation being transferred to culture medium is 80mlIn. Select as another kind, cell suspending liquid can directly be placed on the rolling bottle stirring with 55rpm or the taper of shaking with 40rpmIn bottle, and be clustered in stirred suspension and form that (c), the final volume of culture medium is 80ml to Fig. 3.

Use this continuous propagating method, passage 20 times, the about split ratio wherein at every turn going down to posterity is 1:3. ?Every generation is measured versatility by flow cytometry, and for chromosome 12 and 17, uses FISH (FISH) to surveyThe method of determining is determined caryogram, and two chromosome is accredited as in hES cell as latent instability. CD9, SSEA4, TRA-1-60,The flow cytometry result that TRA-1-81 and CXCR4 express illustrates with scatter diagram form, and shows for versatility markHigh expressed, and show low expression or do not express for differentiation mark (CXCR4), the FISH of chromosome 12 and 17 measures simultaneouslyDemonstrate normal copy number. These data show, H1hES cell can use(nonmammalian enzyme cell solutionFrom method) under conventional continuous passage, remain in suspension culture, and will keep dynamically stirring many in suspension culture systemsCan property and stable caryogram, thus in the process going down to posterity for 20 times, produce 1 × 10⁹Individual cell/original input cell. EDTA also can useSuspend continuously in this, and continued for 6 generations.

Table 3: the cell of the result based on mark CD9, SSEA4, TRA-1-60, TRA-1-81 and CD184 (CXCR4) is many The flow cytometry of energy property is as the function of time

Example 4

The directed differentiation of the human embryo stem cell that the suspension of clone H1 is cultivated

UseBy cell (WA01 cell, WiCell, the Madison of the 40th generation human embryonic stem cell H1WI) promote from plane adhere-wall culture thing, and transfer to suspension culture form. The method of describing in use-case 3 is by cellRemain in dynamic stirring suspension culture systems and continued for 30 generations.

Confirm versatility by front 20 generations as shown in table 3, wherein stable high-caliber versatility mark is protectedBe held in whole culture, as measured in flow cytometry. In order to confirm versatility and to show that it is provided for treating glycosuriaThe ability of sick cell source, cell is intended to summarize the morphogen of Normal Pancreas development or the difference of growth factor by comprisingThe progressively progress of culture medium and be divided into pancreas precursor in dynamically stirring suspension culture systems. This process has produced by highThe diabetes group that PDX1 and NKX6.1 coexpression characterize. In the time that these cells are transplanted, they further become maturationThe functional glucose tissue that stimulates insulin secretion, this tissue can be in response to glucose excreting insulin and keep chain ureaEuglycemia in the diabetes model of assistant rhzomorph induction. Referring to Fig. 4 c and table 4c.

In order to produce these diabetes, the method for describing in use-case 3 is stirred increasing and remaining on dynamicallyIn moving suspension culture systems, the H1 human embryo stem cell in 16 generations breaks up. That summarizes says, cell was increased for 30 generations, measures theseThe versatility in front 20 generations in going down to posterity, cell breaks up in the time of the 16th generation. The pluripotent cell of the form of trooping at 4 DEG C through 3 hours fromMedia transfer is to FBC solution (table 4a). Then cell cluster moves to the biology by SartoriusStedimIn 3 liters of glass suspended biological reactors of inhibitor B-DCU (Goettingen, Germany) control module regulation and control, and according toTable 4b is suspended in differential medium with 0.55 hundred ten thousand cell/mL. Cell is being carried out for temperature, pH and dissolved oxygen (DO)Regulation and control closed sterile soft suspended biological reactor (PH electrode 225mm, model F-635, and dissolved oxygen12mm sensor, model D-145, from BroadleyJamesCorporatio, IrvineCA) middle guarantorHold 14 days.

In whole service, by the CO in total culture volume of≤1.6 liters of regulation and control₂Stream makes culture medium bicarbonateLevel remains on 3.64g/L, and makes pH remain on 7.4. Under the control of Sartorious control system, use CO₂, air and O₂Continous pouring bioreactor headroom, wherein along with the constant gas of 150cc/ minute forward, the 1st stage was 20% dissolvingOxygen set point and the 2nd stage are 30% dissolved oxygen set point. Regulate and control oxygen flow in response to dissolved oxygen content, come in response to pHRegulation and control CO₂Flow. In whole service, by electric heating cover, temperature is remained on to 37 DEG C. In the time that operation starts and at every turnCulture medium is changed (each culture medium that removes 93% of changing), stop impeller (under 70rpm, operate 3 " stainless steel pitch bladeImpeller), and remove culture medium, or pass through through using Terumo^TMPipe welding machine is connected toThe biology of pipe arrangement is anti-Answer the peristaltic pump of the dip-tube in device to add culture medium to maintain closed system. In the time that each stage of differentiation finishes, obtain thinBorn of the same parents'/troop image, collects flow cytometry sample, and finishes 3 days afterwards for the 3rd day the 1st stage and the 2nd stageCXCR4 expresses and sample is measured to (Fig. 4 a). Observe, the negative pluripotent cell group of CXCR4 while initiation from differentiation (table 3, the16 generations) to the CXCR4 group who expresses cell, (98.5% cell is the CXCR4 positive, and Fig. 4 total group that approaches b) changes. The 2nd stageFinish after 3 days, these cells are transformed into almost CXCR4 negative status (7.0% cell is the CXCR4 positive), and to the 3rdStage, while end, cell was almost transformed into CD56 positive completely. In the time that the atomization of the 4th day in the 4th stage finishes,Cell is expressed as 88.5% positive for PDX1, and (Fig. 4 b) and demonstrate and pancreatic endocrine cell (33.5% chromograinPositive) with the consistent expression pattern of mixture of pancreatic progenitor cell (the 65.7%NKX6.1 positive). This phase specificity markExpression pattern instruction is effectively progressively divided into pancreatic cell from multipotency colony. In the time that atomization finishes, produce 2.77 hundred ten thousandCell/mL (4,100,000,000 cells in 1.5 liters), this shows to input the gross mass amplification of 5 noble cells of hES cell at every turn.

In the time of end of run, remove 500mL for centrifugal and washing, and at transplanting, ripe and functional animal mouldIn type, test. Remaining 1000mL cell suspending liquid is existedIn 400 systems (KBI biosystem, DurhamNC)Process with washing in complete closed-loop system, filtration and concentrating cells product. By cellular products with 4,100 ten thousand cell/mLFinal concentration is condensed into the concentrating cells of 50mL from the initial volume of 1 liter. Then use automatic bottle filling machine (Fill-It,TAP, HertfordshireUK) with 1.2ml packing volume, these concentrating cells are distributed in 24 bottles, and by inciting somebody to actionIt is placed in liquid nitrogen freezing apparatus, carry out freezing.

By by standard centrifuge washing and concentrated 500mL noble cells with 500 ten thousand cells of each SCID-Bg mouseDosage is transplanted, place it in kidney peplos under, or be placed on the immunity of subcutaneous implantation (the lower 6 kinds of animals of each condition)Large-scale packaging system (the Theracyte of protectiveness^TM, IrvineCA) in. After implantation, 12 weeks time, implant cellular response in fastingFeed subsequently and express as detected in ELISA (people c-peptide customization ELISAMercodia catalog number (Cat.No.) 10-1141-01)Circulation people C-the peptide (> 0.1ng/mL of the level of signifiance), and in 16-20 week, animal has the circulation c-peptide (table that exceedes 1ng/mL4c)。

After implantation when 27 weeks (190 days), by two kinds of animals of the device with encapsulation immune protective graft separatelyProcess optionally to kill all endogenous mouse β islet cells and to lure with the Streptozotocin (STZ) of single-bolus high-doseLead diabetes (250mg/Kg). After the STZ that is enough to induce obvious diabetes in control-animal processes next twoIn week, the blood sugar level of transplanting animal remains in normal range (NR) (< 150mg/dL). After after implantation, 29 weeks and STZ useTwo weeks, the glucose of two kinds of animals of test stimulated insulin secretion (GSIS), and used and demonstrate and follow in response to glucoseThe remarkable increase of ring people c-peptide. In addition, when each in graft is when within the 209th day after implantation, (29.5 weeks) remove, animalBlood sugar level is sharply increased to > 500mg/dL.

These results show, the cellular products that is used for the treatment of the human embryo stem cell source of diabetes can be by increasing and breaking upThe suspension preparation of stem cell. This product can produce in telescopic stirring closed loop bioreactor system, and can rootManufacture and need to and concentrate to process cellular products with closed loop washing according to business cGMP. The cellular products in this human embryo stem cell sourceCan treat widely usedly as the ability of GSIS ability, regulating and controlling blood sugar, and in the time removing cell therapy, turn back to glycosuria symptomDiabetes in diabetes animal model shown in state.

The composition of table 4aFBC solution

AUSP=American Pharmacopeia

BNF=NF

CFCC=Food Chemistry code

DNA=is inapplicable

EACS=American Chemical Society

Table 4b: nutrient media components and differentiation scheme

(term: the feeding for the first time of time 0=new stage; After time 24,48 or 96 hours=new stage culture mediumTime)

Table 4c:C-peptide is expressed (ng/mL)

C-peptide (ng/mL)	4 weeks	8 weeks	12 weeks	16 weeks	20 weeks	24 weeks	29 weeks
								Kidney peplos implant (N=6)	0.00	0.03	0.19	0.95	2.56
Standard deviation	0.00	0.03	0.17	0.71	1.33
								Theracyte device implant (N=6)	0.00	0.02	0.35	0.58	1.45	2.49	2.85
Standard deviation	0.01	0.01	0.54	0.51	1.02	0.75	0.21

Example 5

Directed differentiation in the adhere-wall culture human embryo stem cell of the suspended form of clone H1

Use EDTA by the cell of the 41st generation human embryonic stem cell H1 (WA01 cell, WiCell, MadisonWI) fromIn plane adhere-wall culture thing, promote, and the method for describing in use-case 2 is transferred to suspension culture form.

Measure the versatility of cell aggregation by the flow cytometry as shown in Fig. 5 a, and observe versatilityThe high expressed of mark CD9, SSEA4, TRA-1-61 and TRA-1-80, this shows that cell is height multipotency. These multipotencys are thinThen born of the same parents are intended to the little molecule of the morphogen driver of summarizing Normal Pancreas development and the difference of growth factor by comprisingThe progressively progress of culture medium and be divided into pancreas precursor in dynamically stirring suspension culture systems. This process produces by pancreas thinThe diabetes group that the coexpression of born of the same parents' transcription factor PDX1 and NKX6.1 characterizes. In the time that these cells are transplanted, theyFurther ripe become the functional glucose tissue that stimulates insulin secretion, the diabetes that this tissue recoverable Streptozotocin is inducedHyperglycaemia in model.

In order to produce diabetes group, remain onThe pluripotent cell of the form of trooping in culture medium turnsMove on to 0.2 liter of glass stirring suspension bioreactor (Dasgip, order of temperature, pH and the dissolved oxygen with via controller regulation and controlRecord DS0200TBSC, Shrewsbury, MA). Pluripotent cell is clustered in bioreactor and is cultivated two days. Now (the 1st rankSection the 0th day), in cell aggregation is suspended in differential medium according to table 5a with about 0.7 hundred ten thousand cell/mL time, changeCulture medium and initiation differentiation. Then cell is kept 14 days in this closed sterile soft suspended biological reactor. In whole differentiationIn, by regulating and controlling 0.3 liter of CO in cumulative volume₂Stream makes culture medium bicarbonate level remain on 3.64g/L, and pH is kept7.4. Under the constant gas of 5 ls/h, there is under the control of Dasgip control system of 30% dissolved oxygen set point Xiang ShengThing reactor head space is filled with CO₂And air. Regulate and control air mass flow in response to dissolved oxygen content, regulate and control CO in response to pH₂Flow.

Table 5a: nutrient media components and differentiation scheme

As used in the present embodiment and whole description, SCIO is Alk5 activator, and it has chemical name: 4-{[2-(the chloro-2-fluorophenyl of 5-)-5-(1-methyl ether) pyrimidine-4-yl] amino }-N-(2-hydroxypropyl) pyridine-3-acid amides and No. CAS:674794-97-9. The chemical constitution of SCIO is in following demonstration:

In whole service, temperature remains on 37 DEG C. Operation start time and change (at every turn more for every subcultureChange the culture medium that removes 95%), impeller stops, and removes culture medium, then passes through through using Terumo^TMPipe welding machine is connected toThe peristaltic pump of the bioreactor dip-tube of pipe arrangement adds culture medium to maintain closed system.

During the 1st stage, test some different feeding settings: (a) causing latter 24 hours replacing culture mediums of differentiation, andLocated not change culture medium at 48 hours; (b) causing latter 24 hours replacing culture mediums of differentiation and locating to add grape at 48 hoursSugar-pill; And (c) whole the 1st stage without glucose change culture medium and cause differentiation latter 24 hours add GDF8Ball, then locates to add grape sugar-pill for latter 48 hours in initiation.

