CN103396987B - A kind of Totipotent stem cell culture method - Google Patents
A kind of Totipotent stem cell culture method Download PDFInfo
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- CN103396987B CN103396987B CN201310324834.3A CN201310324834A CN103396987B CN 103396987 B CN103396987 B CN 103396987B CN 201310324834 A CN201310324834 A CN 201310324834A CN 103396987 B CN103396987 B CN 103396987B
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Abstract
The invention provides a kind of Totipotent stem cell culture method.With the polypeptide of synthetic as extracellular matrix, be laid on Tissue Culture Dish surface, myeloid-lymphoid stem cell be inoculated in aforementioned culture dish surface, inject without feeder layer substratum, then by postvaccinal myeloid-lymphoid stem cell subculture.Method provided by the invention can be supported the amplification of myeloid-lymphoid stem cell and keep Oct4+ cell content to maintain more than 80% all the time.Cultivate through at least 40 days (go down to posterity for 6 times, about 40 times cell is double), after cell still highly expresses the various marker Oct4 amplification cultivation of myeloid-lymphoid stem cell, caryogram is still consistent.
Description
Technical field
A kind of method that the object of the present invention is to provide myeloid-lymphoid stem cell to cultivate, in particular, provides the cultural method of a kind of generate induced pluripotent stem cells (iPSCs).
Background technology
Stem cell is the cell that can be divided into various kinds of cell type.Come from brephic stem cell and the similar brephic myeloid-lymphoid stem cell iPSCs by induction mode generation again, can be divided into as biont overwhelming majority cell type.These two kinds of cells infinitely rise in value in can cultivating in vitro, for regenerative medicine research and industry provide base mateiral.But be that ESCs or iPSCs is easily dead in cell cultures, especially primate is as the stem cell of people.And this death especially occurs in the process gone down to posterity of cell, no matter go down to posterity with the mode of machinery bulk or the mode of being disperseed by enzymic digestion, be all difficult to avoid.
On the other hand, in the culture systems of embryonic stem cell ESCs with the myeloid-lymphoid stem cell iPSCs of induction generation, use matrigel (Matrigel) conduct as bed board material always.But it is uncertain that Matrigel has chemical composition, the problems such as batch quality is unstable.These problems are for being that basic scientific research and Clinical practice all will bring a lot of disadvantage.Therefore be necessary that finding a kind of chemical composition that can substitute Matrigel determines, and be easy to the substitute of producing the extracellular matrix preserved.
NatureBiotechnology describes and adopts restructuring laminin(NatBiotechnol.2010Jun; 28 (6): 611-5) and adopt from Vitronectin extract polypeptide (NatBiotechnol.2010Jun; 28 (6): 606-10.) mode that Matrigel cultivates for stem cell described above is replaced.But restructuring laminin cost is still extremely high; And the polypeptide deriving from two kinds of tests of Vitronectin is prompted specificly to provide the bonding of integrin alphavbeta3, thus make stem cell growth.
Extracellular basilar membrane is the extracellular matrix defining in-vivo tissue border, is usually present in epithelium layer, the bottom of endothelial layer and neurocyte, has physiologic function widely.The architecture basics of basilar membrane forms cross-linked network by different types of extracellular matrix protein interaction and formed.Wherein most important is ln (laminins) and four collagen types (typeIVcollagen), basement membrane structure based on these two kinds of major protein is high conservative, is present in widely in invertebrates and vertebrate animal tissues structure.From auxology, vertebrate ontogeny first time formation basilar membrane is the inner cell mass (innercellmass in blastular (blastocyst) phase, ICM), in, the formation of ICM makes ICM continue to be divided into two confluent monolayer cells-entoderm (hypoblast) and ectoderm (epiblast).This two confluent monolayer cells separates with basilar membrane, dorsad relatively.Epiblast may be used for deriving embryonic stem cell, also represent the myeloid-lymphoid stem cell of the final period in the natural growth course of individuality.Successfully be seeded in the cell that external myeloid-lymphoid stem cell has the structural performance similar with epithelial cell height-have polar contribution completely to be coupled together by be tightly connected (tightjunction).
Summary of the invention
In order to overcome the deficiencies in the prior art, the invention provides Totipotent stem cell culture method.
Totipotent stem cell culture method, comprises the following steps:
1) be NCKHQCTCIDGAVGCIPLCP(SEQIDNO:1 with the aminoacid sequence that comprises of synthetic) polypeptid solution as extracellular matrix, be laid on Tissue Culture Dish surface;
2) myeloid-lymphoid stem cell is inoculated in described Tissue Culture Dish surface;
3) add without feeder layer substratum, carry out the subculture of myeloid-lymphoid stem cell.
As a preferred embodiment of the present invention, the aminoacid sequence of the polypeptide described in step 1) is NCKHQCTCIDGAVGCIPLCP(SEQIDNO:1).
As another kind of preferred version of the present invention, described polypeptid solution concentration is 0.01mg/ml ~ 20mg/ml; Preferred concentration is 0.1-10mg/ml, and more preferred concentration is 0.2-5mg/ml further, more more preferred concentration is 0.5-2mg/ml further.
