CN101538553A - Method for separating, purifying, culturing and proliferating totipotent stem cell from tissue of early aborted fetus of human being - Google Patents

Abstract

The invention discloses a method for separating and purifying totipotent stem cells from the tissues of early aborted fetuses of human beings, including the steps: a. a sample of the placenta tissue of the aborted fetus after the first pregnancy period is collected; b. the umbilical cord tissue or the intestine tissue of the fetus is obtained from the sample gained in the step a; c. cells are obtained from the umbilical cord tissue or the intestine tissue of the fetus gained in the step b; and d. markers of the embryonic stem cell, such as OCT-4, Nanog, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, and the like which are obtained from the cells of the umbilical cord tissue or the intestine tissue of the fetus, are separated, purified and expressed, and can form the stem cell of an embryoid body. The cells obtained by the method of the invention are always in the state of undifferentiation. The invention also discloses the application of the stem cells in forming different human cells, such as cardiac muscles, nerves and islet beta cells or osseus cells. The method avoids the dispute over ethnics which needs to be faced with during the operation of obtaining the totipotent stem cells from the embryo, and more stem cells can be obtained than from blastocyst.

Description

From fetal tissue's separation and purification of human early abortion and the method for cultivating proliferating totipotent stem cell

Technical field

The present invention relates to the method for a kind of separation and purification stem cell, specifically, this method can be from the fetal tissue of early abortion, comprises in the umbilical cord of fetus or the intestinal tissue separation and purification and cultivates proliferating totipotent stem cell.

Background technology

To because of the damaged and handicapped disease of the human tissue organ that various factors caused, as Parkinson's disease, diabetes, traumatic spinal cord injury, cytopathy disease, burn, myocardosis and bone injury etc., modern medicine does not still have effective methods of treatment.But, brought new hope for curing these diseases along with the discovery of stem cell with in recent years to the deepening continuously and develop of stem-cell research.Therefore, the research of human stem cell has become after the Human Genome Project, most active research field in the life science; Also will be the epoch-making innovation of human disease treatment pattern (Guillot et al.2007; Bajada et al.2008).

Stem cell is a kind of from the cell that has lasting or lifelong self ability in embryo, fetus or the adult's histoorgan.It can produce special cell type, forms tissue and organ.According to the precedence difference that occurs in the ontogenetic process, stem cell can be divided into embryonic stem cell (embryonic stemcells) and adult stem cell (adult stem cell).Ability according to differentiation of stem cells can be divided into stem cell myeloid-lymphoid stem cell (pluripotent stem cell) again, myeloid-lymphoid stem cell (multipotentstem cell) and special energy stem cell (unipotent stem cell).

Adult stem cell is meant the myeloid-lymphoid stem cell that exists and specially can stem cell (Young and Black.2004) from children and adult tissue.Adult stem cell can obtain from patient self, thereby does not have the problem of histocompatibility.But the multiplication capacity of adult stem cell and very undesirable to the efficient of polyphyly differentiation.Such as, can't breed in a large number external from the hemopoietic stem cell that umbilical cord blood is collected, its quantity only can be used for children's treatment (Rogers and Casper.2003).

Human embryo stem cell then is a kind of myeloid-lymphoid stem cell.All show with external research in the body: embryonic stem cell can be divided into most cell monoids of human body, such as neurocyte, cardiac muscle, skeletal muscle, endotheliocyte, gastrointestinal tissue, liver cell, pancreatic, bone, cartilage, hematopoietic cell etc.Compare with adult stem cell, embryonic stem cell has unlimited multiplication capacity, and unlimited source (Pera and Trounson.2004) can be provided for the transplantation treatment of cell, tissue or organ.But only can obtain 200-250 cell from blastocyst; And the research of embryonic stem cell is hampered by problems such as ethics, morals, law.Recently, two groups of scientists of the U.S. and Japan declare to utilize " reprogramming " technology successfully to induce the human skin cell to generate embryonic-like stem cell system respectively.The researchist thinks that this invention finally can solve the source of stem cell, finishes embryonic stem cell ethics battle (Takahashi and Yamanaka.2006; Yuet al.2007).But, have different significantly with embryonic stem cell from the stem cell that becomes the somatocyte source.These induce the pleiotropy stem cell line just to have the gene expression characteristics of human embryo stem cell, at external small-scale propagation some months, can induce to generate several functioning cells.But on a lot of characteristics, greatly differ from each other (Chan and Harris.2008) with embryonic stem cell.Therefore, seeking new natural myeloid-lymphoid stem cell source from new approach, avoid stem cell ethics battle again simultaneously, is one of developing direction of stem-cell research now and clinical application.

(mesenchymal stem cell is to have self ability and can be to the stem cell of a plurality of directions differentiation MSC) to mescenchymal stem cell.MSC is found in the marrow the earliest.The current MSC that is used for basis and clinical study is mainly derived from marrow.But, the content extremely low (0.01%～0.001%) of MSC in adult's marrow, and its quantity descends with people's age growth.Further studies confirm that: except that marrow, in the multiple tissues such as reticular tissue of peripheral blood, amniotic fluid, placenta, Cord blood, fat, synovium of joint, skeletal muscle and skin, all find to have (the Anker et al.2003 that exists of MSC; Et al.Romanovet al 2003; Lee et al.2004; McElreavey et al.1991).Research also shows: this class stem cell can self breed as other adult stem, can be divided into mesenchymal cell system, and can be divided into (Wanget al.2004 such as scleroblast, chondroblast, adipocyte, myocardial cell, neurone; Mitchell et al.2003).But this class stem cell is not expressed the distinctive cell surface marker of embryonic stem cell, does not have the embryoid body of formation (embryonic bodies, ability EB) yet.Therefore, the characteristics that not exclusively have embryonic stem cell.

To conceived the 12 week, be referred to as the gestation period 1 from embryo nidation.During this period, the various histoorgan basic formings of fetus, but still be in early development stage.No matter how flourishing science and technology is, also no matter how people are familiar with, spontaneous abortion or artificial abortion always are difficult to avoid, our hypothesis: the stem cell that goes out from fetal tissue's separation and purification of early abortion is than having the potentiality of myeloid-lymphoid stem cell from the isolated stem cell in back that is born.The fetal tissue of early abortion is a kind of iatrogenic waste, and seeking the nature myeloid-lymphoid stem cell from the fetal tissue of early abortion then is to utilize dead fetus, does not have the problem of destroying the live body embryo, i.e. the ethnics Problem of so-called stem cell.

Key is, how to find a kind of method, can be more easily from the fetal tissue of pregnant first phase artificial abortion successfully separation and purification go out stem cell and the stable undifferentiated state that keeps stem cell.When the separation and purification from different early abortion fetal tissues easily and reliably of this method, freezing preservation and recovery myeloid-lymphoid stem cell, set up the myeloid-lymphoid stem cell storehouse and just become possibility, be the damaged and handicapped diseases of the various human tissue organs of cellular replacement therapy just also, as Parkinson's disease, diabetes, traumatic spinal cord injury, cytopathy disease, burn, myocardosis and bone injury etc., bring hope.