For as each reactor of listing in 5b of table, start in process, middle and carry out cell count while finishing. ?1 stage, while end, sampled protein expression mode by flow cytometry. (starting to be divided in setting at condition AWithin after germinal layer 24 hours, change culture medium, and do not change culture medium at ensuing 48 hours) cell of lower differentiation demonstrates and passes throughInduction differentiation mark (CD99 and CXCR4) and reduce versatility mark (CD9) and express (b) measured the best knot of Fig. 5Really. Definitive entoderm form the CXCR4 of being combined with the lower expression of CD9 and CD99 while finishing compared with high expressed and pancreas geneThe lower expression compared with the gene of the replacement organ destiny after high expressed and instruction differentiation relevant (Fig. 5 d and Fig. 5 are e). SpecificallyGround, did not change culture medium or glucose is not added to a large amount of feeding forms in the 1st stage in the whole first stage of differentiationOne or both in culture medium lower CXCR4 level while causing for the 1st stage finished, non-when this and four-stage differentiation finishThe morphology of the aggregate of Chang Butong is correlated with, and (Fig. 5 c). Specifically, in the time that the 4th stage finished, condition B and C have lower pancreasGene expression (NKX6.1 and CHGA) and higher non-pancreas gene expression (CDX2 and SOX2), as passed through flow cytometry institute(Fig. 5 d and the table 5b) measuring. These find to confirm that by qRT-PCR (e), this is because condition A demonstrates more remarkable than condition C to Fig. 5Higher pancreas gene expression, condition B is between A and C. In addition, condition C is expressed remarkable higher levels of instruction and is substituted non-pancreasGland destiny, for example CDX2, AFP and albumin (Fig. 5 gene e). These data show, last 48 hours that form at DE are not moreThe high CXCR4 of homogeneity that changes culture medium and produce expresses definitive entoderm (DE) and can be transformed into after a while pure pancreas entoderm group.

In the time that four-stage differentiation finishes, by removing from bioreactor according to the cell of condition A differentiation, with comprisingThe MCDB131 culture medium washing of 0.1%FAF-BSA, and implant in SCID-Bg mouse. Each mouse all kidney peplos just underFang Yizhi 5,000,000 cells. Implant every 4 weeks afterwards, draw blood, and measure blood sugar and c-peptide. To implanting latter 12 weeks,Detect that by ELISA people c-peptide level is higher than 1ng/mL, and in the time of 16 weeks, c-peptide horizontal is 2.5ng/mL (figure5f). After implantation 20 weeks, with fasting state, then with the c-peptide of feed state measurement animal. Glucose is processed induction circulation peopleThe 0.93ng/mL of c-peptide from fasting state is significantly increased to 2.39ng/mL in feed state, and (g), this shows to transplant thin Fig. 5Born of the same parents are ripe effectively to be organized for functional GSIS. In addition, when animal is used Streptozotocin (STZ) (compared with people β cell,Mouse β cell is more responsive and preferentially destroyed by STZ to STZ) when inducing diabetic disease states, there is effective group of functional GSISThe animal of the graft of knitting keeps normal blood sugar level, and unlike the untreated control that develops into obvious diabetes, (Fig. 5 h).These results show, the animal that has hES noble cells graft by functional human fetal pancreatic tissue transplantation thing protection is exempted to suffer from STZ and luresThe diabetes of leading.

Table 5b: cell count and flow cytometry data

Example 6

The directed differentiation of the microcarrier adhere-wall culture human embryo stem cell of the suspended form of clone H1

PreparationMicrocarrier bead (C3) (Sigma-AldrichCoLLC, St.Louis, MO, catalog number (Cat.No.)C3275) glass, being coated with by 400mg pearl being immersed in to the 20ml volume silicon of the PBS (DPBS) that comprises 15mlDulbeccoIn flicker bottle, within 4 hours to 24 hours, cultivate.By the change that is chemically connected to cross-link dextran matrixProperty collagen thin layer composition.On denatured collagen layer be subject to various protease (comprise trypsase andCollagenase) impact of digestion, and provide in maintaining maximum cell activity, functional and integrality cell from micro-The ability that carrier removes.

After soaking, pearl is carried out to autoclaving and also rinse with aseptic DPBS, and make its Eddy diffusion be supplemented with 10 μIn the mouse embryo fibroblast conditioned medium (MEF-CM) of MY-27632. Then with the density of 100mg pearl/flask, pearl is turnedMove on to 125mlIn glass rolling bottle (CorningIncorporated, Corning, NY). To contain pearl and haveThe circulator of the MEF-CM of Y-27632 at 37 DEG C at the CO of humidity 5%₂Balance at least 60 minutes in incubator.

Use TrypLE^TM(LifeTechnologiesCorporation, GrandIsland, NY) (temperature at 37 DEG CEducate 8 minutes to form single-cell suspension liquid) by the cell of the 44th generation human embryonic stem cell H1 (WA01 cell, WiCell,MadisonWI) promote from plane adhere-wall culture thing. Then washed cell be suspended in the MEF-CM with Y-27632In, and in static (static) incubation cycle, allow 1,100 ten thousand hES cell attachments to continue 6 hours to pearl. Then will haveThe MEF-CM of Y-27632 adds rolling bottle to so that final culture volume is 75mL, in glass rolling bottle with the impeller speed of 50rpmDegree stirs cell and pearl. Cell is grown 5 days by this way, and culture medium is changed 50mLMEF-CM every day. In culture 5 daysAfterwards, flask comprises 53 × 106 cells (± 12 × 10SD). In contrast, 1,000,000 H1hES cells are inoculated into and are coated withThe Matrigel of 1:30 dilution^TMAnd keep culture medium to change on the 6 hole tissue culturing polystyrene culture dishes of MEF-CM every day.

In multipotency culture, after 5 days, be then intended to summarize the little of Normal Pancreas development form generation element by containingThe progressively progress of the different culture media of the one or both in molecule and growth factor and make these cells dynamically stir and suspendIn culture systems, be divided into pancreas precursor. As the method for summarizing Normal Pancreas development, test two kinds of culture medium prescriptions; OneKind form DE with activin A and Wnt3A, and another kind of with MCX compound and GDF8 form DE (respectively show 6a withIn table 6b). Change culture medium every day, and characterize sample by RT-PCR and the flow cytometry of determining cell characteristics. ObtainGet cell and differ figure on microcarrier, and before Cell Differentiation causes as time of the cellular morphology of multipotency cultureProcess is shown in Fig. 6 a. The time course that culture differentiation is shown is shown in Fig. 6 b. Also obtain by experiment at multiple time points placeGet cell count, and for the culture medium prescription in plane culture or microactuator suspension carrier culture, result is depicted as surfaceLong-pending (is cell/cm in Fig. 6 c²) or the function of culture volume (being cell/mL in Fig. 6 d).

At multiple somes place of whole process, carry out characterize cells by flow cytometry and RT-PCR. The first stage of differentiationAnd the flow cytometry result that forms definitive entoderm is shown the cellular expression of CXCR4 (Y-axis) and CD9 (X-axis) in Fig. 6 eDot chart, and this result is also expressed as total expression of every kind of mark in Fig. 6 f. Result shows, under all conditions,Basic most cells forms definitive entoderm, and the increase of expressing as CXCR4 and the minimizing of versatility surface marker CD9 are limitFixed. In addition, more effectively forming with following processing hierarchal order of definitive entoderm occurs: MCX/GDF8 microcarrier > MCX/GDF8 plane > WNT3A/AA microcarrier > WNT3A/AA plane. Really demonstrate cell is had to culture medium specificity effect, thisBecause demonstrate the lower expression (figure of CERBERUS (Cer1), GOOSECOID and FGF17 with the cell that MCX/GDF8 processes6g). But all treatment conditions all demonstrate the similar of definitive entoderm gene C D99, CXCR4, FOXA2, KIT and SOX17Expression (Fig. 6 g and table 6c). These processes produce by the coexpression of pancreatic cell transcription factor PDX1 and NKX6.1 and characterizeDiabetes group. In the time that these cells are transplanted, they are further ripe is that functional glucose stimulates insulin to divideSecrete tissue, the hyperglycaemia in the diabetes model of this tissue recoverable Streptozotocin induction.

As used in following table 6a, B27 isReplenishers (LifeTechnologiesCorporation,Carlsbad,CA)。

As used in this example, MCX compound is 14-third-2-alkene-1-base-3,5,7,14,17,23,27-, seven azepines fourRing [19.3.1.1～2,6-～.1～8,12.～] heptacosane-1 (25), 2 (27), 3,5,8 (26), 9,11,21,23-ninth of the ten Heavenly StemsAlkane-16-ketone, it has following formula (formula 1):

Other ring-type aniline-pyridine triazines also can replace above-mentioned MCX compound to use. This compounds includes but not limited to14-methyl-3,5,7,14,18,24,28-, seven azepine Fourth Rings [20.3.1.1～2,6～.-1～8,12～] octacosane-1(26), 2 (28), 3,5,8 (27), 9,11,22,24-nonane-17-ketone and 5-are chloro-1,8,10,12,16,22,26,32-eight nitrogenAssorted five rings [24.2.2.1～3,7～-1～9,13～.1～14,18～] 30-3 (33), 4,6,9 (32), 10-, 12,14(31), 15,17-nonane-23-ketone. These compounds illustrate (formula 2 and formula 3) hereinafter:

Exemplary suitable compound is open in U.S. Patent Application Publication 2010/0015711, because this patent relates toAnd MCX compound, relevant ring-type aniline-pyridine triazine, and their synthetic, its disclosure is incorporated in full.

In this embodiment the 4th the stage use Cyp26 inhibitor for N-{4-[2-ethyl-1-(1H-1,2,4-triazole-1-yl) butyl] phenyl }-1,3-BTA-2-amine is for its No. CAS 201410-53-9 and have following structure:

This Cyp26 inhibitor is also referred to as " Cypi ". The structure of this Cyp26 inhibitor and the synthetic U.S. Patent number 7 that is disclosed in,378,433, because relating to Cyp26 inhibitor, it synthesizes with it, and the disclosure of this patent is incorporated herein in full.

Table 6a: culture medium prescription and differentiation scheme

Table 6b: culture medium prescription and differentiation scheme

Table 6c

Example 7

The subclone WB0106 of H1 (WA01) hES clone is used for to this example. WB0106 is at WiCellResearchIt is inoculation material that Institute (Madison, WI) derives from the H1 that is called DDL-13. The WB0106 subclone of H1 system derives fromThaw to Matrigel in the 23rd generation^TMSuprabasil mTESR1^TMDDL-13 bottle in culture medium, and use subsequently EDTAGo down to posterity. WB0106 is freezing in the 28th generation, and based on normal dyeing body group type (FISH and G-band), be divided into pancreatic progenitor cellAbility, and form the ability of trooping and increasing in suspension culture, these researchs are selected.

Then WB0106WCB bottle is thawed in T225 flask (CorningIncorporated, Corning, NY)Matrigel^TMIn suprabasil culture medium, and in the time going down to posterity for the first time, cell increases in multiple T225 flasks. SecondWhen inferior going down to posterity, in conjunction with the cell from multiple T225 flasks, and use it for the single 2 layers of CellStack of inoculation^TM(CS2). OneDawn CS2 70% converges,Piping components lid and adjacent pump pipe arrangement are attached to culture medium port with shutdown system. With After pipe arrangement shutdown system, by Terumo welding machine, bag or bottle are welded above, and use peristaltic pump transfer liquid volume(culture medium PBS^-/-、Or suspension cell).

For promoting cell from CS2, by PBS-for cell/-washing once, then useHalf intensity solution placeReason, with PBS-/-dilution, and incubation 4 minutes to 5 minutes. Then removeAnd application enzyme solutions after 3Minute, flicking CS2 is to promote cell to promote. To be supplemented with 0.5%BSA and comprise 10 micromole Rho inhibitors of kinases Y-One bottle of culture medium of 27632 is pumped in CS2 to rinse and deactivation remnantsThen collect flushing liquor. Add andCollect the second flushing liquor volume, and merge with the first flushing liquor. Then by 200mL 2.0 × 10⁸-2.5×10⁸Individual cell turnsMove on to 1 layerIn and at the CO of humidity 5%₂In incubator at 37 DEG C incubation 2 hours. Use is attached at twoIndividualC-Flex between culture medium port^TMThe closed loop of pipe arrangement and pump pipe arrangement, suspends cell by peristaltic pumpLiquid under 75rpm, grinds 5 minutes so that aggregation homogenising. Closed loop pipe arrangement is replaced with to aseptic 0.2 micron filter to allowGas exchange, and willAt 37 DEG C, in the CO2 of humidity 5% incubator, be incubated overnight. After being incubated overnight(12 hours to 22 hours, 18 hours the bests),In cell form the circular spheroidal aggravation of pluripotent cell(trooping).

To contain the culture medium that is supplemented with 0.5%BSA that suspension cell troops together with being supplemented with 0.5%BSA and maintenance0.4 liter of fresh culture of 55-65rpm fromTransfer to 1 liter of disposable rolling bottle (Corning;Corning, NY) in. After shifting twenty four hours, the CO by 1 liter of disposable rolling bottle from humidity 5%₂Incubator removes, and permitsSedimentation 5-10 minute is permitted to troop. Then by culture medium sucking-off, until 200mL is retained in container, then by extra 400mL newFresh culture medium adds rolling bottle to. In the time within the 2nd day, finishing (shifting latter 48 hours), repeat this process.