As another preferred version of the present invention, described polypeptid solution is as extracellular matrix, being laid on Tissue Culture Dish surface method is, the polypeptid solution described in 0.1mL is drawn with liquid-transfering gun, move in Tissue Culture Dish, rock, shake up, be uniformly distributed in bottom culture dish, then the Tissue Culture Dish that described polypeptid solution covers placed 3 ~ 12 hours at 37 DEG C ± 1 DEG C.Polypeptide is fixed on to cultivate by suction types such as covalent linkage, electrostatic, hydrophobic interactions to be used on the surface.
As further preferred version of the present invention, step 2) in, through following treatment step before myeloid-lymphoid stem cell inoculation:
1) thaw; Or the myeloid-lymphoid stem cell among continuous print is cultivated does not need to thaw;
2) shearing enzyme or EDTA is used to make myeloid-lymphoid stem cell depart from former culture surface.The myeloid-lymphoid stem cell group obtained after process is inoculated in described Tissue Culture Dish surface.
As another preferred version of the present invention, described without feeder layer substratum in step 3), comprises following component: water, mineral substance, VITAMIN, amino acid, glucose, fibroblast growth factor, TGF-β, Transferrins,iron complexes, Regular Insulin or rhIGF-1.
Preferably, myeloid-lymphoid stem cell culture temperature is 36 DEG C ~ 37.5 DEG C, described in changing every day without feeder layer substratum 1 time, within 7 days, go down to posterity once.
Preferably, described myeloid-lymphoid stem cell is generate induced pluripotent stem cells.
The polypeptide of the use in the present invention supports that the amplification of iPSCs does not have studied mistake in appropriate culture medium (as mTeSR, B27 substratum, or conditioned medium).We find, above with NCKHQCTCIDGAVGCIPLCP(SEQIDNO:1) polypeptide of sequence can support the amplification of myeloid-lymphoid stem cell and keep Oct4+ cell content to maintain more than 80% all the time.Cultivate through at least 40 days (going down to posterity for 6 times, about 40 double doubling of cell), after cell still highly expresses the various marker Oct4 amplification cultivation of myeloid-lymphoid stem cell, caryogram is still consistent.
Beneficial effect of the present invention: the invention enables in non-trophoblast system myeloid-lymphoid stem cell can reach criterion for clinical use completely for the requirement of basilar membrane, and involved material can cheaply on a large scale be synthesized in not containing the environment of any zooblast by manual type, greatly simplify the basement membrane components of myeloid-lymphoid stem cell culture systems.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the present invention is further described.
Fig. 1 illustrates at NCKHQCTCIDGAVGCIPLCP(SEQIDNO:1) cultured continuously 30 days is later in sequence colony morphology figure.In figure, scale is 200um.
Fig. 2 is the graph of a relation not breaking up probability and add ROCKinhibitor amount.
Embodiment
IPSCs(is referred to as PSC, myeloid-lymphoid stem cell) the cultural method substratum that needs into fiber feeder cell or make contact to fibroblast feeder cells to be to maintain undifferentiated state by stem cell.We are with the cultivation for myeloid-lymphoid stem cell of polypeptide that can promote cell adhesion now, myeloid-lymphoid stem cell can in repeatedly going down to posterity indefinite maintenance undifferentiated state.
Systematically testing after more than 200 peptide species fragments, obtain the substrate polypeptide that iPSCs Growth of Cells can be supported to adhere to.These polypeptide one or more (comprising derivative) are also proved to be the growth can supporting iPSCs, and are in undifferentiated state.Only have continuous several times (going down to posterity more than 10 times) and substrate coverture that stem cell is in undifferentiated state can be continued and could define and form core polypeptide sequence described in the present invention.This matrix is enough to support that iPSCs is in undifferentiated state in the medium forever.At the bottom of these substratum, oneself is proved and can supports undifferentiated ES cell proliferation many generations, at least 6 generations, and this is by this strong evidence of cultivating indefinitely of support at the bottom of these substratum.Some in these data are shown in Fig. 1.
By obviously supporting that myeloid-lymphoid stem cell (PSCs) adheres to the polypeptide of amplification by after being chemically bonded to culture surface above, test the expression of the myeloid-lymphoid stem cell marker of (the 35th day) after 5 times of going down to posterity further.
For the condition of detection ES cell colony and the cultivation suitability of long term maintenance people ES cell culture, TeSRl substratum and culture condition best before (situation that just we grasp uses conditioned medium) are compared.Find that TeSRI substratum can maintain people ES cell and be in so a kind of undifferentiated state, even if the cell namely more than 90% still continues positive in Oct4 after long-term cultivation.This detected result is shown in Fig. 2.
Going down to posterity in maintenance process, similar the same under other culture systems, the clone of obviously differentiation is manually rejected.