Summary of the invention

For this reason, we adopt a kind of simple relatively method from the fetal tissue of pregnant first phase artificial abortion successfully separation and purification go out stem cell.

Separation and purification of the present invention comprises from the method for the myeloid-lymphoid stem cell of people's early abortion fetal tissue: the fetal placental tissue sample of a, the period 1 miscarriage of collection gestation; B, from the sample of step a, separate to obtain fetal cord or intestinal tissue; C, from the fetal cord of step b or intestinal tissue, obtain fetal cord or intestinal tissue cell; Embryonic stem cell mark OCT-4 is expressed in separation and purification d, the fetal cord that obtains from step c or the intestinal tissue cell, SSEA-3, and SSEA-4, the stem cell of TRA-1-60 and TRA-1-81, these stem cells can form embryoid body.

Method of the present invention also comprises: the purifying stem cell that e, frozen steps d obtain; F, the frozen purifying stem cell of recovery.

The inventive method comprises that also keeping these stem cells is in undifferentiated state.

The inventive method at first is the various fetal tissues of collecting pregnant first phase miscarriage.The collection of tissue is the method by artificial abortion or spontaneous abortion.From these iatrogenic refuses of collecting, look for fetal cord or intestinal tissue then.After finding these tissues, these tissues are handled.Its step comprises that these organize the cell that obtains these tissues with collagenase digesting.Wash isolated cells with physiological saline then.If digesting these, the collagenase of any kind organize the cell that obtains these tissues all can adopt; Type i collagen enzyme such as 1mg/ml.

More specifically, the described gestation of the inventive method step a period 1 aborted fetus is organized as the fetal placental tissue of gestation 8 thoughtful 12 all artificial abortion or spontaneous abortion.

In step c, earlier with fetal cord or intestinal tissue with the physiological saline washing of sterilization, be cut into small pieces again, with collagenase digesting to cellular constituent behind state, centrifugal acquisition cell precipitation, standby with the physiological saline washing.

In the steps d during separation and purification stem cell, can adopt the mode of chromatography column purifying, earlier will express OCT-4 with immunomagnetic bead technique, SSEA-3, SSEA-4, the stem cell purifying of TRA-1-60 and/or TRA-1-81 (Durcova et al.1998), the stem cell with wash-out is transferred to the cell cultures flask culture again, obtains the stem cell strain of purifying.

Also can adopt subculture in vitro separately best cultivation purifying stem cell, fetal cord or intestinal tissue cell that step c is obtained suspend with the stem cell nutrient solution, shift to cultivate and go down to posterity, and culture condition is, at 37 ℃ and 5%CO ₂Condition under cultivated 3-7 days or cell proliferation accounts for culturing bottle or more than 70% of culture dish floorage, shift to go down to posterity at least 5 times, obtain the stem cell of purifying.

Described stem cell nutrient solution is by the α-MEM substratum of volumn concentration 88%, 10% foetal calf serum or bFGF (0.1 μ g/ml), and 100X green grass or young crops/Streptomycin sulphate of 1%, the plain B of 1% 100X two property enzymes forms.Other nutrient solution or culture condition, the vigor that if can keep cell also can adopt.

Stem cell is the cell with lasting or lifelong self ability; And other histocyte vitro culture after for some time with natural death.The method that cells in vitro cultivate to be regenerated comprises: at first will breed to accounting for culturing bottle or the cell of culture dish floorage more than 70% washs with phosphoric acid buffer; Use trypsinase-EDTA solution-treated then, stick on the cell suspension of culturing bottle or culture dish bottom during with growth; With the cell transfer of these suspensions to centrifuge tube, centrifugation; With fresh nutrient solution cell is suspended again again and dilute; Fetal tissue's cell with dilution reapposes new culturing bottle or culture dish at last, continue to cultivate under the condition of 5% CO2 and 37 ℃ with the stem cell nutrient solution, renewed bright nutrient solution once in every 3-5 days, until cell proliferation to accounting for culturing bottle or more than 70% of culture dish floorage.Repeat above-mentioned steps again and show consistent like fibrous morphocytology feature to the cell in cultivating.Other nutrient solution or culture condition, the vigor that if can keep cell also can adopt.

Keep the method that these stem cells are in undifferentiated state and comprise above-mentioned stem cell nutrient solution, or in the stem cell nutrient solution of serum-free, add some cytokine, as Prostatropin (basic fibroblast growth factor, bFGF), under the condition of 5% CO2 and 37 ℃, cultivate.Other nutrient solution or culture condition, the undifferentiated state that if can keep cell also can adopt.

The method that keeps in cold storage after the separation and purification and cultivate the stem cell of propagation then is at first will breed to accounting for culturing bottle or the cell of culture dish floorage more than 70% washs with phosphoric acid buffer; Use trypsinase-EDTA solution-treated then, stick on the cell suspension of culturing bottle or culture dish bottom during with growth; With the cell transfer of these suspensions to centrifuge tube, centrifugation.Again isolating stem cell is placed 4 ℃, suspend again with the stem cell stock solution then, be transferred to the pipe that keeps in cold storage.The pipe that keeps in cold storage that stem cell is housed is positioned over-70 ℃, and 24 hours, the pipe that keeps in cold storage that stem cell will be housed at last was transferred to the liquid nitrogen container standing storage.The stem cell stock solution contains 40% foetal calf serum, 50% α-MEM substratum and 10% DMSO.The vigor that other stem cell stock solution if can be kept cell also can adopt.

The method of storing stem cell of thawing is that the pipe that keeps in cold storage that will stem cell be housed takes out from liquid nitrogen container, is placed on 37 ℃ immediately and thaws; Centrifugation then; And wash with phosphoric acid buffer.The cell that will thaw is placed on culturing bottle or training again, supports ware stem cell nutrient solution, and cultivation is 24 hours under the condition of the CO2 5% and 37 ℃.Then, replace fresh nutrient solution, continue under the condition of 5% CO2 and 37C °, to cultivate; Renewed bright nutrient solution once in every 3-5 days.Other nutrient solution or culture condition, the vigor that if can recover to store stem cell also can adopt.

Another object of the present invention provides a kind of myeloid-lymphoid stem cell from people's early abortion fetal tissue, is separated obtaining by the described method of claim 1.The stem cell of these separation and purification can be expressed the embryonic stem cell mark that undifferentiated embryonic stem cell is expressed, as OCT-4, and SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81; And some transcription factor, as OCT-4, Telemerase and Nanog; And can form embryoid body.