In the time within the 3rd day, finishing (from CS2 transfers to rolling bottle 72 hours), useProcess isolated cell troop withGo down to posterity and further amplification. Cause by remove 1 liter of disposable rolling bottle from the CO2 incubator of humidity 5% process of going down to posterity. WillFlask is placed on the circulator plate in Biohazard Safety Equipment to keep the unit for uniform suspension of cell. Cell suspending liquid is passed through100mL pipette removes from rolling bottle, and is evenly distributed on four 175mL taper Merlon pipe (ThermoFisher-Nalgene; Buffalo, NY) between, and with 80-200rcf centrifugal 5 minutes. The culture medium that sucking-off was used and interference cell notGranulation. Then 25mL be there is no to the DPBS (DPBS of calcium or magnesium^-/-) add each pipe to, and by Cell binding to circular coneIn pipe and under 80-200rcf centrifugal 5 minutes. By DPBS^-/-sucking-off from conical tube, and add 50% of 30mL/50％DPBS^-/-solution is to pipe. Cell cluster is aspirated 1-3 time, and then turn at intermittence 4 minutes, then at 80-Under 200rcf centrifugal 5 minutes. Then as far as possible fully sucking-offAnd not interference cell granulation, and continuous and warmWith ground flicking conical tube 3 minutes to 5 minutes, until cell suspending liquid presents uniform milky. To comprise 10 micromole RhoThe 10mL culture medium that is supplemented with 0.5%BSA of inhibitors of kinases Y-27632 add to cell suspending liquid and grind 2 to 4 times withMake remnants'Inactivation. Be supplemented with 0.5%BSA's by what comprise 10 micromole Rho inhibitors of kinases Y-2763290mL culture medium adds to through 40 microns of cell filter screen (BDFalcon; FranklinLakes, NJ) cell and suspensionLiquid.

Use NC-100(ChemoMetecA/S, Allerod, Denmark) determines 100mL volumeFiltration cell suspension in cell density, and add extra culture medium to comprise 10 micromole Rho kinase inhibitionIn the culture medium that is supplemented with 0.5%BSA of agent Y-27632, obtain 1 × 10⁶The final cell concentration of individual cell/mL. Then willThe cell suspending liquid of 225mL (22,500 ten thousand cells) is transferred to 1 liter of disposable rolling bottle and incubation 1 hour, and not in humidityIn 5% CO2 incubator, stir. Then flask is removed from incubator, and on circulator plate in Biohazard Safety Equipment with100rpm stirs 1-3 minute. In the time of cell mixing suspension, being supplemented with of 10 micromole Rho kinase inhibition Y-27632 will be comprisedThe extra 225mL culture medium of 0.5%BSA adds cell suspending liquid to. Then rolling bottle is turned back to the CO2 incubator of humidity 5%In continue 30 minutes. Then flask is removed from incubator, and stir with 100rpm on circulator plate in Biohazard Safety EquipmentMoving 1-3 minute. In the time of cell mixing suspension, the 0.5%BSA that is supplemented with of 10 micromole Rho kinase inhibition Y-27632 will be comprisedExtra 150mL culture medium add cell suspending liquid to so that final volume is 600mL, and flask is turned back in incubatorStirred suspension. ?Dissociate and locate for latter 24 hours and 48 hours, allow cell cluster at 5 minutes in 10 minutesBe deposited to drag. Guarantee to make any minimization of loss of trooping, by stirring, the 400mL culture medium of crossing is removed from flaskAnd replace with fresh culture. Use this process, H1 cell is transformed into suspension culture from suprabasil adhere-wall culture thing and doesFor cell cluster.

InitiallyProcess latter 72 hours, repetitive cell is trooped and is dissociated with the process of rolling bottle inoculation (going down to posterity) to protectHold cell in suspension for repeatedly go down to posterity (test specification: 1 generation in generation to 10). Follow said process, different, firstAfter individual 24 hours, do not remove culture medium, and add 200mL fresh culture. ?Dissociate 48 hours afterwardsPlace, allows to be clustered in 5 minutes to 10 minutes and is deposited to drag, sucking-off 600mL, and 400mL fresh culture is addedBe added to flask.

These suspensions are gone down to posterity and the cell cultivated then can be frozen and store for using in the future. For for the preparation of frozenSuspension amplifying cells, cell cluster is used as mentioned aboveDissociate and go down to posterity for suspension, different is thinBorn of the same parents are not through 40 microns of cell filter screens. Determine the cytometer of the 100mL cell suspending liquid being produced by each 1 liter of disposable flaskNumber. Then in conjunction with cell suspending liquid under 80-200rcf centrifugal 5 minutes. Then as far as possible fully remove from centrifuge tubeCulture medium and not interference cell granulation. Then add cold (< 4 DEG C) in mode dropwise(StemCellTechnologies, Inc., Vancouver, BC, Canada) to realize the ultimate density of 15,000 ten thousand cell/mL, andTransfer to 1.8mLCryovial (CorningIncorporated, Corning, NY) or 15mLMiltenyiIn the process of freezer bag (MiltenyiBiotecInc.Auburn, CA), cell solution is remained in ice bath.

Then as described belowly in controlled rate freezer unit, suspension amplifying cells is chilled in bottle with high density. By chamberBe chilled in advance 4 DEG C and keep this temperature until sample flasket temperature reaches 6 DEG C. Then by room temperature with 2 DEG C/min reduce untilSample reaches-7 DEG C. Once sample flasket reaches-7 DEG C, chamber is cooling until chamber reaches-45 DEG C with 20 DEG C/min. Then allowRoom temperature with 10 DEG C/min of of short duration risings until room temperature reaches-25 DEG C, then that chamber is further cooling straight with 0.8 DEG C/minReach-45 DEG C to sample flasket. Then with 35 DEG C/min of cooling chamber temperature until chamber reaches-160 DEG C. Then room temperature is protectedBe held in-160 DEG C at least 10 minutes, after this, bottle is transferred to gas phase liquid nitrogen storage device.

In order to inoculate stirred-tank bioreactor, high density freeze-stored cell is removed from liquid nitrogen storage device, thaw alsoAnd for inoculating 3 liters of glass biological reaction device (DASGIP of sealing; Julich, Germany). By four or five bottles from gasPhase liquid nitrogen storage device removes, and is directly placed in 37 DEG C of water-baths 105 seconds. Then will thaw by 2ml glass pipetVial content is transferred to 50ml conical tube. Then will contain 0.5%BSA and be supplemented with 10 micromole Rho inhibitors of kinases9ml culture medium (IH3 or the Essential8 of Y-27632^TMCulture medium (" E8^TM")) add in pipe in mode dropwise. Then makeCell under 80-200rcf centrifugal 5 minutes. From pipe sucking-off supernatant, add and contain 0.5%BSA and be supplemented with 10 micromoles10ml fresh culture (IH3 or the E8 of Rho inhibitors of kinases Y-27632^TM), and the volume that contains cell is pipetted to cultivationGroup-transfer bottle (SaniSure, Moorpark, CA) in. Then join by the aseptic C-flex being welded by peristaltic pumpPipe is directly pumped into bottle content in bioreactor. Preparing in multipotential stem cell inoculation, bioreactor is trained with 1.5LSupport base (IH3 or the E8 that are supplemented with 0.5%BSA and comprise 10 micromole Rho inhibitors of kinases Y-27632^TM) preparation, be preheated to37 DEG C, stir with 70rpm, regulate and control to 6.8-7.1pH by CO2, wherein dissolved oxygen set point be 30% (CO2, air, O2 andN2 regulation and control). And then after inoculation, bioreactor is sampled for cell count, adjust and cultivate as requiredMatrix is long-pending to obtain 0.225 × 10⁶The final cell concentration of individual cell/mL.

The cell being inoculated in stirred-tank bioreactor forms cell cluster in continuous agitator tank, and remains on anti-Answer the versatility culture medium in device (to be supplemented with IH3 or the E8 of 0.5%BSA^TM) in three days altogether. Change culture medium every day, whereinBe removed and 1.5 liters of fresh cultures are added with the culture medium of mistake along with 1-1.3 rises, within latter 24 hours, carry out part in inoculationCulture medium is changed. Latter 48 hours of inoculation, removes 1.5-1.8 and rises the culture medium of using and add 1.5 liters of fresh cultures.Within latter 72 hours, locate in inoculation, by removing the culture medium crossed of 90% use and add differential medium and cause pluripotent cell and divideChange by (table 7).

Once you begin segmentation atomization, cell temperature (37 DEG C), pH (for being divided into 7.4) and dissolved oxygen (forThe 1st stage was 10%DO set point, and all be 30%DO set point At All Other Times, CO2, O2, N2 and air regulation) through regulation and controlClosed sterile soft suspended biological reactor in keep 12 days or more days. In whole atomization, change at every subcultureTime, impeller stops 5 minutes to 20 minutes, then removes culture medium to allow the sedimentation of trooping by dip-tube. Use by passingTerumo^TMPipe welding machine is connected to C-Flex^TMThe peristaltic pump of the dip-tube of pipe arrangement by the culture medium in bioreactor from sealingBottle or bag remove or add to wherein to keep closed system. Once enough culture mediums are added to container with complete submergence leafWheel, just switches on again to impeller and heater.

For monitoring bio reactor process, aspirate the media samples that comprises cell cluster every day to determine cell quantityAnd activityAs shown in Figure 7. During this process, observe the general amplification of cell, becauseThe 3rd day 4 stages, 0.225 × 10⁶The inoculum of individual living cells/mL increases to produce average 0.92 × 10⁶Individual living cells/mL. ?In the process that bioreactor is inoculated and pluripotent cell is trooped and cultivate, by cell being remained on to acid set point (pH7.0-6.8), the average cell output in the 4th the 3rd day stage is increased to average 1.3 × 10⁶Individual cell/mL (Fig. 7).

Except counting every day, pass through NOVAFLEX(NovaBiomedical,Corporation，Waltham, MA) bioreactor culture basic pattern product are analyzed. Can observe, according to reactor set point, the 0th stageIn the pH stable state standard pH 7.4 total with respect to most of culture mediums of culture medium be acid, and bioreactor culture basePH is because cellular metabolism declined by the 0th stage (Fig. 8). These results and lactic acid concn increase and glucose level declinesTrend correlation (Fig. 9 and Figure 10) while end for the 6th day by breaking up. In a word, the cell in these data Indicator Reaction devices passes throughThe first two stage (1-6 days) growth of the 0th stage and differentiation the most rapidly and be glucose consumption. But, from the 3rd rankDuan Xiangqian, cellular metabolism (lactate levels of reduction and the glucose level of increase) in reactor declines, this and the 3rd rankThe peak value of section cell quantity, the decline of cell density is relevant in the 4th phase process subsequently.

For the phase specificity of determining pH and metabolism changes the variation of whether mating mrna expression pattern. Use appointment manyEnergy property, definitive entoderm (DE), intestinal tube (GT) or four applying biologicals in the 4th stage (S4)Low-density array (LifeTechnologiesCorporation, Carlsbad, CA) carry out the test of bioreactor cell sample, by result and workFor the past of contrast does not break up H1 (WB0106) hES cell sample and compares so that the expression standard on all operations and arrayChange.

Use these arrays, for differentiation each stage determine gene expression. Also observe, thaw to bioreactorIn inoculation material cell the 1st day the 0th stage and the 3rd day the 0th stage (after bioreactor inoculation 24 hours and 72 littleTime: Figure 11, Figure 12, Figure 13 and Figure 14) demonstrate not differentiation gene expression pattern. These results and flow cytometry result are closeRelevant, it demonstrates CD9, SSEA4, TRA-1-60 and the TRA-1-81 of high expression level, but does not have CXCR4/CD184 (figure15 and table 8). Although the flow cytometry of gene expression and qRT-PCR determination method demonstrate for versatility gene (CD9,NANOG, POU5F1, SOX2, TDGF and ZFP42) with the effective and stable expression pattern of stable multipotency state consistency, but alsoDuring it should be noted that the 0th phase process before directed differentiation, the appropriateness of the gene expression of GATA4, GSC, MIXL1 and T butVariable increase, and CER1, FGF17 in some samples, FGF4 and GATA2 express and increase >=100 times (Figure 16 and Figure 17).

In the time completing for the 0th stage (reactor is inoculated latter 72 hours), signaling is trained to the differentiation that comprises MCX and GDF8Support in base (table 7). This culture medium is changed rear twenty four hours, notices remarkable change (Figure 18 and the Tu of gene expression pattern19), approximately 700 times of increases expressing such as FOXA2, and CER1, EOMES, FGF17, FGF4, GATA4, GATA6, GSC,1000 times of increases that MIXL1 and T express. The expression indicator cells of these increases changes by mesendoderm destiny. Also shouldWork as attention, CDX2 level increased the 1st day the 1st stage and (expresses and increase by 470 compared with the control compared with neoblastDoubly), but this is the instantaneous increase of expressing, and CDX2 level declined 94% from the 1st day the 1st stage to the 3rd day the 1st stage,Turn back to and induce those suitable levels (Figure 14, Figure 19 and Figure 21) of observing before differentiation.

Within 72 hours after being exposed to DE differential medium, locate, the specification of cellular expression and definitive entoderm consistent pointCloth, this is because CXCR4 level reaches peak value and FOXA2 and SOX17 to exceed historical control > 1000 times express. With fixedShape entoderm is consistent, shall also be noted that gene C ER1, EOMES, FGF17, FGF4, GATA4, GATA6, GSC, MIXL1 and T fromThe elevated levels decline (Figure 20 and Figure 21) that the 1st the 1st day stage was observed.

The change of the gene expression of observing by qRT-PCR is relevant to the result of observing by flow cytometry. AlsoHomogeneity when the negative pluripotent cell group of CD9 expression/CXCR4 (Figure 15) while observing from differentiation initiation finished to the 1st stageCXCR4 expresses cell mass (98.3% cell is the CXCR4 positive, ± 1.9SD) approaching transformation completely (Figure 22).