Fig. 2 is the relation of not breaking up probability and adding ROCKinhibitor amount.ROCKinhibitorY27632 can reduce the dependency of mankind's myeloid-lymphoid stem cell for extracellular matrix.We find that the Y27632 of 1-5mM can improve the non-differentiation degree of myeloid-lymphoid stem cell on above peptide material further.
embodiment1
: mankind's myeloid-lymphoid stem cell of induction ties up to the growth under this culture system, goes down to posterity and breaks up
Peptide sequence as extracellular matrix is: NCKHQCTCIDGAVGCIPLCP(SEQIDNO:1).Its polypeptid solution concentration is 2mg/ml.Draw the polypeptid solution of 1mL with liquid-transfering gun, immigration area is 10cm
2in culture dish, slowly rock, shake up, make it to be uniformly distributed in bottom culture dish, then the culture dish that polypeptid solution covers is placed 12 hours at 37 DEG C ± 1 DEG C.Polypeptide is fixed on to cultivate by suction types such as covalent linkage, electrostatic, hydrophobic interactions to be used on the surface.
Step 2) in, the generate induced pluripotent stem cells before inoculation is through following treatment step: 1) thaw.Generate induced pluripotent stem cells among continuous print is cultivated does not need to thaw.2) digest.Shearing enzyme or EDTA is used to make cell detachment culture surface.Then be seeded in new in the culture surface being attached with aforementioned polypeptides.
Generate induced pluripotent stem cells group after process is inoculated in abovementioned steps 1) culture dish in.
Substratum in step 3), comprises following component: water, mineral substance, VITAMIN, amino acid, glucose, fibroblast growth factor, pipecolinic acid, TGF-β, Transferrins,iron complexes, Regular Insulin or rhIGF-1 (insulin-likegrowthfactor).Generate induced pluripotent stem cells culture temperature is 37 DEG C, and every day, replaced medium 1 time, went down to posterity once for 7 days.
Confirm that clone MMHPS-iPSCs-001 caryogram after this culture system increased for 5 generations is normal.Confirm that clone MMHPS-iPSCs-001 forms teratoma after 6 generations.Not containing any feeder layer cells and the non-differential growth level that can not maintain people ES cell containing the substratum of conditioned medium in cultivating more than 80% cell maintain undifferentiated degree in all stages.The adding of inhibitors of apoptosis Y27632 (2.5mM) makes not break up (using Oct4 positive mark) iPSCs stem cell population and brings up to more than 85%.
SEQUENCELISTING
The micro-matrix biopolymers Science and Technology Ltd. in <110> Hangzhou
<120> Totipotent stem cell culture method
<160>1
<170>PatentInversion3.5
<210>1
<211>20
<212>PRT
<213>ArtificialSequence
<220>
<223>peptide
<400>1
AsnCysLysHisGlnCysThrCysIleAspGlyAlaValGlyCysIle
151015
ProLeuCysPro
20
Claims (10)
1. a Totipotent stem cell culture method, comprises the following steps:
1) be NCKHQCTCIDGAVGCIPLCP(SEQIDNO:1 with the aminoacid sequence of synthetic) polypeptid solution as extracellular matrix, be laid on Tissue Culture Dish surface;
2) myeloid-lymphoid stem cell is inoculated in described Tissue Culture Dish surface;
3) add without feeder layer substratum, carry out the subculture of myeloid-lymphoid stem cell.
2. the method for claim 1, is characterized in that, described polypeptid solution concentration is 0.01mg/ml ~ 20mg/ml.
3. method as claimed in claim 2, it is characterized in that, the preferred concentration of described polypeptid solution is 0.1-10mg/ml.
4. method as claimed in claim 2, it is characterized in that, the preferred concentration of described polypeptid solution is 0.2-5mg/ml.
5. method as claimed in claim 2, it is characterized in that, the preferred concentration of described polypeptid solution is 0.5-2mg/ml.
6. the method for claim 1, it is characterized in that, described polypeptid solution is as extracellular matrix, the method being laid on Tissue Culture Dish surface is, draws the polypeptid solution described in 0.1mL with liquid-transfering gun, moves in Tissue Culture Dish, rock, shake up, be uniformly distributed in bottom culture dish, then the Tissue Culture Dish that described polypeptid solution covers is placed 3 ~ 12 hours at 37 DEG C ± 1 DEG C.
7. the method for claim 1, is characterized in that, step 2) in, through following treatment step before myeloid-lymphoid stem cell inoculation:
1) thaw; Or the myeloid-lymphoid stem cell among continuous print is cultivated does not need to thaw;
2) shearing enzyme or EDTA is used to make myeloid-lymphoid stem cell depart from former culture surface.
8. the method for claim 1, it is characterized in that, described without feeder layer substratum in step 3), comprises following component: water, mineral substance, VITAMIN, amino acid, glucose, fibroblast growth factor, TGF-β, Transferrins,iron complexes, Regular Insulin or rhIGF-1.
9. method as claimed in claim 8, it is characterized in that, myeloid-lymphoid stem cell culture temperature is 36 DEG C ~ 37.5 DEG C, described in changing every day without feeder layer substratum 1 time, within 7 days, go down to posterity once.
10. the method for claim 1, is characterized in that, described myeloid-lymphoid stem cell is generate induced pluripotent stem cells.
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