The stem cell of these separation and purification can directed differentiation become various dissimilar cells under certain condition of in vitro culture, as the myocardial cell, and neurocyte, pancreatic or osteocyte.

These can directed differentiation myeloid-lymphoid stem cell might provide the source of Transplanted cells as myocardosis, Parkinson's disease, traumatic spinal cord injury, diabetes and bone injury etc. to because of the damaged and handicapped disease of the human tissue organ that various factors caused.

Based on separation and purification of the present invention, the cryopreservation resuscitation method from the myeloid-lymphoid stem cell of people's early abortion fetal tissue, a further object of the present invention provides a kind of method of setting up the myeloid-lymphoid stem cell storehouse, comprising:

A, the fetal placental tissue sample of the early abortion of collecting is done necessary loimology inspection;

B, separation and purification stem cell from different fetal placental tissues in accordance with the method for claim 1;

The evaluation of classifying of the histocompatibility antigen of c, stem cell strain that step b is obtained;

D, set up stem cell strain catalogue according to the classification qualification result of step c;

E, the stem cell strain that step b is obtained keep in cold storage by the described method of claim 10.

The present invention also comprises the myeloid-lymphoid stem cell storehouse of setting up according to the method described above.

Adopt embryonic stem cell to carry out Transplanted cells, the same with the allosome organ with bone marrow transplantation, face patient produces immunological rejection because of the difference of histocompatibility antigen to the donor tissue organ problem.The problem that also is related to the key of success of embryonic stem cell clinical treatment.Address this problem and have two kinds of approach: the one, adopt molecular biology method that stem cell is transformed, generation can be transplanted to the stem cell line of Different Individual; The 2nd, set up the embryonic stem cell bank of similar existing now marrow storehouse and infant umbilical cord blood bank.Preceding a kind of approach is had got long long way to go; Then a kind of approach goes out myeloid-lymphoid stem cell in our institute's invention technology separation and purification from iatrogenic waste makes it to become possibility.

The foundation in myeloid-lymphoid stem cell storehouse makes adopts cellular transplantation therapy because of the damaged and handicapped disease of the human tissue organ that various factors caused, and becomes a kind of potential means that can expect as myocardosis, Parkinson's disease, traumatic spinal cord injury, diabetes and bone injury etc.

Description of drawings

Fig. 1, immunohistochemical methods detect in the pregnant period 1 aborted fetus intestinal tissue of being pregnent expresses OCT-4 (Figure 1A), TRA-1-60 (Figure 1B), TRA-1-81 (Fig. 1 C), SSEA-1 (Fig. 1 D), SSEA-3 (Fig. 1 E) and SSEA-4 (Fig. 1 F) wait the stem cell of embryonic stem cell mark.

Fig. 2, immunohistochemical methods detect in the pregnant period 1 aborted fetus umbilical cord tissue of being pregnent expresses OCT-4 (Fig. 2 A), TRA-1-60 (Fig. 2 B), TRA-1-81 (Fig. 2 C), SSEA-1 (Fig. 2 D), SSEA-3 (Fig. 2 E) and SSEA-4 (Fig. 2 F) wait the stem cell of embryonic stem cell mark.

Fig. 3, the cell morphological characteristic that when the stem cell of fetal cord or intestinal tissue separation and purification is cultivated, shows, Fig. 3 A be the 3rd generation cell, Fig. 3 B then be the 12nd generation cell.

Fig. 4, all has the ability that forms embryoid body from the stem cell of fetal cord or intestinal tissue separation and purification.Fig. 4 A and B are presented at the cell cluster that forms naturally in the culturing process.Fig. 4 A zoom is 40 times, and Fig. 4 B then is 200 times.Fig. 4 C and D demonstration are transferred to low adherent culture dish with cultured cells, cultivate the embryoid body that forms with the embryoid body nutrient solution.Fig. 4 C zoom is 40 times, and Fig. 4 D then is 100 times.

Fig. 5, immunohistochemical methods detect three the expressed germinal layer marks of embryoid body that form when stem cell is cultivated, and Fig. 5 B is Nestin (ectoderm), and Fig. 5 C is AFP (entoderm), and Fig. 5 D is SAM (mesoderm) and contrast (Fig. 5 A).

Fig. 6, from the growth characteristics of the stem cell of fetal cord or intestinal tissue separation and purification.

Fig. 7, immunohistochemical methods detect the stem cell expression of the 3rd generation SSEA-1 (Fig. 7 A), OCT-4 (Fig. 7 B), SSEA-3 (Fig. 7 C), SSEA-4 (Fig. 7 E), TRA-1-60 (Fig. 7 D) and the TRA-1-81 embryonic stem cell marks of cultivating such as (Fig. 7 F).

Fig. 8, immunohistochemical methods detect the stem cell expression of the 12nd generation SSEA-1 (Fig. 8 A), OCT-4 (Fig. 8 B), SSEA-3 (Fig. 8 C), SSEA-4 (Fig. 8 E), TRA-1-60 (Fig. 8 D) and the TRA-1-81 embryonic stem cell marks of cultivating such as (Fig. 8 F).

Fig. 9, RT-PCR detect the OCT-4 of the stem cell expression in the 1st to the 12nd generation of cultivating, Nanog, the mRNA of embryonic stem cell marks such as Telemerase.

Figure 10, induce the morphological feature that is divided into pancreatic from these stem cell directionals.Figure 10 A and Figure 10 B are the morphological feature of inducing the islet cells group in the III stage in the process; 100 times of Figure 10 A zoom, 400 times of Figure 10 B zoom.Figure 10 C and Figure 10 D are the morphological feature of inducing the islet cells group in the IV stage in the process; 100 times of Figure 10 C zoom, 400 times of Figure 10 D zoom.

Figure 11, immunohistochemical methods detect directional induction and are divided into pancreatic expression pancreatic specificity marker thing (Figure 11 A is Insulin, and Figure 11 B is Nestin).200 times of zoom.

Figure 12, directional induction are divided into pancreatic can be external to glucose concn reacting condition, excreting insulin.

Figure 13, induce the morphological feature that is divided into the myocardial cell from these stem cell directionals.100 times of Figure 13 A zoom; 200 times of Figure 13 B image multiplications

Figure 14, immunohistochemical methods detect the cell expressing myocardial cell's of directional induction differentiation specificity marker thing (Figure 14 A is β-MHC, and Figure 14 B is cTNI).

Figure 15, RT-PCR detect the myocardial cell express alpha-MHC of directional induction differentiation, ANF, the mRNA of MLC2v and Nkx2.5.

Figure 16, induce the morphological feature that is divided into neurocyte from these stem cell directionals.100 times of Figure 16 A zoom, 400 times of Figure 16 B zoom.

Figure 17, immunohistochemical methods detect the specificity marker thing (Figure 17 A is Nestin, and Figure 17 B is β-tubulin, and Figure 17 C is that MAP-2 and Figure 17 D are MPB) of the cell expressing neurocyte of directional induction differentiation.