Complete definitive entoderm form (the 1st stage) afterwards, by culture medium be replaced by contain FGF7 (for induce former beforeIntestines form morphogen) culture medium (the 2nd stage). Consistent with the formation of former anterior intestine, HNF4 α and GATA6 expressionIncrease the 1st day the 2nd stage and the 3rd day, and the 3rd day gene with high level expression in the 1st stage (CXCR4, EOMES,FGF17, FGF4, MNX1, PRDM1, SOX17 and VWF) in the time that finishing, demonstrates the expression (Figure 23) of minimizing in the 2nd stage. Anterior intestine baseBecause the expression of (AFP, PDX1 and PROX1) increases (Figure 24).

After cell has been cultivated 72 hours in the 2nd stage culture medium, culture is transformed into the 3rd stage culture medium (table7). Once in this culture medium, cellular expression is expressed measured entoderm pancreas with PDX1 and FOXA2 as shown in Figure 25The mark (90.9% ± 11.9SDPDX1 positive and 99.2% ± 0.6SDFOXA2 positive) that pedigree is consistent. These results are logicalCross the data validation of the sample of analyzing for the qRT-PCR of gene expression. The gene expression of PDX1 in 24 hours from the 2nd stageWithin the 3rd day, finish (compared with H1 38,000 times) and finished (compared with H1 200,000 times) to the 1st day the 3rd stage and increase by 500, andAfter the 3rd the 3rd day stage, within 48 hours, again double (compared with H1 435,000 times). It is designated that these data demonstrate cellFor pancreas destiny (Figure 26). The gene of conventionally expressing in pancreas by increase level (ARX, GAST, GCG, INS, ISL1,NEUROD1, NGN3, NKX2.2, NKX6.1, PAX4, PAX6, PTF1A and SST) host further support this observed result, asShown in Figure 26. In addition, also observe low-down or do not have OCT4/POU5F1 to express the (2-10% of contrast or true by qRT-PCRFixed 32-37 sample Cts) and for the high expression level of other entoderm pedigree marks AFP, ALB and CDX2-, enterOne step show the specification of cell mass in bioreactor with and from relative plastics intestinal tube destiny to pancreas the turn of the wheel.

As shown in figure 27, in the time that the atomization in the 3rd day the 4th stage finishes, cell keeps high-caliber PDX1 and FOXA2Express and further develop and pancreatic endocrine cell (28.1% ± 12.5SD chromograin positive) and pancreatic progenitor cellThe consistent expression pattern of mixture of (58.3% ± 9.7SDNKX6.1 positive). This phase specificity marker expression pattern refers toShow from multipotency colony and be effectively progressively divided into diabetes. Use and further confirm thin by streaming from the data of qRT-PCRThe result that born of the same parents' art is observed. The gene of conventionally expressing in pancreas (ARX, GAST, GCG, IAPP, INS, ISL1, MAFB,NEUROD1, NGN3, NKX2.2, NKX6.1, PAX4, PAX6, PTF1A, and SST) host all demonstrate the expression water of increaseFlat. (Figure 28).

At the pH to multiple process variables such as different vaccination material, the 0th stage culture medium, the 0th stage culture medium andWhen the use of defoamer is tested, the expression pattern of observing in Figure 27 is in service being consistent repeatedly. To inoculation materialMultiple source test, and every kind effectively produces and has > 90%FOXA2, > 75%PDX1 and > 50%NKX6.1Pancreas entoderm destiny (Figure 29). In addition, it should be noted that in the time that cell was grown in following culture medium in the 0th stage, biological anti-Answer in the expression pattern of device product and there is no significant difference: be supplemented with the inner culture medium of the customization that is called " IH3 " of 0.5%BSA, orBe supplemented with the culture medium of the commercially available acquisition of 2%BSA: Essential8^TM, be supplemented with 0.5%BSA (Figure 30). Exist when detecting pHThe 0th stage was done the used time in cultivating, it should be noted that in the 0th stage with the cell of relatively low pH (6.8) growth with respect toOn average operate in the amplification (Fig. 7) in bioreactor with increase, but not aobvious in the 3rd day the 4th stage cell distributionWork changes (Figure 31). In addition, observe with 94ppm and use defoamer C emulsion (Sigma catalog number (Cat.No.) A8011) to reduce by bubblingThe bubble producing, finishes cell distribution (table 9 and the figure to the 4th stage the 3rd day cell but seem not affect from the 0th stage32)。

In the time that each bioreactor differentiation finishes, by product cell cryopreservation. Cell is had to 3.63g/L sodium acid carbonateMCDB131 in or having in the MCDB131 of 3.63g/L sodium acid carbonate, glucose (final 8mM) and 1xGlutamaxWashing, then transfer to by the 57.5%MCDB131 with 2.43g/L sodium acid carbonate, 30% without allos KSR, 10%DMSO andCold (< 4 DEG C) frozen culture medium (ultimate density is 25mM) of 2.5%HEPES composition. Then use cooling distribution by cell freezingIn controlled rate freezer unit (CRF), this cooling distribution remains on cell cluster at ambient temperature in frozen culture medium and holdsContinuous maximum 15 minutes, the temperature that is reduced to 4 DEG C continued 45 minutes, is further reduced to-7.0 DEG C (sample) with 2.00 DEG C/min.Sample is then cooling fast, by room temperature with the rate reduction of 25.0 DEG C/min to-45.0 DEG C. Then by by room temperature with10.0 DEG C/min are increased to-25.0 DEG C of increases (chamber) that afford redress. Then with 0.2 DEG C/min of cooling sample until temperature reachTo-40.0 DEG C. Then chamber is cooled to-160 DEG C with the speed of 35.0 DEG C/min, and at this temperature, keeps 15 minutes. ?When CRF runs abort, sample is moved to gas phase liquid nitrogen storage vessel.

Can cell be thawed by removing from gas phase liquid nitrogen storage device and bottle being transferred to 37 DEG C of water-baths. By littleBottle in water-bath gently turn be less than 2 minutes, until little ice crystal is stayed in bottle. Then vial content is transferred to 50ml circleThe MCDB131 culture medium that cone and use have 2.43g/L sodium acid carbonate and 2%BSA was dropwise diluted to finally in two minutesVolume is total up to 20ml. Then pass throughDetermine total cell quantity, and cell suspending liquid is transferred to superThe low culture dish that adheres to continues 1 hour. Then by cell, the culture medium from 50ml circular cone separates, and removes supernatant and by cellBe resuspended in the 4th stage culture medium for studying in analysis or body.

Alternatively, after thawing, the cell that packs bottle into is transferred to empty 125mL glassRolling bottle(Corning, Incorporated, Corning, NY), and will contain the 10mL of 2.43g/L sodium acid carbonate and 2%BSAMCDB131 culture medium adds flask in mode dropwise. Final volume is adjusted into the same medium of 80mL. Pass throughDetermine total cell quantity, and cell suspending liquid is stirred and spent the night (12 hours with 40rpm to 65rpmTo 28 hours). Then cell is characterized or uses it in body and study.

The composition of IH3 illustrate below and U.S.'s published application 2013/0236973 in show method obtain, byRelate in this patent the cell culture medium that cell is suitable, its disclosure is incorporated herein by reference in full. In IH3 culture mediumThe amount of BSA can change.

Table 7

Table 8

Table 9

Material：

People embryo does (hES) clone H1, (WA01 cell, WiCell, MadisonWI)

PBS (catalog number (Cat.No.) 14190, Invitrogen)

Y-27632 (Axxora catalog number (Cat.No.) ALX-270-333, SanDiego, CA)

EDTA, (Lonza, catalog number (Cat.No.) 17-7-11E)

·-(ChemoMetecA/S, catalog number (Cat.No.) YC-T100, Allerod, Denmark)

Not with 6 hole culture dishes of tissue culture processing (BectonDickinson, catalog number (Cat.No.) Falcon351146,FranklinLakes,NJ)

·(Sigma, catalog number (Cat.No.) 12604-013, St.Louis, MO)

PH and dissolved oxygen (DO) bioreactor probe (PH electrode 225mm, model F-635, andDO12mm sensor, model D-145, from Broadley-James, IrvineCA)

Large-scale packaging system (the Theracyte of immune protective^TM,IrvineCA)

Mm people C-peptide ELISA (MERCODIA catalog number (Cat.No.) 10-1141-01)

·Glutamax^TM, MCDB131 and ITS-XInvitrogen

·FAF-BSA(Proliant)

Retinoic acid, glucose 45% (2.5M), SANT (Shh inhibitor) is (Sigma)

·GDF8(Peprotech)

·MCX

·FGF7(R&DSystems)

LDN-193189 (bmp receptor antagonist) (Stemgent)

TPPB (PKC activator) (ChemPartner)

MCDB131 customizes culture medium

Example 8

Maturation and functionalization that the pancreas ancestral that frozen bioreactor produces troops

Study for each bioreactor in order to produce enough cells, by one of H1hES (WB0106) cellThe 31st generation master cell bank bottle thaw. Use EDTA go down to posterity by cell under adherent conditionIn culture medium, increaseSo that at Matrigel^TMUpper several times go down to posterity, until produce sufficient cell to inoculate five Matrigel^TMCoating 2 layers(CS2). Once the attached cell growing in CS2 70% converges,Piping components lid and adjacent pumpPipe arrangement is attached to culture medium port with shutdown system. After shutdown system, utilize C-Flex^TMBy Terumo welding machine by bag orBottle welding is upper, and uses peristaltic pump transfer liquid volume (culture medium PBS^-/-、Or suspension cell).

For promoting cell from CS2, the Du Shi phosphate buffered saline (PBS) (PBS by cell with calcic not or magnesium^-/-) washing oneInferior, then useHalf intensity solution-treated, with the PBS of equal parts^-/-dilution, and incubation 4 minutes to 5 minutes.Then removeSolution, and after application enzyme solutions 3 minutes, flicking CS2 is to promote cell lifting. To containOne bottle of 10 micromole Rho inhibitors of kinases Y-27632Be pumped in CS2 to rinse and deactivation remnantsThen collect flushing liquor. Add and collect the second flushing liquor volume, and merging with the first flushing liquor. At 2 litersFinal volume in by 1.6-2.0 × 10⁹Individual cell removes from CS2. By every layer 2.0108 to 2.5 × 108⁸Individual cell is transferred toFour CS2 or eight 1 layer of CellStack^TMIn, and with the volume of every layer of 200mL the CO in humidity 5%₂In incubator 37Incubation 2 hours at DEG C.

Use is attached atBetween culture medium portThe closed loop of pipe arrangement and adjacent pump pipe arrangement,By peristaltic pump, cell suspending liquid is ground 5 minutes so that aggregation homogenising under 75rpm. Then willAt the CO of humidity 5%₂In incubator, at 37 DEG C, be incubated overnight and continue 18 hours. Then by 2 liters thin from CellStacksBorn of the same parents and culture medium are fresh together with 1.5 liters of each bioreactorsCulture medium merges and transfers to two 3 liters of DASGIPIn bioreactor, each 1 liter. Before causing differentiation, useCulture medium keeps cell other two days, at biologyReactor is inoculated latter 24 hours and is carried out complete medium replacing. Then cause differentiation and directed described in table 10. By cellFor (the drift or be regulated to 6.8 or 7.2 by CO2 for pluripotent cell, and thin for differentiation of temperature (37 DEG C), pHBorn of the same parents are regulated to 7.4) and dissolved oxygen (30%DO set point, CO₂Air regulation) regulation and control closed sterile soft suspended biological reactor inKeep 14 days or 15 days (2 days mTeSR altogether₂+ 12 days or segmentation in 13 days differentiation). Before every subculture is changed, impeller is stoppedStop 5-20 minute to allow the sedimentation of trooping. Use Terumo by passing^TMPipe welding machine is connected to C-Flex^TMPipe arrangement (Cole-ParmerNorthAmerica, VernonHills, IL) the peristaltic pump of dip-tube remove or add culture medium to keep envelopeClosed system. Once add enough culture mediums with submergence impeller, just impeller and heating jacket switched on again.

Use these methods to cause production run in 3 liters of reactors. In service at the first reactor, at multipotency culture mediumA few days ago test two different pH set points. Reactor 1 is set to pH by 5% fixed co2 gas infusion rates7.2, so along with reactor environment is passed acidifying in time due to the metabolic activity of cell, pH is by lower to " drift ". Will be anti-7.2 the pH that answers that device 2 is set to regulate and control by CO2 gas level. In service at the second reactor, for reactor 1, pH is establishedBe decided to be 6.8, and be set as 7.2 for reactor 2, both all pass through CO₂Gas level regulation and control.

For monitoring bio reactor process, in the time that each stage of differentiation finishes, obtain cell cluster and pass through streamingCell art is measured (table 11, table 12). The negative pluripotent cell group of CD9 expression/CXCR4 while observing from differentiation initiation is to fixedHomogeneity CXCR4 when shape entoderm has formed expresses approaching completely of cell mass (96.9-98.1% cell is the CXCR4 positive)Change.

The result of observing by flow cytometry is relevant to the result of the paired samples from analyzing by rt-PCR. ?The whole process of the allelic expression of the segmentation differentiation from versatility to pancreas destiny, sample is tested. Fixed in initiationBefore differentiation, for the assortment of genes being associated with versatility or early differentiation destiny, anti-by the biology on low-density arrayAnswer device cell cluster to test mRNA.