Figure 18, RT-PCR detect the neurocyte of directional induction differentiation and express Bf1, NF, the mRNA of Noggin.

Figure 19, from these stem cell directionals induce the differentiation osteoblastic morphological feature.Figure 19 A and B are at the morphological feature of cultivating the 14th day osteocyte, 400 times of Figure 19 A zoom; Figure 19 B amplifies 100 times.Figure 19 C and D are at the morphological feature of cultivating the 22nd day osteocyte; 100 times of zoom.

The formation of bone brief summary in the osteocyte of Figure 20, von Kossa dyeing detection directional induction differentiation.100 times of Figure 20 A zoom; 200 times of Figure 20 B zoom.

Figure 21, immunohistochemical methods detect the osteocyte of inducing differentiation and express later stage osteogenesis mark (ONC).100 times of Figure 21 A image multiplications; 200 times of Figure 21 B image multiplications

Above-mentioned accompanying drawing is black and white picture, and in order to be illustrated more clearly in the cells and characteristic of stem that the inventive method obtains, the colour picture that the contriver submits Fig. 1～Fig. 8, Figure 10～Figure 14, Figure 16, Figure 17, Figure 19～Figure 21 simultaneously to as a reference.

Embodiment

Embodiment 1: from the separation and purification of the myeloid-lymphoid stem cell of people's early abortion fetal tissue, cultivate propagation and identify

1.1 material and method

1.1-1. reagent

(α-MEM), foetal calf serum (FCS) and trypsinase-EDTA (trypsin-EDTA) solution are available from Invirogen (U.S.) for α-MEM substratum.The plain B of green grass or young crops/Streptomycin sulphate and two property enzymes, type i collagen enzyme and sheep anti-mouse antibody-HRP mixture is available from Sigma (U.S.).Anti-OCT-4, SSEA-1, SSEA-3, SSEA-4, the monoclonal antibody of TRA-1-60 and TRA-1-81 is available from R﹠amp; D System (U.S.).Anti-AFP, the monoclonal antibody of Nestin and SMA is available from Sigma (U.S.) and Dako (Denmark).Reverse transcription (RT) test kit is available from Promega (U.S.); DNA Taq enzyme reagent kit is available from Qiogen (Germany).The primer that is used for RT-PCR is then synthetic by Invirogen.

1.1-2. collect sample and isolating fetal histocyte

Collect the fetal placental tissue that gestation period 1 (＞8 week＜12 week) obtains by artificial abortion operation.In the vessel of tissue collecting behind autoclaving, and deliver to Cell Culture Lab immediately and handle.Earlier tissue is placed on the culture plate of 100X15mm, from the fetal placental tissue, carefully seeks tissues such as the umbilical cord of fetus or intestines with tweezers and scissors for surgery.Discard remaining tissue then.

Tissues such as the fetal cord that searches out or intestines are washed 3 times with autoclaved physiological saline.With scissors for surgery washed tissue is shredded and is transferred to the centrifuge tube of 15ml again.Digested 1 hour in 37 ℃ of water-baths with type i collagen enzyme (1mg/ml).Then with sample with whizzer 4 ℃ with 800rpm centrifugal 15 minutes, abandoning supernatant obtains cell precipitation.Again with cell with autoclaved physiological saline washed twice.

1.1-3. cell cultures

Fetal tissue's cell of washing is suspended with the stem cell nutrient solution, and be transferred to the Tissue Culture Flask of 75cm.The stem cell nutrient solution of 50ml is by α-MEM substratum of 44ml, the foetal calf serum of 5ml, and the plain B of the 100X green grass or young crops/Streptomycin sulphate of 0.5ml and the 100X of 0.5ml two property enzymes forms.

1.1-4. regenerating, cultivates cells in vitro

Tissue Culture Flask is placed the CO2 incubator, under the condition of 37 ℃ and 5%CO2, cultivated 3-7 days or cell proliferation accounts for culturing bottle or more than 70% of culture dish floorage, be considered as a generation.Then cell culture fluid is discarded, use twice of phosphoric acid buffer washed cell.Stick on the cell suspension of culturing bottle bottom again in the time of will growing with trypsinase-EDTA solution of 0.25%, and with the cell transfer of these suspensions to centrifuge tube, 800rpm centrifugation under the room temperature.Use the stem cell nutrient solution with cell suspension at last, and about 10% cell transfer to the Tissue Culture Flask continuation of another 75cm2 is cultivated.This process repeats 5 to 7 times at least.

1.1-5. keeping of stem cell undifferentiated state

Keep method that these stem cells are in undifferentiated state and comprise and use above-mentioned stem cell nutrient solution, or replace foetal calf serum in the above-mentioned stem cell nutrient solution, cultivate under the condition of the CO2 5% and 37 ℃ with bFGF (0.1 μ g/ml).

1.1-6. stem cell is frozen

In the process that each cell cultures is regenerated, with the cell of 800rpm centrifugation under room temperature stem cell stock solution (40% foetal calf serum, 50% α-MEM substratum and 10% DMSO) suspend, being transferred to keeps in cold storage managed and places-70 ℃ and spend the night, and was transferred to the liquid nitrogen container prolonged preservation in second day.

1.1-7. thawing and the recovery of cell viability of stem cell

The pipe that keeps in cold storage that stem cell is housed is taken out from liquid nitrogen container, be placed on 37 ℃ immediately and thaw; Centrifugation then, and with the phosphoric acid buffer washing once.The cell transfer that to thaw is to culturing bottle again, cultivates 24 hours under the condition of 5% CO2 and 37 ℃ with the stem cell nutrient solution.Replace fresh nutrient solution and continue under the condition of 5% CO2 and 37 ℃, to cultivate, and every 3-5 days renew bright nutrient solution once.

1.1-8. immunohistochemistry and cellular immunization chemistry

With fetal cord or an intestinal tissue part 10% formalin fixed that searches out.Paraffin embedding is prepared into 4 μ m slabs then.During immunohistochemical analysis,, carry out direct immunization peroxidase stain (Bullock Gr and Petrusz P.1982) by standard operation to paraffin-embedded section.At first, organize endogenous peroxidase activity and non-differential protein binding site respectively with 1% hydrogen peroxide treatment and contain the solution sealing 30 minutes of 10% normal goats serum.Add OCT-4 then, SSEA-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 monoclonal antibody are in 4 ℃ of incubated overnight.Second day, with behind the PBS washing slice with the goat anti-mouse igg-HRP mixture room temperature of dilution in 1: 2000 under insulation 1 hour.PBS develops a film, and makes binding antibody colour developing 5 minutes with diaminobenzidine (DAB).Specificity Quality Control contrast comprises with PBS or normal mouse IgG (Sigma company product) replaces specific antibody.

The cellular immunization chemical detection is carried out direct immunization peroxidase stain (Javois1999) by standard operation.At first with the embryoid body cultivated or cell transfer to cell culture carrier, 37 ℃, cultivated 2-3 days under the 5%CO2 condition.Then slide glass is fixed 10 minutes in-20 ℃ of acetone.Endogenous peroxidase activity and non-differential protein binding site are respectively with 1% hydrogen peroxide treatment and contain the solution sealing 30 minutes of 10% normal goats serum.Then with OCT-4, SSEA-1, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 monoclonal antibody are in 4 ℃ of incubated overnight.Second day with after the PBS washing, with insulation under the goat anti-mouse igg-HRP mixture room temperature of dilution in 1: 2,000 1 hour.PBS develops a film, and makes binding antibody colour developing 5 minutes with diaminobenzidine (DAB).Specificity Quality Control contrast comprises with PBS or normal mouse IgG (Sigma company product) replaces specific antibody.

1.1-9.RT-PCR

With cultivate each for the part cell transfer be stored at-70 ℃ stand-by.During detection, adopt Trizon reagent (Invirogen) to extract total RNA of sample.The synthetic of cDNA then adopted reverse transcription (RT) test kit of Promega company, and by specification carries out.OCT-4, the PCR primer of Nanog and Telomerase is respectively: OCT-4 (gagcaaaacccggaggagt (sense), ttctctttcgggcctgcac (antisense); 310bp); Nanog (gcttgccttgctttgaagca (sense), ttcttgactgggaccttgtc (antisense); 225bp); HTERT (cagctcccatttcatcagca (sense), cgacatccctgcgttcttg (antisense); 114bp); β-actin (cgcaccactggcattgtcat (sense), ttctccttgatgtcacgcac (antisense), 207bp) (Beattie et al.2005).The condition of the PCR of OCT-4 is 94 ℃, 30 seconds, and 55 ℃, 1 minute, 72 ℃, 35 circulations in 1 minute; The condition of the PCR of Nanog is 94 ℃, 30 seconds, and 57 ℃, 1 minute, 72 ℃, 35 circulations in 1 minute; The condition of the PCR of Telomease is 94 ℃, 30 seconds, and 53 ℃, 1 minute, 72 ℃, 35 circulations in 1 minute; The condition of the PCR of β-actin is 94 ℃, 30 seconds, and 54 ℃, 1 minute, 72 ℃, 35 circulations in 1 minute; The product of PCR then adopts painted 2% agar of Golden to detect, and with the RT-PCR product of β-actin as internal standard.

1.2 result

1.2-1. immunohistochemical methods is determined the embryonic stem cell-like in the fetal tissue

Fig. 1 shows in the fetus intestinal tissue of miscarrying with the pregnant period 1 of being pregnent of immunohistochemical methods method detection discovery the stem cell of expressing the body early embryo stem cell markers.OCT-4 wherein, SSEA-3, SSEA-4, marks such as TRA-1-60 and TRA-1-81 are all positive, and SSEA-1 (being the mark of mouse embryonic stem cell) is then negative.Express OCT-4, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 positive cells be positioned at intestinal mucosa cells around.There is stem cell in the fetus intestinal tissue that this discovery proof is miscarried in early days with embryonic stem cell feature.Zoom is 100 times.

Fig. 2 shows in the fetal cord tissue of miscarrying with the pregnant period 1 of being pregnent of immunohistochemical methods method detection discovery the stem cell of expressing the body early embryo stem cell markers.OCT-4 wherein, SSEA-3, SSEA-4, marks such as TRA-1-60 and TRA-1-81 are all positive, and SSEA-1 (being the mark of mouse embryonic stem cell) is then negative.Express OCT-4, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 positive cells mainly are positioned at Whartons jelly.There is stem cell in the fetal cord tissue that this discovery proof is miscarried in early days with embryonic stem cell feature.Zoom is 100 times.

1.2-2. the morphological feature of embryonic stem cell-like

Fig. 3 shows from the fetal cord or the intestinal tissue separation and purification of the pregnant period 1 miscarriage of being pregnent and the cell morphological characteristic of the embryonic stem cell-like after cultivating.These cells demonstrate similar fibroblastic cellular form uniformly.Fig. 3 A be the 3rd generation cell, Fig. 3 B then be the 12nd generation cell.The zoom of Fig. 3 A and Fig. 3 B is 100 times.

1.2-3. the stem cell of separation and purification can form embryoid body

Fig. 4 shows from the fetal cord of the pregnant period 1 miscarriage of being pregnent or the stem cell of intestinal tissue separation and purification and cultivation all have the ability that forms embryoid body.Fig. 4 A and B are presented at the cell cluster that forms naturally in the culturing process.Fig. 4 A zoom is 40 times, and Fig. 4 B then is 200 times.Fig. 4 C and D demonstration are transferred to low adherent culture dish with cultured cells, cultivate the embryoid body that forms with the embryoid body nutrient solution.Fig. 4 C zoom is 40 times, and Fig. 4 D then is 100 times.

Fig. 5 shows that detecting discovery with the immunohistochemical methods method can express entoderm (α-fetoprotein with these separation and purification and the formed embryoid body of culturing stem cells, AFP, alpha-fetoprotein, Fig. 5 C), mesoderm (smooth muscle actin, SMA, smooth muscle cell specific proteins, Fig. 5 D) and ectoderm (Nestin, neuronspecific enolase, Fig. 5 B) three germinal layer cell expressing specificity marker things.The then negative contrast of Fig. 5 A (in immunohistochemical methods detects, not adding anti-AFP, the specific antibody of SMA or Nestin).Zoom is 100 times.

1.2-4. the growth characteristics of the embryonic stem cell-like of separation and purification

Fig. 6 shows from the growth curve of the stem cell of the fetal cord of the pregnant period 1 miscarriage of being pregnent or intestinal tissue separation and purification and cultivation.Cultivating in 3 days about 100 times of the cell number average increment of four Different Individual separation and purification.

1.2-5. evaluation to the stem cell immunohistochemical methods after the separation and purification cultivation

Fig. 7 and Fig. 8 showed cell immunohistochemical methods respectively detect the 3rd and the 12nd generation stem cell expression body early embryo stem cell markers of cultivating.As the result the same (Fig. 1 and Fig. 2) of the immunohistochemical methods of intestinal tissue and umbilical cord tissue, OCT-4 wherein, SSEA-3, SSEA-4, marks such as TRA-1-60 and TRA-1-81 are all positive, and SSEA-1 is then negative.Show that these stem cells are in undifferentiated state always under culture condition of the present invention.Zoom is 100 times.