Can be observed, compared with undifferentiated H1 control group, keep for gene multipotency from the cell of bioreactorProperty feature expression (POU5F1, NANOG, SOX2 and ZFP42) and demonstrate the minimum induction to Gene Differentiation feature or do not haveThere are induction (AFP and FOXA2: express and increase < 50 times; FOXD3, GATA2, GATA4, GSC, HAND2, MIXL1 and T: express and increase< 10 times) (Figure 33). But, once cell contact with the 1st day the 1st stage differential medium, gene expression pattern with CDX2,CER1, FGF17, FGF4, FOXA2, GATA4, GATA6, GSC, MIXL1, MNX1 and Brachyury (T) expression is increased toBe greater than and do not break up 100 to 1000 times, H1hES cell and significantly change (Figure 34). To the 3rd day the 1st stage end (definitive entodermForm) time, CD9, CDX2, FGF4, MIXL1, NANOG, POU5F1 and Brachyury (T) had with respect to the 1st day the 1st stageThe expression reducing, and characteristic definitive entoderm gene (such as CD99, CER1, CXCR4, FGF17, GATA4, GATA6, KIT,OTX or SOX17) expression reach peak value (Figure 35).

In the time that the 1st stage finished, cell culture medium is replaced by the culture medium that contains FGF7 from the culture medium that contains GDF8.Notice some different gene expression patterns: some gene expression increase in the process in the 2nd stage (AFP, ATOH1,HHEX, OSR1, PDX1, PROX1, SOX2 and SOX9), express and reduce (HAND1 and SOX17), the high expressed (HNF4 of all-the-time stableα), or low expression/do not express (CDX2, GAST, NKX2.2, NKX6.1 and PTF1a) (Figure 36 a-e). These pattern instructions, rightIn the mesoderm mark (HAND1 and SOX17) reducing, the cell in reactor becoming anterior intestine (AFP, ATOH1, HHEX,HNF4 α, OSR1, PDX1, PROX1, SOX2 and SOX9) express. But while end to the 2nd stage, cell is not yet designated as moreRipe intestines or pancreas destiny (CDX2, GAST, NKX2.2, NKX6.1 and PTF1a).

While end to the 3rd stage, cell has been specified and has been expressed measured pancreas pedigree as PDX1, this PDX1 express by with notDifferentiation contrast is compared, and mRNA increases > 76-98% of 100,000 times (Figure 36) and the expression PDX1 by Flow Cytometry AssayCell illustrates (table 11). Also observe other genes (GAST, NKX2.2, NKX6.1, PROX1, PTF1a and SOX9) to pancreasWith the induction of other genes (such as AFP and CDX2) of intestines, this shows that cell has started to specify more ripe destiny.

While end to the atomization of the 3rd day of the 4th stage or the 4th day, cell demonstrates and pancreatic endocrine cellThe consistent expression pattern of mixture of (the 47-54% chromograin positive) and pancreatic progenitor cell (the 33-52%NKX6.1 positive).This phase specificity marker expression pattern is indicated the effectively progressively differentiation from multipotency colony to pancreatic progenitor cell, and ancestral is thin for this pancreasBorn of the same parents are characterised in that compared with undifferentiated H1 human embryo stem cell, PDX1 (> 1 × 10⁶Doubly induction) and other pancreas genes(ARX, GCG, GAST, INS, ISL, NEUROD1, NGN3, NKX2.2, NKX6.1, PAX4, PTF1a and SST's > 1000 times lureLead) high expression level, and OCT4/POU5F1 express approach total loss (Figure 37).

In the time that atomization finishes, produce 0.08-0.45 × 10⁶Cell/mL (Figure 38: cell count every day). Then willThe cell cryopreservation producing in this process, or pass through TheraCyte^TMDevice is directly implanted in animal body through subcutaneous, or is placed onKidney peplos below. For freeze-stored cell, transferred to by the 57.5%MCDB131 with 2.43g/L sodium acid carbonate, 30% nothingThe frozen culture medium (ultimate density is 25mM) of KSR, the 10%DMSO of allos and 2.5%HEPES composition. Once cell cluster existsEnvironment temperature low suspension, in frozen culture medium, moved to cryovial controlled rate freezer unit (CRF) in 15 minutes.Then room temperature was reduced to 4 DEG C in 45 minutes, and was further reduced to-7.0 DEG C (sample) with 2.00 DEG C/min. ThenSample is cooling fast, by room temperature with the rate reduction of 25.0 DEG C/min to-45.0 DEG C. Then by by room temperature with10.0 DEG C/min are increased to-25.0 DEG C of increases (chamber) that afford redress. Then with 0.2 DEG C/min of cooling sample until temperature reachTo-40.0 DEG C. Then chamber is cooled to-160 DEG C with the speed of 35.0 DEG C/min, and at this temperature, keeps 15 minutes. ?When CRF runs abort, sample is moved to gas phase liquid nitrogen storage vessel.

After cell has been stored in gas phase liquid nitrogen, by removing from storage device the cell that thaws, and transferred to 37DEG C water-bath. By bottle in water-bath gently turn be less than 2 minutes, until little ice crystal is stayed in bottle. Then by vial contentTransfer to MCDB131 culture medium that 50ml circular cone and use have 2.43g/L sodium acid carbonate and a 2%BSA in two minutes byDrip and be diluted to final volume and be total up to 20ml. Then pass throughDetermine total cell quantity, and cell is outstandingSupernatant liquid is transferred to the ultralow culture dish that adheres to and continues 1 hour. Then by cell, the culture medium from 50ml circular cone separates, and removes supernatantLiquid and cell is resuspended in the 4th stage culture medium. Then cell is passed through to TheraCyte^TMDevice is implanted subcutaneously movingIn object or be implanted to kidney peplos below, or cell is incubated overnight in adhering to culture dish ultralow, is then implanted to animalIn body.

Blood sugar and the C-peptide level of every surrounding monitoring animal after graft is implanted. Use TheraCyte^TMNon-in deviceFrozen diabetes is processed or by cell being directly placed on to the animal maturation processed kidney peplos below with to 16When week in, expresses and exceedes 1ng/mLC-peptide, and (Figure 39 a and Figure 39 are d) to reach 2ng/mLC-peptide when implanting afterwards 20 weeks. In addition,In the time processing to eliminate β cell function with STZ, transplanted animal maintains euglycemia until graft is removed, and this showsGraft can watch for animals and exempt to suffer from the diabetes of single high dose STZ induction (Figure 39 b).

Also in the animal with freeze-stored cell processing, observe this pattern. After thawing, cultivate 1 hour freeze by havingThe animal of depositing the kidney peplos graft processing of diabetes (1207B) has respectively average in the time of 16 weeks and 20 weeksThe C-peptide of 0.56ng/mL and 1.09ng/mL, and after thawing the cell of overnight incubation (1207C) in the time of 16 weeks and 20 weeks respectively(Figure 39 d) to have the C-peptide of average 0.81ng/mL and 1.35ng/mL. Use TheraCyte^TMFrozen pancreas precursor in device is thinIn born of the same parents process animal to 16 when week, has the 1ng/mLC-of exceeding peptide, and with non-frozen contrast similar, can be after STZ processes(0.98ng/mL, Figure 39 c) for the C-peptide of one week expression treatment level. These results show, in the time testing in animal model, freezeDeposit diabetes and can play equivalent effect with non-frozen contrast.

Table 10

Points for attention:

In the time that Glutamax is not used in replenishers, the basal medium in above table 10 can be optional in the 1-5 stageGround comprises 5mM glucose.

In table 10, optionally add Cypi ([100nM]) in the 4th stage shown in above.

Table 11

Table 12

The calculating of the shear stress being experienced in stirred-tank bioreactor by cell aggregation

Determine 2.7 liters that are mixed with the stirring speed of 70rpm in 3lDASGIP bioreactor by cell aggregationThe shear stress experiencing in DASGIP stirring suspension bioreactor. In order to calculate shear stress values, make following statedSuppose.

Suppose：

1. the maximum shear stress being applied on cell aggregation is not the result of turbulent vortices.

2. the maximum shear stress being applied on cell aggregation is not aggregation-aggregation or the collision of aggregation-impellerResult.

3. the shear stress that baffle plate (, dip-tube and probe) applies is not processed in these calculate.

For calculating object herein, use following term and the physical parameter of listing.

Term：

Parameter：

Culture medium parameter
			Density (ρ)	1000	kg/m3
Viscosity (μ)	8.50E-04<	Pa s
			Kinematic viscosity ()	8.50E-07<	m2/s

The culture medium parameter of listing and bioreactor parameter are applied to following formula.

Formula：

Reynolds number:

$\mathrm{Re}=\frac{{\mathrm{\&rho;ND}}_i^2}{\mathrm{\&mu;}}$

The maximum shear stress (Cherry and Kwon, nineteen ninety) on aggregation

The power (ε) that per unit mass consumes

$\mathrm{\&epsiv;}=\frac{P}{V_L\mathrm{\&rho;}}$

Consumed power (P)

$P=P_N{N}^3{D}_i^5\mathrm{\&rho;}$

For unbaffled agitator tank, power number calculates the Empirical Equation based on deriving from Nagata (1975).

$P_N=\frac{K_1}{\mathrm{Re}}+K_2{\&lsqb;\frac{10+1.2{\mathrm{Re}}^{0.66}}{10+3.2{\mathrm{Re}}^{0.66}}\&rsqb;}^{K_4}$

Wherein

$\begin{array}{c}{K}_1=14+\frac{W}{D_t}\&lsqb;670{(\frac{D_i}{D_t}-0.6)}^2+185\&rsqb;\&rsqb;\\ K_2={10}^{K_3}\end{array}$

$K_3=1.3-4{\&lsqb;\frac{W}{D_t}-0.5\&rsqb;}^2-1.14\frac{D_i}{D_t}$

$K_4=1.1+4\left(\frac{W}{D_t}\right)-2.5{\&lsqb;\frac{D_i}{D_t}-0.5\&rsqb;}^2-7{\&lsqb;\frac{W}{D_t}\&rsqb;}^4$

Calculate in 2.7LDASGIP bioreactor and be applied on cell aggregation extremely with the stirring speed of 70rpmFew 2.5dyn/cm²Maximum shear. The shear stress that comprises the outermost cell experience highest level of trooping. Shear stressValue highly depends on the hypothesis of statement.

Example 9

The differentiation of human embryo stem cell from clone WA01 to definitive entoderm: MCX/GDF8 is in the effect suspending cultivating

To contain 3.64g/ml sodium acid carbonate from being clustered in of pluripotent human embryonic stem cell line H1 (NIH code: WA01)With in the MCDB-131 culture medium of 5.5mM glucose (catalog number (Cat.No.) A13051DJ, Invitrogen, CA) in conical flask/shaking flask, turnBottle or uncoated ultralow combination or not with in 6 orifice plates of tissue culture processing with 0.25 × 10⁶Cell/ml to 2 × 10⁶IndividualThe inoculation of cell density within the scope of cell/ml, this culture media supplemented have 2% not the BSA of fatty acids (catalog number (Cat.No.) 68700,Proliant,IA)、1XGlutamax^TM(catalog number (Cat.No.) 35050-079, Invitrogen, CA), other 2.5mM glucose (catalogueNumber G8769, Sigma) and 1:50, the ITS-X (catalog number (Cat.No.) 51500056, Invitrogen, CA) of 000 original liquid concentration. ForThe object of this application, supplementary MCDB-131 culture medium will be called " the 1st stage basal medium " by this way. This culture mediumIn any one processing in the following stated material for the first day that is clustered in differentiation: 3 μ MMCX (GSK3B inhibitor, 14-third-2-alkene-1-base-3,5,7,14,17,23,27-, seven azepine Fourth Rings [19.3.1.1～2,6～.1～8,12～] heptacosane-1(25), 2 (27), 3,5,8 (26), 9,11,21,23-nonane-16-ketone, U.S. Patent application 12/494,789, with way of referenceBe incorporated in full herein) and 100ng/mlGDF-8 (catalog number (Cat.No.) 120-00, Peprotech), or 3 μ MMCX only, or 20ng/mlWNT-3A (catalog number (Cat.No.) 1324-WN-002, R&DSystems, MN) adds 100ng/ml activin A (catalog number (Cat.No.) 338-AC, R&DSystems, MN), or 20ng/mlWNT-3A only. At second day, by cell transfer to be supplemented with 100ng/mlGDF8 orFresh the 1st stage basal medium of 100ng/ml activin A. Collect sample for (and then adding basis training zeroBefore foster base adds replenishers) as many as differentiation start multiple time points place within the scope of latter 72 hours carry out flow cytometry,PCR and western blot analysis.

After differentiation 3 days, under each condition, express cell surface marker by using flow cytometry to measureThe percentage of the cell of CXCR4, CD99 and CD9 is recently determined the efficiency that produces definitive entoderm. These data are (in Figure 40 a-dShown in FACS figure and as table 13, sum up) instruction, in suspending and cultivating, in the situation that not there is not TGF-'beta ' family memberThe first day of differentiation adds 3 μ MMCX to be equivalent to adding 100ng/mlGDF-8 or 20ng/ml at first day 3 μ MMCXWNT-3A adds that the level that activin A obtains while processing cell produces definitive entoderm.

Table 13

Example 10

The differentiation of human embryo stem cell from clone WA01 to definitive entoderm: MCX compound concentration is suspension is cultivated Dose response

Will from pluripotent human embryonic stem cell line H1 (NIH code: WA01) be clustered in as described in example 9 above the 1stIn stage basal medium in conical flask/shaking flask or rolling bottle with 0.25 × 10⁶Cell/ml to 2 × 10⁶Within the scope of individual cell/mlCell density inoculation. At the 1st stage basal medium that contains 1.5,2,3 or 4 μ MMCX for first day of differentiation, andWithin the 2nd day, troop with the 1st stage fresh basal medium processing that contains 100ng/mlGDF-8. Do not carry out culture medium at the 3rd dayChange. Collect sample for carry out flow cytometry and pcr analysis in the time that differentiation finishes on the 3rd day.