1.2-6. to the molecular biological evaluation of the stem cell of separation and purification

Fig. 9 shows the early transcription gene OCT-4 that the stem cell in the 3rd to the 12nd generation that the RT-PCR detection is cultivated is expressed constantly, the mRNA of Nanog and Telomerase.As internal standard, show that the from the 3rd to the 12nd generation of amount of these early transcription genes and Telomerase Expression is close with the RT-PCR product of β-actin simultaneously, show that these stem cells are in undifferentiated state under the culture condition of this invention always.

1.3. conclusion

Umbilical cord of collecting from the pregnant period 1 abortion tissue of being pregnent or intestinal tissue be the separation and purification stem cell that goes out to have the embryonic stem cell characteristic successfully.This kind stem cell (1) can form embryoid body in vitro culture; (2) has unlimited multiplication capacity: 100 multiplications were just arranged in 3 days, and this multiplication capacity goes down to posterity and still keeps this multiplication capacity after a year and a half; (3) immunohistochemistry, cell tissue chemistry and molecular Biological Detection find that all distinctive cell surface marker of all human embryo stem cells of these cell expressings and expression keep embryonic stem cell proliferation ability and totipotent early transcription gene Oct4, Nanog and Telomerase.Show that these stem cells may have similar myeloid-lymphoid stem cell potentiality.

Embodiment 2: the vitro differentiation of stem cell----pancreatic

2.1 material and method

2.1-1. reagent: purchase phosphoric acid buffer (PBS) from Invitrogen company; α-minimalessential medium (α-MEM); Dulbecco modified Eagle ' s medium (DMEM); The F12 substratum, foetal calf serum (FCS); Knockout serum; Green grass or young crops/Streptomycin sulphate; Amphotericin B; L-glutamin; 0.25%Trypsin-EDTA; B27-supplement, bFGF.Purchase insulin from Sigma company, transferrin, selenium chloride, fibronectin and putrescine.The anti-specific antibody of anti-Insulin and Nestin is available from Dako (Denmark).The Insulin enzyme linked immunological kit is available from Abbott (U.S.).

2-1-2. method: the document (Lumeksky et al.2006) that people such as the method main reference Lumelsky of the directed differentiation of pancreatic deliver.This method is divided into five stages with the vitro differentiation process.In Phase I, undifferentiated stem cell is cultivated amplification.In the II stage, allow the stem cell of amplification form embryoid body.The embryoid body directed differentiation is become the cell mass of similar pancreas islet in the III stage.IV and V stage further are divided into the beta cell with function with islet cells group.Details are as follows:

2.1-2-1. frozen stem cell is taken out from liquid nitrogen container, and 37 ℃ thaw rapidly; At room temperature centrifugal 800 changeed abandoning supernatant 10 minutes; The PBS of cell precipitation with 1ml suspended again, be transferred to the aseptic centrifuge tube of 15-ml, the PBS at room temperature centrifugal 800 that adds 9ml changes washing in 10 minutes once.

2-1-2-2. abandoning supernatant; The stem cell nutrient solution of cell precipitation with 1ml suspended, be transferred to 75-cm2 culturing bottle 5%CO2 in cell culture incubator, cultivate under 37 ℃ of conditions.

2-1-2-3.4-5 after it was cultivated, cell proliferation covered the culturing bottle bottom surface of 70-90%.Nutrient solution is discarded; With the PBS of 10-20ml with twice of cell washing.The 0.25%Trypsin-EDTA that adds 5ml was then hatched 5 minutes down for 37 ℃.

2.1-2-4. with the cell transfer that the suspends aseptic centrifuge tube to 15-ml, at room temperature centrifugal 800 change and separated in 10 minutes; And once with the PBS centrifuge washing of 10ml.

2.1-2-5. the extremely low adherent 6-hole of cell transfer culture dish with washing, with embryoid body nutrient solution (80% knockout dMEM, 20% foetal calf serum, the L-glutamine of 1mM, 0.1M β-mercaptoethanol and 1% nonessential amino acid) 5%CO2 in cell culture incubator, cultivated under 37 ℃ of conditions 6-7 days; Cultivate second day these stem cell and both formed embryoid body.Changed liquid once in per 3 days.

2.1-2-6. embryoid body is transferred to the culture dish of 10-cm2, with 1: 1 DMEM/F12 nutrient solution add insulin (10mg/L)-Transferrin (6.7ng/L)-Selenium (5.5mg/L) (ITS) and the Fivronectin of the L-glutamine of 1mM and 5 μ g/L cultivated 7 days.

2.1-2-7. cell is suspended with 0.25% Trypsin-EDTA and be transferred to the 10-cm2 culture dish, add N2 with 1: 1 dMEM/F12 nutrient solution and replenish the liquid (Insulin of 500 μ g/ml, the Transferrin of 10mg/ml, 0.63 the progesterone of μ g/ml, the Selenium of the Putrascine of 1611 μ g/ml and 0.52 μ g/ml) and the B27 nutrient solution, the L-glutamine of 1mM and the bFGF of 10ng/ml form class islet cells group.Cultivated 6-7 days, changed liquid once in per 3 days.

2.1-2-8. 6-7 days, change bFGF the Nicotimide of 10mM into, and under the low sugar condition, cultivated 4 days.Islet cells group is transferred to 24 well culture plates to continue to cultivate 4 days with above-mentioned nutrient solution.

2.1-2-9. change nutrient solution into do not contain serum high sugar or low sugar nutrient solution, the pancreatic of observing differentiation changes the insulin secretion that produces to glucose concn.

2.1-2-10. carry out morphological observation with the inversion opticmicroscope.

2.1-2-11., adopt anti-Insulin of specificity and Nestin antibody to press the pancreatic that the described cellular immunization group of 1.1-8 method is identified differentiation in the differentiation V stage.

2.1-2-12. collect the nutrient solution of 2.1-2-9, the specification sheets that adopts the Insulin enzyme-linked immunologic detecting kit to press test kit detects the Insulin concentration in the nutrient solution.

2.2 result

The morphological feature that is divided into islet cells group is induced in Figure 10 demonstration from these stem cell directionals.Figure 10 A and Figure 10 B are the morphological feature of inducing the islet cells group in the III stage in the process; 100 times of Figure 10 A zoom, 400 times of Figure 10 B zoom.Figure 10 C and Figure 10 D are the morphological feature of inducing the islet cells group in the IV stage in the process; 100 times of Figure 10 C zoom, 400 times of Figure 10 D zoom.

Figure 11 shows the specificity marker thing that detects the islet cells group expression beta cell of differentiation with immunohistochemical methods.Figure 11 A is Insulin, and Figure 11 B is Nestin.200 times of zoom.

Figure 12 shows that the pancreatic group of directional induction differentiation can secrete the Regular Insulin of higher concentration to the glucose of high density.Glucose culture solution than low concentration has significant statistical significance (P=0.036).The pancreatic that these differentiation are described has the glycometabolic function of adjusting.