Then pass through hundred of the cell that uses flow cytometry to measure expression cell surface marker CXCR4, CD99 and CD9Divide and recently determine the efficiency that produces definitive entoderm. These data are (as shown in the FACS figure in Figure 41 A-D and as total in table 14Knot) instruction, in suspension culture, add to be less than the concentration of 2 μ M the definitive entoderm positive cell that MCX generation reduces gradually(as the CD9 positive cell of the CXCR4 positive cell of lower percentage and higher percent confirms). In addition, higher than 4 μ M'sUnder concentration, MCX shows the illeffects to cell, and this causes the cytoactive declining. But, by increasing BSA concentration,The effect of MCX can be regulated and controled into make to use >=4 micromolar concentration. On the contrary, when using together with lower BSA concentrationTime, can produce definitive entoderm by≤1.5 micromolar concentration.

Table 14

Example 11

The differentiation of human embryo stem cell from clone WA01 to definitive entoderm: culture medium replacement frequency is suspension is cultivated Effect

Will be from pluripotent human embryonic stem cell line H1 (NIH code: WA01) be clustered in as described in Example 9 the 1stStage culture medium in conical flask/shake bottle between 0.25x10⁶Cell/ml to 2x10⁶Under the interior density of scope of individual cell/mlInoculation. Troop and process and use to contain and break up for the 2nd day with the 1st stage basal medium of 3 μ MMCX that contains differentiation in the 1st dayThe 1st stage fresh culture processing of 100ng/mlGDF-8. Accepted to change culture medium at the 3rd day according to culture; For listSolely container, did not carry out culture medium and changes at the 3rd day. Collect articles for use for flow cytometry and PCR in the time that differentiation finishes on the 3rd dayAnalyze.

Then under each condition by use flow cytometry to measure to express cell surface marker CXCR4, CD99 andThe percentage of the cell of CD9 is recently determined the efficiency that produces definitive entoderm. The results are shown in the FACS figure in Figure 42 A and Figure 42 BAnd be summarized in table 15.

Table 15

Example 12

The differentiation of human embryo stem cell from clone WA01 to definitive entoderm: use GlutaMAX suspension culture ^TM

Will from pluripotent human embryonic stem cell line H1 (NIH code: WA01) be clustered in conical flask/shaking flask or rolling bottle with0.25×10⁶To 2 × 10⁶Cell density inoculation within the scope of cell/ml.

Implement this embodiment to be suspended in and to add or deduct Glutamax by trooping^TMThe 1st stage basal mediumIn (describing to some extent 9 in embodiment 9), determine whether produce definitive entoderm needs Glutamax^TMFill-in, this culture medium existsThe first day of differentiation was supplemented with 3 μ MMCX, and was supplemented with the 1st fresh base of stage that contains 100ng/mlGDF-8 at the 2nd dayBasal culture medium. Do not carry out culture medium replacing at the 3rd day. Collect sample thin for carry out streaming in the time that differentiation finishes on the 3rd dayBorn of the same parents' art and pcr analysis.

Under each condition by using flow cytometry to measure to express cell surface marker CXCR4, CD99 and CD9The percentage of cell is recently determined the efficiency that produces definitive entoderm. Data and the results are shown in the FACS figure in Figure 43 A and Figure 43 BAnd be summarized in table 16.

Table 16

Example 13

The differentiation of human embryo stem cell from clone WA01 to definitive entoderm: sodium acid carbonate concentration suspend cultivate Effect

Will be from the 1st rank described in example 9 that are clustered in of pluripotent human embryonic stem cell line H1 (NIH code: WA01)In segment base basal culture medium (containing 3.64g/l sodium acid carbonate), or in the 1st stage of the modification that contains 2.43g/l sodium acid carbonateIn basal medium in conical flask/shaking flask or rolling bottle with 0.25 × 10⁶To 2 × 10⁶Cell density within the scope of cell/ml connectsKind. Troop with containing MCX described in example 12 and the 1st stage basal medium processing of GDF-8. Collect sample forIn the time that finishing on the 3rd day, differentiation carries out flow cytometry. Also catch phase difference image in the every day of differentiation.

Then pass through hundred of the cell that uses flow cytometry to measure expression cell surface marker CXCR4, CD99 and CD9Divide and recently determine the efficiency that produces definitive entoderm. Data are shown in the FACS figure in Figure 44 A and Figure 44 B and are summarized in table 17In. In suspension culture, and when cell differentiation phase (cellular expression of average 97.35% in the culture medium that contains 3.64g/LCXCR4) compare, the sodium acid carbonate level (average 87.4% cellular expression CXCR4) that is low to moderate 2.43g/L seems more poor efficiency real estateRaw definitive entoderm. In addition, can be observed, in the time that the 1st stage finished, the difference of bicarbonate level and the poor morphology out-phase of troopingClose, as passed through phase contrast microscope viewed (Figure 44 C and Figure 44 D). In addition, it should be noted that under high carbon acid hydrogen salt levelThe cell of differentiation forms than looser the trooping of cell of breaking up under 2.43g/L bicarbonate.

Table 17

Example 14

In telescopic bioreactor processes, producing pancreas ancestral by people's induced multi-potent stem cells troops

Cell therapy is by every dose of a large amount of (> 10 of needs⁸) cell. This example illustrates can induction multipotential stem cellThe process of (iPS cell), the quality that the current cell therapy manufacturing practice of mass ratio of described induced multi-potent stem cells may needLarge 3 to 5 orders of magnitude.

In this embodiment, use iPS clone-UTC (to derive from before in U.S. Patent application 13/330,931The umbilical cord tissue cell (U.S.'s published application 2013/0157365) of describing, because this patent relates to derivative IPS clone, its public affairsOpening content is incorporated to herein in full). This cell uses plasmid transfection to obtain on mice embryonic feeder cells in nothing " impression " mode,And go down to posterity frozen at the 15th time.

By these freeze-stored cells, by source material bottle directly being thawed to from LifeTechnologiesThe Essential8 of Corporation (GrandIsland, NY)^TMCulture medium (E8^TM) in people recombinate laminin (comeFrom Biolamina, Stockholm, the hrLaminin of Sweden, catalog number (Cat.No.) LN-521) above produce inner inoculation material and produceA series of cell banks. This thaws and the material that increases is called " preparation master cell bank " (preparation MCB), and it is as the inoculation material in following storehouseMaterial. Use preparation MCB, then produce 3 continuous cell banks, prepare MCB, MCB and working cardial cell storehouse (WCB). Then willA WCB bottle thaws, and uses EDTA to go down to posterity on hrLaminin amplification at E8^TMIn carry out three times and go down to posterity. First willCell is inoculated into T225 flask (Corning by thawing; Corning, NY) in, then go down to posterity in multiple T225 flasks. SoAfter multiple T225 flasks are gone down to posterity and in conjunction with to inoculate single 1 layer of CellStack^TM(CS1). Once the cell in CS1 convergesClose, by PBS-for cell/-washing once, then useHalf intensity solution-treated, with PBS-/-dilution, andIncubation 4 minutes to 5 minutes. Then removeAnd after application enzyme solutions 3 minutes, flicking CS1 was to promote carefullyBorn of the same parents promote. By the E8 that is supplemented with 0.5%BSA and comprises 10 micromole Rho inhibitors of kinases Y-27632^TMAdd in CS1 withRinse and deactivation remnantsThen collect flushing liquor and add and collect the second flushing liquor volume, and with firstFlushing liquor merges.

By cell in the culture medium that is supplemented with 0.5%BSA and contain 10 micromole Rho inhibitors of kinases Y-27632With 1 × 10⁶The concentration of cell/mL, transfers to 1 liter of disposable rolling bottle (Corning with the number that rises of 225mL; Corning, NY) in.Allow cell CO in humidity 5% in static suspension liquid₂In incubator, troop and continue 60 minutes, then under 55-65rpm, stirMoving 5 minutes, and it is extra to add the 225mL that is supplemented with 0.5%BSA and contains 10 micromole Rho inhibitors of kinases Y-27632Culture medium. Allow cell in addition sedimentation 30 minutes in static culture thing, then will be supplemented with 0.5%BSA and contain 10 micro-The extra culture medium of 150mL of mole Rho inhibitors of kinases Y-27632 adds in rolling bottle. After this, by cell in humidity 5%In CO2 incubator with 50-70rpm continuous stirring. After twenty four hours, rolling bottle is removed from incubator, and allow to troopSedimentation 5-10 minute. Then by culture medium sucking-off, until 200mL is retained in container, then by fresh cultured extra 400mLBase adds in rolling bottle. In the time within the 2nd day, finishing (shifting latter 48 hours), repeat this process.

Then initiallyProcess latter 72 hours, repetitive cell is trooped to dissociate and is inoculated the mistake of (going down to posterity) with rolling bottleJourney with keep cell in suspension for repeatedly go down to posterity (test specification: 1 generation in generation to 10).

Use this process, UTCiPS cell is transformed into suspension culture as cell mass from suprabasil adhere-wall culture thingCollection then increases in suspension. Then the cell cryopreservation these suspensions being gone down to posterity and cultivate also stores for using after a while. ForFor the preparation of frozen suspension amplifying cells, cell cluster is used as mentioned aboveDissociate, different is cellNot through 40 microns

Cell filter screen. Then to counting from the cell of each 1 liter of disposable flask, combination as required, andUnder 80-200rcf centrifugal 5 minutes. Then as far as possible fully remove supernatant and not interference cell granulation. Then with dropwiseMode is added cold (< 4 DEG C)To realize the ultimate density of 15,000 ten thousand cell/mL, and transferring to1.8mLCorning cryovial (Corning; Corning, NY) or 15mLMiltenyi freezer bag (MiltenyiBiotecInc.Auburn, CA) process in cell solution is remained in ice bath.

Then as described belowly in controlled rate freezer unit, suspension amplifying cells is chilled in bottle with high density. By chamberBe chilled in advance 4 DEG C and keep this temperature until sample flasket temperature reaches 6 DEG C. Then room temperature is slowly reduced with 2 DEG C/minUntil sample reaches-7 DEG C. Once sample flasket reaches-7 DEG C, chamber is cooling until chamber reaches-45 DEG C with 20 DEG C/min. ThenAllow room temperature with 10 DEG C/min of of short duration risings until room temperature reaches-25 DEG C, then that chamber is further cold with 0.8 DEG C/minBut until sample flasket reaches-45 DEG C. Then with 35 DEG C/min of cooling chamber temperature until chamber reaches-160 DEG C. Then by room temperatureDegree remain on-160 DEG C at least 10 minutes, after this, bottle is transferred to gas phase liquid nitrogen storage device.

In order to inoculate stirred-tank bioreactor, high density freeze-stored cell is removed from liquid nitrogen storage device, thaw alsoAnd for inoculating 0.2 liter of glass biological reaction device (DASGIP of sealing; Julich, Germany). By cryovial from gas phase liquidNitrogen storage device removes, and is directly placed in 37 DEG C of water-baths 105 seconds. Then by the 2mL glass pipet bottle that will thawContent is transferred to 50mL conical tube. Then will contain 0.5%BSA, be supplemented with 10 micromole Rho inhibitors of kinases Y-276329mLE8^TMAdd pipe in mode dropwise. Then make cell under 80fcf to 200rcf centrifugal 5 minutes. Afterwards, by supernatantLiquid is sucking-off from pipe, and add contain 0.5%BSA and be supplemented with the 10ml of 10 micromole Rho inhibitors of kinases Y-27632 newFresh E8. By celliferous bag this volume pipette media transfer bottle (Sanisure, Moorpark, CA) in,And by the aseptic C-flex pipe arrangement being welded by peristaltic pump, bottle content is directly pumped in bioreactor. Many in preparationIn the inoculation of energy stem cell, bioreactor is supplemented with 0.5%BSA with 0.15L and comprises 10 micromole Rho inhibitors of kinases Y-27632 E8^TMPreparation, is preheated to 37 DEG C, stirs with 70rpm, passes through CO₂Regulate and control to 6.8pH to 7.1pH, wherein dissolved oxygen is establishedFixed point is 30% (CO₂, air, O₂And N₂Regulation and control). And then after inoculation, bioreactor is sampled for carefullyBorn of the same parents' counting, adjusts culture volume as required to obtain 0.225 × 10⁶The final cell concentration of individual cell/mL.

Be inoculated into the cell shape cell cluster in continuous agitator tank in stirred-tank bioreactor. After inoculation, willCell cluster remains on the E8 that is supplemented with 0.5%BSA in reactor^TMIn culture medium three days. Change culture medium every day, inoculate itLatter 24 hours, remove culture medium and 0.15 liter of fresh culture of interpolation that 90% use is crossed. Latter 48 hours of inoculation, removesThe culture medium that 90% use is crossed and 0.15 liter of fresh culture of interpolation. Within latter 72 hours, locate in inoculation, by removing 90% useCulture medium and interpolation differential medium cause pluripotent cell differentiation (table 18).

Once you begin segmentation atomization, cell temperature (37 DEG C), pH (for being divided into 7.4) and dissolved oxygen (forThe 1st stage was 10%DO set point, and all be 30%DO set point At All Other Times, CO₂、O₂、N₂And air regulation) through regulation and controlClosed sterile soft suspended biological reactor in keep 12 days or more days. In whole atomization, change at every subcultureTime, impeller stops 5 minutes to 20 minutes, then removes culture medium to allow the sedimentation of trooping by dip-tube. Use by passingTerumo^TMPipe welding machine is connected toThe peristaltic pump of the dip-tube of pipe arrangement by the culture medium in bioreactor from sealingBottle or bag remove or add to wherein to keep closed system. Once enough culture mediums are added to container with complete submergence leafWheel, just switches on again to impeller and heater.