2.3 conclusion

The stem cell with embryonic stem cell characteristic that umbilical cord of collecting from the pregnant period 1 abortion tissue of being pregnent or intestinal tissue separation and purification go out can directional induction be divided into the pancreatic with the glycometabolic function of adjusting in vitro culture.

Embodiment 3: the vitro differentiation of stem cell----myocardial cell

3.1 material and method

3.1-1 reagent: purchase phosphoric acid buffer (PBS) from Invitrogin company; α-minimalessentialmedium (α-MEM); Dulbecco modified Eagle ' s medium (DMEM); Foetal calf serum (FCS); Knockout serum; Green grass or young crops/Streptomycin sulphate; Amphotericin B; L-glutamin; 0.25%Trypsin-EDTA; β-mercaptoethanol; Nonessential amino acid; Anti-β-myoglobulin heavy chain (β-myosin heavy chain, MHC) antibody and anti-troponin I (cardiac troponinI, cTnl) antibody is available from Chemicon (U.S.); The primer of RT-PCR: α-myosin heavy chain (myosin light chain ventricular, α-MHC) (ggggacagtggtaaaagcaa (sense), tccctgcgttccactatctt (antisense); 512bp), Nkx2.5 (ggtggagctggagaagacaga (sense), cgacgccgaagttcacgaagt (antisense); 536bp) (Passier et al.2005).Atrial natriuretic peptide (Atrial natriuretic factor, ANF) (gaaccagaggggagagacagag (sense), ccctcagcttgctttttaggag (antisense), 406bp) with cardiac myosin light chain 2v (myosin light chain 2v, MLC2v) (firstround:gcgccaactccaacgtgttct (sense); Gtgatgatgtgcaccaggttc (antisense); Nested PCR:aggaggccttcactatcatgg (sense); Gtgatgatgtgcaccaggttc (antisense); 444bp) (Mummery et al.2003).The condition of the PCR of α-MHC is 94 ℃, 1 minute, and 55 ℃, 1 minute, 72 ℃, 35 circulations in 1 minute; The condition of the PCR of Nkx2.5 is 94 ℃, 1 minute, and 57 ℃, 1 minute, 72 ℃, 35 circulations in 1 minute; The condition of the PCR of ANF is 94 ℃, 1 minute, and 53 ℃, 1 minute, 72 ℃, 35 circulations in 1 minute; MLC2v the first time PCR condition be 94 ℃, 1 minute, 62 ℃, 1 minute, 72 ℃, 35 circulations in 1 minute; The condition of nest-type PRC is 94 ℃, 1 minute, and 62 ℃, 1 minute, 72 ℃, 35 circulations in 1 minute; The condition of the PCR of β-actin is 94 ℃, 30 seconds, and 60 ℃, 1 minute, 72 ℃, 35 circulations in 1 minute; The product of PCR then adopts painted 2% agar of GOLDEN to detect, and with the RT-PCR product of β-actin as internal standard.

3.1-2 method: the document (He JQ et al.2003) that people such as the method main reference He JQ of myocardial cell's directed differentiation deliver.

3.1-2-1. frozen stem cell is taken out from liquid nitrogen container, and 37 ℃ thaw rapidly; At room temperature centrifugal 800 changeed abandoning supernatant 10 minutes; The PBS of cell precipitation with 1ml suspended again, be transferred to the aseptic centrifuge tube of 15-ml, the PBS at room temperature centrifugal 800 that adds 9ml changes washing in 10 minutes once.

3.1-2-2. abandoning supernatant; The stem cell nutrient solution of cell precipitation with 1ml suspended, be transferred to 75-cm2 culturing bottle 5%CO2 in cell culture incubator, cultivate under 37 ℃ of conditions.

3.1-2-3.4-5 after it was cultivated, cell proliferation covered the culturing bottle bottom surface of 70-90%.Nutrient solution is discarded; With the PBS of 10-20ml with twice of cell washing.The 0.25%Trypsin-EDTA that adds 5ml was then hatched 5 minutes for 37 ℃.

3.1-2-4. with the cell transfer that the suspends aseptic centrifuge tube to 15-ml, at room temperature centrifugal 800 change and separated in 10 minutes; And once with the PBS centrifuge washing of 10ml.

3.1-2-5. with the washing cell transfer to 6-hole culture dish, with the embryoid body nutrient solution (Dulbecco ' s modified Eagle ' s substratum adds 20% knockout serum, the L-glutamine of 1mM, 0.1M β-mercaptoethanol and nonessential amino acid) 5%CO2 in cell culture incubator, cultivate to form embryoid body under 37 ℃ of conditions.

3.1-2-6. cultivate after 4-6 days, the big I of embryoid body reaches 300-2000 μ m.Then embryoid body is transferred to the 10-cm2 culture dish, cultivates, change liquid every day once with differentiation culture liquid (DMEM adds 15% FCS, 2mML-glutamine and 1% nonessential amino acid).

3.1-2-7. with being inverted the morphologic variation of observation by light microscope.

3.1-2-8. immunohistochemical methods is identified: adopt β-MHC and cTnl antibody to press the myocardial cell that the described cellular immunization group of 1.1-8 method is identified differentiation.β-MHC is the structure found among the myocardial cell and the composition of contractile protein, and cTnl then be myocardium specific proteins, a kind of myocardial cell's specificity marker thing (Aubin JE.1998).

3.1-2.9.RT-PCR detect α-MHC, Nkx2.5, the expression of the mRNA of ANF and MLC2v..Nkx2.5 is the transcription factor of source capsule Qu Block together, and heart development plays an important role in early days.ANF secretes a kind of albumen of regulating dielectric balance by the myocardial cell, is myocardial cell's early sign thing.α-MHC is at the structure of myocardial cell's discovery and the composition of contraction.At the adult cardiomyocytes cell expressing.MLC2v only expresses (Segev et al.2005) the myocardial cell that contraction is arranged.

3.3 result

Figure 13 demonstration is induced the morphological feature that is divided into the myocardial cell from these stem cell directionals.100 times of Figure 13 A zoom; 200 times of Figure 13 B image multiplications.

Figure 14 shows that immunohistochemical methods detects directional induction differentiation myocardial cell expression specificity mark.Figure 14 A is β-MCH, and Figure 14 B is cTnI.200 times of zoom.

Figure 15 shows that RT-PCR detects the mRNA of myocardial cell's expression specificity mark of directional induction differentiation.

3.4 conclusion

The stem cell with embryonic stem cell characteristic that umbilical cord of collecting from the pregnant period 1 abortion tissue of being pregnent or intestinal tissue separation and purification go out can directional induction be divided into the myocardial cell in vitro culture.