For monitoring bio reactor process, aspirate the media samples that comprises cell cluster every day to determine cell quantityAnd activityAs shown in figure 45. During this process, observe the general amplification of cell, becauseThe 4th the 3rd day stage, 0.225 × 10⁶The inoculum amplification of living cells/mL generates 0.65 × 10⁶Living cells/mL (Figure 45).

Except counting every day, pass through NOVAFLEX(NovaBiomedical,Corporation，Waltham, MA) bioreactor culture basic pattern product are analyzed. Can be observed, according to the reactor set point in the 0th stage(pH6.8), in whole the 0th stage, the pH of the 0th stage culture medium be acid (pH6.8) (Figure 46). Observe the 0thIn stage culture medium, under relatively low lactate level and high glucose level, the acid set point in the 0th stage seems to reduceThe metabolic activity of cell. Once cell starts differentiation until the 3rd stage finished, the nearly all grape in cell consumption culture mediumSugar (Figure 47) and produce high-caliber lactic acid (Figure 48). The 1st and the process in the 2nd stage in observe the extra of cell densityIncrease (Figure 45).

Whether mate as passed through the measured mrna expression of qRT-PCR in order to determine that the phase specificity of pH and metabolism changesThe phase change of pattern, carries out the following stated. Use and specify versatility, definitive entoderm (DE), intestinal tube (GT) or the 4th stage(S4) four applying biological system low density array (Life^TM, Carlsbad, CA). Result be rendered as with as whole serviceDoubly poor that UTCiPS cell sample compares that do not break up with the contrast of the normalized expression on array.

Use these arrays, determine gene expression in each stage of differentiation. Then observe, thaw to bioreactorIn inoculation material cell in the 0th stage, the 1st day, the 2nd day and the 3rd day (after bioreactor inoculation 24 hours, 48 hoursWith 72 hours: Figure 49 and Figure 50) demonstrate not differentiation gene expression pattern. These results and the close phase of flow cytometry resultClose, it demonstrates CD9, SSEA4, TRA-1-60 and the TRA-1-81 of high expression level, but does not have CXCR4/CD184 (figure51). These flow cytometries and qRT-PCR data demonstrate versatility gene (CD9, NANOG, POU5F1, SOX2, TDGF andZFP42) strong and stable expression pattern, but the gene that characteristic is not expressed in atomization (CD99, CDH2,CDX2、CER1、CXCR4、EOMES、FGF17、FGF4、FOXA2、GATA2、GATA4、GATA6、GSC,HAND2、HNF4α、KIT、MNX1, MIXL1, PRDM1, PTHR1R, SOX17, SOX7, T, TMPRSS2 and VWF) expression, this and stable multipotency state oneCause.

In the time completing for the 0th stage (reactor is inoculated latter 72 hours), signaling is trained to the differentiation that comprises MCX and GDF8Support in base (table 18). This culture medium is changed rear twenty four hours, notices remarkable change (Figure 49 and the Tu of gene expression pattern50, contrast the multiple compared and express with not breaking up), increase such as the expression of FOXA2, HAND2, PRDM1, PTH1R and SOX1710 times, the expression of CER1, FGF4, GATA4, GATA6, GSC and MNX1 increases > 100 times, and EOMES, FGF17, MIXL1 and TExpression increase > 1000 times. The expression indicator cells of these increases changes by mesendoderm destiny. Shall also be noted thatCDX2 level increased the 1st day the 1st stage (express and increase by 2700 times compared with the control) compared with neoblast, soAnd this is the instantaneous increase of expressing, and 1 stage of CDX2 level to the, within the 3rd day, decline 97%, before breaking up with induction, seeThose suitable levels of examining (Figure 49 and Figure 50, with do not break up contrast the multiple of comparing and express).

Within 72 hours after being exposed to the 1st stage differential medium, locate, cellular expression is consistent with the specification of definitive entodermDistribution, this is because CXCR4 level locates to reach peak value exceeding approximately 400 times of historical controls, FOXA2 is to exceed 136 times of contrastsExpress, and SOX17 expresses to exceed 470,000 times of historical controls. Consistent with definitive entoderm, be also noted that the 1st rankWhen section finishes, the gene expression of (the 3rd day) CER1, EOMES, FGF4, GSC, MIXL1 and T is from observing the 1st day the 1st stageElevated levels decline (Figure 49 and Figure 50, with do not break up contrast the multiple expression of comparing).

These that utilize gene expression that qRT-PCR observes change and result phases of observing by flow cytometryClose. Homogeneity when the negative pluripotent cell group of CD9 expression/CXCR4 (Figure 51) while observing from differentiation initiation finished to the 1st stageCXCR4 expresses cell mass (98.6% cell is the CXCR4 positive) approaching transformation completely (Figure 52).

Complete definitive entoderm form (the 1st stage) afterwards, by culture medium be replaced by contain FGF7 (for induce former beforeIntestines form morphogen) culture medium. Consistent with the formation of former anterior intestine, HNF4 α and GATA6 expression are in the 2nd stageThe 1st day and the 3rd day increase, and the gene of the 3rd day the 1st stage with high level expression (CXCR4, EOMES, FGF17, FGF4,MNX1, PRDM1, SOX17 and VWF) (Figure 50 and Figure 53 contrast with not breaking up in the time that the 2nd stage finished, to demonstrate the expression of minimizingThe multiple of comparing is expressed). The expression of anterior intestine gene (AFP, HHEX, PDX1 and PROX1) increases that (Figure 53 and does not break up taking a pictureThe multiple of ratio is expressed).

After cell has been cultivated 72 hours in the 2nd stage culture medium, culture is transformed into the 3rd stage culture medium (table18). Once in this culture medium, cell is just expressed the interior embryo of measuring with the qRT-PCR determination method as by for gene expressionThe consistent mark of layer pancreas pedigree. The gene expression of PDX1 exceedes 12,000 times of contrasts to the in the time that the 3rd day the 2nd stage finishedThe 3rd day 3 stages exceeded 739,000 times of contrasts while end increased by 60 times. These data indicator cellses are designated as pancreas destiny(Figure 54). Support this observed result, compare with not breaking up to contrast, the gene of conventionally expressing in pancreas (ARX, GAST,GCG, INS, ISL1, NEUROD1, NGN3, NKX2.2, NKX6.1, PAX4, PAX6, PTF1A and SST) host's the table of increaseReach level, as shown in Figure 54 and Figure 55. What is interesting is, also observe, for other mark of entoderm AFP, ALB and CDX2Thing does not have OCT4/POU5F1 to express (37 sample Ct of qRT-PCR) and high level expression. This instruction, in bioreactorCell mass first from multipotential stem cell group be divided into relatively overall enteron aisle pipe and be then further divided into pancreas destiny (Figure 54 andFigure 55).

In the time that quadravalence section atomization finishes, cell keep high-caliber PDX1 (be defined as 95.6% positive by FACS,Be defined as exceeding contrast approximately 1,000,000 times of induction by qRT-PCR) and FOXA2 (being defined as 99.5% positive by FACS)Express. Cell demonstrates and pancreatic progenitor cell (being defined as the 39.2%NKX6.1 positive by FACS) and pancreatic endocrine cell(being all defined as the 9.4%PAX6 positive, the 12.4% chromograin positive, the 15.2%NKX2.2 positive by FACS) group is consistentExpression pattern. It is thin that this phase specificity marker expression pattern instruction is effectively progressively divided into pancreas precursor from multipotency colonyBorn of the same parents. Confirm these results of observing with flow cytometry by qRT-PCR. Shall also be noted that and conventionally in pancreas, expressGene (ARX, GAST, GCG, IAPP, INS, ISL1, MAFB, NEUROD1, NGN3, NKX2.2, NKX6.1, PAX4, PAX6,PTF1A, and SST) host all there is the expression of increase the 3rd day the 4th stage. (Figure 55). As a reference, Mei GejieWhen section finishes, the representative micrograph (4 times) of cell cluster is shown in Figure 56.

Table 18

Table 18a

Table 18b

Material：

People embryo does (hES) clone H1, (WA01 cell, WiCell, MadisonWI)

PBS (catalog number (Cat.No.) 14190, Invitrogen)

Y-27632 (Axxora catalog number (Cat.No.) ALX-270-333, SanDiego, CA)

EDTA, (Lonza, catalog number (Cat.No.) 17-7-11E)

·-(ChemoMetecA/S, catalog number (Cat.No.) YC-T100, Allerod, Denmark)

Not with 6 hole culture dishes of tissue culture processing (BectonDickinson, catalog number (Cat.No.) Falcon351146,FranklinLakes,NJ)

·(Sigma, catalog number (Cat.No.) 12604-013, St.Louis, MO)

PH and dissolved oxygen (DO) bioreactor probe (PH electrode 225mm, model F-635, andDO12mm sensor, model D-145, from Broadley-James, IrvineCA)

Large-scale packaging system (the Theracyte of immune protective^TM,IrvineCA)

People C-peptide ELISA (MERCODIA catalog number (Cat.No.) 10-1141-01)

·Glutamax^TM, MCDB131 and ITS-XInvitrogen

·FAF-BSA(Proliant)

Retinoic acid, glucose 45% (2.5M), SANT (Shh inhibitor) is (Sigma)

·GDF8(Peprotech)

·MCX

·FGF7(R&DSystems)

LDN-193189 (bmp receptor antagonist) (Stemgent)

TPPB (PKC activator) (ChemPartner)

Example 15

The differentiation of human embryo stem cell from clone WA01 to definitive entoderm: MCX/GDF8 is as cell cycle regulating agent In the effect suspending in cultivating

To contain 3.64g/ml sodium acid carbonate from being clustered in of pluripotent human embryonic stem cell line H1 (NIH code: WA01)With in the MCDB-131 culture medium of 5.5mM glucose (catalog number (Cat.No.) A13051DJ, Invitrogen, CA) in taper shaking flask with0.5×10⁶The inoculation of individual cell/ml, this culture media supplemented have 2% not the BSA of fatty acids (catalog number (Cat.No.) 68700, Proliant,IA)、1XGlutamax^TM(catalog number (Cat.No.) 35050-079, Invitrogen, CA), other 2.5mM glucose (catalog number (Cat.No.) G8769,Sigma) and 1:50, the ITS-X (catalog number (Cat.No.) 51500056, Invitrogen, CA) of 000 original liquid concentration. For this exampleObject, supplementary MCDB-131 culture medium will be called the 1st stage basal medium or " pure " culture medium by this way. GSK3BInhibitor, 14-third-2-alkene-1-base-3,5,7,14,17,23,27-, seven azepine Fourth Ring [19.3.1.1～2,6～.1～8,12～] heptacosane-1 (25), 2 (27), 3,5,8 (26), 9,11,21,23-nonane-16-ketone will be called " MCX ", United States Patent (USP) ShenPlease 12/494,789, be incorporated in full herein with way of reference.

One processing below the first day of differentiation is used in six kinds of conditions is trooped: (1) is pure, and (2) 3 μ MMCX add100ng/mlGDF-8 (catalog number (Cat.No.) 120-00, Peprotech), (3) are 3 μ MMCX only, (4) only 100ng/mlGDF-8, (5)20ng/mlWNT-3A (catalog number (Cat.No.) 1324-WN-002, R&DSystems, MN) adds 100ng/ml activin A (catalog number (Cat.No.)338-AC, R&DSystems, MN) or (6) only 20ng/mlWNT-3A.

Causing differentiation latter 24 and 48 hours, under every kind of condition in described condition, change culture medium. In these times,Cell in condition 1,2,3 and 4 is replaced by fresh the 1st stage basal medium that is supplemented with 100ng/mlGDF8, and incite somebody to actionCell in condition 5 and 6 is replaced by fresh the 1st stage basal medium that is supplemented with 100ng/ml activin A.

Initiation differentiation one hour before, and cause differentiation 5 hours, 23 hours, 29 hours, 47 hours or 71 hours afterwards(being called " time 0 "), suspension sample is transferred to not in the six hole culture dishes with tissue culture processing, and with EdU (EdU kit, LifeTechnologiesCorporation, Carlsbad, CA) incubation one hour. ThenAfter initiation differentiation, 0,6,24,30,48 or 72 hours time located to assess by flow cytometry the cell of EdU incubation, measured thinThe interim cell percentage (Figure 81 to Figure 87) of G0/G1 phase, S phase or G2/M in born of the same parents' cycle.

After this scheme, observe the interim cell percentage of G0/G1 phase, S phase or the G2/M of cell cycle significantlyDifference (Figure 82 to Figure 87), and noticing, compared with four kinds for the treatment of conditions of cell and other that MCX and MCX+GDF8 process, knotThe EdU minimizing of closing approaches 40% (Figure 81). This minimizing of EDU combination increases by 38% by the sample of processing for MCX+GDF8G0/G1 cell and the cell of processing for MCX only increase 54%G0/G1 cell and mate. Causing latter 6 hours of differentiation, EDUIn conjunction with these changes and being increased in GDF8, WNT3A, WNT-3A+ activin A or pure cultivation to the transformation of G0/G1In the cell of base processing, do not observe. On the contrary, use the thin of GDF8, WNT-3A, WNT-3A+ activin A or pure medium treatmentThe minimum that born of the same parents show the percentage of the cell with EDU combination reduces (mean value, 48.1%, SD ± 1.2) and causes and divideChange average 13% minimizing (standard deviation, ± 5%) of cell quantity in latter six hours G0/G1, as shown in Figure 81 and Figure 82.

Compared with other treatment conditions, for observing in process after a while with the cell that MCX or MCX+GDF8 processThe same difference of the gap between G0/G1 value. After the time 0 30 hours, the cell that MCX or MCX+GDF8 process with use WNT-The cell of 3A+ activin A, GDF8, WNT-3A or pure medium treatment compare in G0/G1, have few 43% to 45% thinBorn of the same parents. Gap between G0/G1 cell percentage remains on and causes differentiation and locate for latter 48 hours, and this is because with MCX or MCX+GDF8The 71.9-75.5% cell of processing is in the G0/G1 of cell cycle, and 48.5%GDF8,55.8%WNT3A, 57.7%WNT-The cell of 3A+ activin A or 49% pure medium treatment is in G0/G1. Except observing in EDU combination and G0/G1 distributionThe difference arriving, when compared with the cell of WNT3A+ activin A, GDF8, WNT-3A or pure medium treatment, MCX or MCX+The cell that GDF8 processes is in the interim cell (figure with many 15-33% of S of cell cycle for 30 and 48 hours after the time 084 and Figure 85).

These data (be shown in CD99, CD9 in Figure 57 to Figure 80 and Figure 88 a to Figure 88 f, CDH1, CDH2, CDX2, CER1,CXCR4、FGF17、FGF4、FOXA2、GATA4、GATA6、GSC、KIT、MIXL1、MNX1、NANOG、OTX2、POU5F1、The gene expression of SOX17, SOX7 and T) instruction, in suspension culture, the interpolation of differentiation first day has or does not have TGF-β familyThe MCX of the member GDF8 of family produces can be with in the time that first day 20ng/mlWNT-3A adds that 100ng/ml activin A is processed cellThe definitive entoderm that the definitive entoderm obtaining is compared, the gene expression while end as formed by definitive entoderm is measured. But, with form the process of definitive entoderm in difference in the viewed cell cycle consistent, observe intermediate base because ofDifferential expression. In the sample of processing with MCX or MCX+GDF8, gene T (short-tail class (brachyury)), GATA4 and CDX2 withSubstantially higher than the cell with WNT-3A+ activin A or other three test condition processing in first 24 hours of differentiationLevel induce that (Figure 88 b, Figure 88 c and Figure 88 are d). On the contrary, when compared with initiator cell group or other four test conditions,The expression of versatility gene (NANOG and POU5F1/OCT4) is aobvious through 24 hours in the sample of processing with MCX or MCX+GDF8(Figure 88 e) in work minimizing. When with cause differentiation within latter 24 hours, locate test other four conditions compared with time, gene (such as FGF4,FOXA2 and SOX17) magnitude of induced expression is much lower in MCX or MCX+GDF8 sample, but by 48 hours, all samplesAll with suitable horizontal expression FGF4, FOXA2 and SOX17. (Figure 88 c and Figure 88 are e).

Example 16

Use extendible suspension atomization to produce ectoderm and mesoderm tissue

This example illustrates and can increase and break up multipotential stem cell (PSC) to realize extendible manufacture process, thereby producesThe process of raw ectoderm tissue or mesoderm tissue.

Two kinds of clones are suspended to amplification so that the inoculation material of these researchs: H1 (WA01) hES clone-WB0106 to be providedSubclone, and by umbilical cord tissue cell (UTC) produce induced multi-potent stem cells (iPSC). Institute in embodiment as in the previousState, by suspend amplification cell be chilled in controlled rate freezer unit with high density, then thaw with 0.225 × 106 cell/3 liters of glass biological reaction device (DASGIP of inoculation sealing under the final cell concentration of mL; Julich, Germany) or disposable 3Liter single use bioreactor (EMDMilliporeCorporation, Billerica, MA). Be inoculated intoCell in stirred-tank bioreactor forms cell cluster in continuous agitator tank, and remains on the versatility in reactorCulture medium (is supplemented with the E8 of 0.5%BSA^TM) in three days altogether. Within latter 72 hours, locate in inoculation, by cell cluster is transferred to and mouldedExpect that disposable conical flask (PETG125mL flask, catalog number (Cat.No.) 4112, ThermoScientificRochesterNY) comesIn its corresponding differential medium (table 19), cause pluripotent cell differentiation, thus formation mesoderm/heart tissue (1) or ectoderm/Nerve fiber (2).

Once initiating stage atomization, by cell shaker platform (MAXQ416hp, ThermoScientific,RochesterNY) above in the CO2 of humidity 5% incubator, keep ten (10) days with 100rpm. Differentiation cause after the 1stMy god, the 3rd day, the 5th day and the 7th day, culture medium in flask with as the fresh culture replacing of preparation described in table 19. ForThen the differentiation that starts of reference adopts qRT-PCR before after causing the 3rd day, the 5th day, the 7th day and the 10th day that breaks upSample.

In order to determine that can ectoderm or mesoderm specificity in mrna expression pattern change and detect by qRT-PCRArrive, use three applying biological system low density arrays of specifying versatility, definitive entoderm (DE) and the 6th stage (S6)(Life^TM, Carlsbad, CA), and using result with as normalized expression contrast suitably do not break up multipotential stem cell sampleProduct compare.

Use these arrays, determine that the multipotency of cultivating is thin in ectoderm (Figure 89) or mesoderm (Figure 90) differential mediumBorn of the same parents' gene expression pattern. Can be observed, the cell breaking up in shaking flask under arbitrary condition was the amplification of the 3rd day to the 10th dayIn cultivation, show the multipotency gene table of minimizing for the versatility gene as NANOG, POU5F1/OCT4, TDGF1 and ZFP42Reach, as measured in versatility array. The expression of CXCR4 is from being divided into ectoderm or mesoblastic hES or iPS cellIn sample, increase to some extent. These results are relevant to qRT-PCR data, and it illustrates the Gene Differentiation feature of high expressed. Use ectodermThe cellular expression of differential medium processing cause the ARX, the NEUROD that are determined by qRT-PCR for 3 to 10 days after differentiation, NKX6.1,100 times of PAX6 (>) and 1000 times of ZIC1 (>) increase level (Figure 91). These data are confirmed by FACS array, and it demonstratesStart to cause being divided into ectoderm destiny three (3) days afterwards, iPSC cell and hES cell all keep SOX2 (versatility and nerveBoth genes of all needing of stem cell) high expressed, but the expression of losing POU5F1/OCT4 (gene that versatility is required),Increase PAX6 (gene of nerve and Endocrine Differentiation) simultaneously and express (Figure 92).

Also observe the similar differentiation power with the cell of mesoderm differential medium processing. In the process of differentiation in 10 daysIn, multipotency gene expression declines (Figure 90), for the early stage instantaneous mesendoderm destiny of the 3rd day (CER1, EOMES, CKIT andVWF) gene expression characteristics is observed early evoking, and these gene expression doses to the within 10 days, drop to approach baseline (figure93). Also observe, causing after differentiation 3,5,7 and 10 days, the expression of characteristic mesoderm gene demonstrates in advance and increasesGene expression (being CDH2, CDX2, GATA6, HNF4 α, MNX1, PRDM1 and SOX17 in Figure 93). At iPS cell sample andHES cell sample is all observed the gene induced of model identical in both, and this shows that atomization is directed and in essence notSpontaneous.

These in the gene expression of observing by qRT-PCR change with observed to result phase by phase contrast microscopeClose, and the frozen portions of trooping is carried out to immunostaining (immunstained). In mesoderm differentiation suspension culture the10 days, 10 approximately 1 beginnings in trooping spontaneously " were beated ", this show cell be divided into cardiac muscular tissue (Figure 94, Zuo Tu,The 10th day, informal voucher). Some painted cross sections of trooping demonstrate the streaky end-to-end 'beta '-tubulin that instruction muscle formsDyeing pattern (Figure 94, right figure).

When compared with being divided into mesoblastic trooping (Figure 94), for (Figure 95, the left side of trooping that is divided into ectoderm destinyFigure) observe significantly different form pattern. Trooping than the cell that is divided into mesoderm destiny more in the differentiation of whole ectodermLarge and more intensive, and ectoderm noble cells is expressed sum beta tubulin still less. Those express beta tubulin reallyCell demonstrates the neuron staining feature of dendroid pattern (Figure 95, right figure, white arrow) more.

Be combined with qRT-PCR and FACS data, these results show, in suspension, the cell of stock and amplification can be determinedTo being divided into mesoderm or ectoderm destiny with renewable mode in suspension culture.

Table 19

Table 20

Although describe and show the present invention in conjunction with various concrete materials, program and example, but should be appreciated that thisThe bright particular combinations being not limited to for this object selected materials and program. It will be apparent to one skilled in the art that can be to theseDetails is made multiple change. Description and example only should be considered to as exemplary, true scope of the present invention and essence by withAttached claims limit. All bibliography, patent and the patent application of mentioning in the application is all in full with way of referenceBe incorporated herein.

Claims (21)

1. a method for amplification and differentiation pluripotent cell in dynamically stirring suspension culture systems, described method is included in planeIn adhere-wall culture thing, pluripotent cell is cultivated into the cell cluster of gathering, and described in breaking up in dynamically stirring suspension culture systemsPluripotent cell is trooped, and wherein said differentiation step comprises use Cyp26 inhibitor.

2. method according to claim 1, wherein said method increases interim thin of G0/G1 in the described cell cycleBorn of the same parents' percentage.

3. method according to claim 1, wherein said cultivation comprises the bovine serum albumin(BSA) that comprises approximately 0.1% to approximately 2%Environment.

4. method according to claim 3, wherein said environment also comprises Rho inhibitors of kinases.

5. method according to claim 1, wherein said pluripotent cell is adherent.

6. a method for the interim cell percentage of the G0/G1 of increase in the cell cycle, described method is included in and comprises approximatelyIn the environment of 0.1% to approximately 2% bovine serum albumin(BSA), pluripotent cell is increased into the cell of gathering in plane adhere-wall culture thingTroop, described multipotential stem cell is trooped and be transferred to dynamic suspension liquid from described plane adhere-wall culture thing, and using little moleculeOptionally with cultivating described cell in the supplementary culture medium of TCG 'beta ' family member in described dynamic suspension liquid.

7. method according to claim 6, wherein said little molecule is that MCX and wherein said TGF 'beta ' family member areGDF-8。

8. method according to claim 6, wherein said amplification comprises the environment that comprises Rho inhibitors of kinases.

9. pluripotent cell is divided into a method for definitive entoderm, described method is included in the ox that comprises approximately 0.1% to approximately 2%In sero-abluminous environment, pluripotent cell is increased into the cell cluster of gathering in plane adhere-wall culture thing, by described multipotencyStem cell troops and be transferred to dynamic suspension liquid from described plane adhere-wall culture thing, and is using MCX and GDF8 or WNT3A and workChange in the supplementary culture medium of plain A and cultivate described cell in described dynamic suspension liquid.

10. method according to claim 9, wherein said amplification comprises the environment that comprises Rho inhibitors of kinases.

11. 1 kinds of methods that increase and break up multipotential stem cell in dynamic agitation suspension culture systems, described method is included inIn plane adhere-wall culture thing, multipotential stem cell is increased into the cell cluster of gathering, described multipotential stem cell is trooped from described flatIn face adhere-wall culture thing, be transferred to dynamic suspension liquid, and break up described pluripotent cell group in dynamically stirring suspension culture systemsCollection, wherein said cell increases into the cell cluster of gathering in the environment that comprises approximately 0.1% to approximately 2% bovine serum albumin(BSA).

12. methods according to claim 11, wherein break up described pluripotent cell troop to produce diabetes group,Neural precursor group or cardiac muscle cell's precursor group.

13. methods according to claim 11, wherein said multipotential stem cell is selected from induced multi-potent stem cells, people's umbilical cord groupKnit the cell of cell, parthenote, human embryo stem cell (hES) and the amniotic fluid-derived in source.

The cellular products of 14. 1 kinds of portable source of human stem cell, the cellular products of described portable source of human stem cell comprises by rootThe described differentiation diabetes group who prepares according to the method described in claim 11.

15. 1 kinds of methods that increase in dynamically stirring suspension culture systems and break up pluripotent cell, described method comprises:

A. in the environment that comprises approximately 0.1% to approximately 2% bovine serum albumin(BSA) in plane adhere-wall culture thing by thin described multipotencyBorn of the same parents increase into the cell cluster of gathering,

B. use enzyme or chelating agent that described pluripotent cell is trooped and from described plane adhere-wall culture thing, is transferred to dynamic suspension trainingSupport thing,

C. described cell cluster is remained in dynamic stirring suspension culture systems, and

D. in dynamically stirring suspension culture systems, break up described pluripotent cell and troop to produce diabetes group, nervePrecursor group or cardiac muscle cell's precursor group,

Wherein said differentiation environment comprises 30% the oxygen scope of about anoxic to about environment, the lipid in 0.1% to approximately 2% scope orTheir combination.

16. methods according to claim 15, wherein said stem cell troop express CD9, SSEA4, TRA-1-60 andTRA-1-81, but the expression to CXCR4 lacked.

17. methods according to claim 15, wherein also comprise Rho inhibitors of kinases for the described environment increasing.

18. methods according to claim 15, wherein said differentiation environment is approximately 0.1% to approximately 2% bovine serum albumin(BSA).

19. methods according to claim 15, wherein said pluripotent cell is selected from induced multi-potent stem cells, human umbilical tissueThe cell of cell, parthenote, human embryo stem cell (hES) and the amniotic fluid-derived in source.

20. methods according to claim 15, wherein said method produces diabetes, described diabetesExpress β cell transcription factor.

21. methods according to claim 15, wherein said transcription factor is PDX1 and/or NKX6.1.