Embodiment 4: the vitro differentiation of stem cell----neurocyte

4.1 material and method

4.1-1. reagent: purchase phosphoric acid buffer (PBS) from Invitrogen company; α-minimalessential medium (α-MEM); Dulbecco modified Eagle ' s medium (DMEM); The F12 substratum, foetal calf serum (FCS); Knockout serum; Green grass or young crops/Streptomycin sulphate; Amphotericin B; L-glutamin; 0.25% Trypsin-EDTA; B27-supplement, bFGF.Purchase insulin from SIGMA company, transferrin, selenium chloride, fibronectin, putrescine.Anti-neurone microtubule-associated protein-2 (mcrotubul-associated ptotein-2, MAP-2) antibody, anti-myelin basic protein (Myelin basic protein, MPB) antibody, anti-Nestin antibody and anti-beta tubulin, the antibody of β-tubulin) is available from Sigma (U.S.).The primer of RT-PCR: Bf1:(actcaaaactcgctgggcaac (sense), cgtgggggaaaaagtaactgg, 226bp) (Yan et al.2005); Noggin:(taatggatccatggagcgctgccccagcctag (sense), gcagaagcttctagcacgagcacttgcactcg (antisense), 771bp); Neurofilament protein (Neurofilament, NF): gagtgaaatggcacgataccta (sense), ttcctctccttcttcaccttc (antisense), 473bp) (Kim et al.2005).The condition of the PCR of Bf1 is 94 ℃, 30 seconds, and 57 ℃, 1 minute, 72 ℃, 35 circulations in 1 minute; The condition of the PCR of Noggin is 94 ℃, 1 minute, and 57 ℃, 1 minute, 72 ℃, 35 circulations in 2 minutes; The condition of the PCR of NF is 94 ℃, 1 minute, and 55 ℃, 1 minute, 72 ℃, 35 circulations in 1 minute.

4.1-2. method:

4.1-2-1. frozen stem cell is taken out from liquid nitrogen container, and 37 ℃ thaw rapidly; At room temperature centrifugal 800 changeed abandoning supernatant 10 minutes; The PBS of cell precipitation with 1ml suspended again, be transferred to the aseptic centrifuge tube of 15-ml, the PBS at room temperature centrifugal 800 that adds 9ml changes washing in 10 minutes once.

4.1-2-2. abandoning supernatant; The stem cell nutrient solution of cell precipitation with 1ml suspended, be transferred to 75-cm2 culturing bottle 5%CO2 in cell culture incubator, cultivate under 37 ℃ of conditions.

4.1-2-3.4-5 after it was cultivated, cell proliferation covered the culturing bottle bottom surface of 70-90%.Nutrient solution is discarded; With the PBS of 10-20ml with twice of cell washing.The 0.25%Trypsin-EDTA that adds 5ml was then hatched 5 minutes for 37 ℃.

4.1-2-4. with the cell transfer that the suspends aseptic centrifuge tube to 15-ml, at room temperature centrifugal 800 change and separated in 10 minutes; And once with the PBS centrifuge washing of 10ml.

4.1-2-5. with the washing cell transfer to 6-hole culture dish, with the embryoid body nutrient solution (Dulbecco ' s modified Eagle ' s substratum adds 20% knockout serum, the L-glutamine of 1mM, 0.1M β-mercaptoethanol and nonessential amino acid) 5%CO2 in cell culture incubator, cultivate to form embryoid body under 37 ℃ of conditions.

4.1-2-6. embryoid body is transferred to the 10cm2 culture dish, in the DMEM-10%FCS nutrient solution, allows embryoid body launch.

4.1-2-7. changed nutrient solution into contain ITSFn (insulin 5 μ g/ml, transferring 50 μ g/ml, selenium chloride 30nM, fibronectin 5 μ g/ml) DMEM/F12 (1: 1) nutrient solution in second day, cultivated 6-8 days; Changed a not good liquor in per two days.To select the Nestin positive cell.

4.1-2-8. cell is suspended with 0.25%Trypsin-EDTA, washing; Be transferred to another 10cm2 culture dish then, with containing the DMEM/F12 nutrient solution of N2 and bFGF with propagation Nestin positive cell.

4.1-2-9. be divided into the morphology of neurocyte with the inversion observation by light microscope.

4.1-2-10. adopt the anti-MAP-2 of specificity, MPB, Nestin and β-tubulin antibody press the neurocyte (Aubin JE.1998) that the described cellular immunization group of 1.1-8 method is identified differentiation.MAP-2 is tree-shaped prominent neurocyte specificity neurone microtubule-associated protein; MPB is by the myelin basic protein that breaks up oligodendrocyte (oligodendrocytes) expression completely; Nestin is the intermediate filament protein (Intermediate filament structural protein) of neural group cell expressing; β-tubulin is the important structural protein of neurone, is the mark of neurone differentiation.

4.1-2-11.RT-PCR the neurocyte that breaks up is identified in detection genetic expression.Noggin is the neurocyte specific gene of expressing in neuronal development process.NF is the important structural protein of neurocyte, in order to identify that the neurocyte .Bf1 that has broken up is the transcription factor of forebrain cell expressing.

4.3 result

Figure 16 demonstration is induced the morphological feature that is divided into neurocyte from these stem cell directionals.100 times of Figure 16 A zoom, 400 times of Figure 16 B zoom..

Figure 17 shows that immunohistochemical methods detects the specificity marker thing (Nestin (A), β-Tubulin (B), MAP-2 (C) and MPB (D)) of inducing the differentiation neurocyte from these stem cell directionals, 200 times of zoom.

Figure 18 shows that RT-PCR detects the mRNA of the specificity marker thing of (12 days, 24 days and 36 days) when being divided into neurocyte directional induction differentiation neurocyte.

4.4 conclusion

The stem cell with embryonic stem cell characteristic that umbilical cord of collecting from the pregnant period 1 abortion tissue of being pregnent or intestinal tissue separation and purification go out can directional induction be divided into neurocyte in vitro culture.

Embodiment 5: vitro culture differentiation----osteocyte of stem cell

5.1 material and method

5.1-1. reagent: purchase phosphoric acid buffer (PBS) from Invitrogen company; α-minimalessential medium (α-MEM); Dulbecco modified Eagle ' s medium (DMEM); Foetal calf serum (FCS); 0.25%Trypsin-EDTA; Green grass or young crops/Streptomycin sulphate; Amphotericin B; Knockout serum; L-glutamin; Nonessential amino acid and β-mercaptoethanol; Purchase L-asorbic acid (AA) from Sigma company; Dexamethasome (DEX); Hexamethyldisilazane (HMDS) and sodium-β-glycerophosphate (β gp); The vonKossa staining kit is available from Polysciences Inc (U.S.), and anti-Bone Gla protein (osteocacin, OCN) monoclonal antibody is available from R﹠amp; D System (U.S.).

5.1-2. method: the document (Karp et al.2006) that people such as the method main reference Karp JM of the directed differentiation of osseous tissue deliver.

5.1-2-1. frozen stem cell is taken out from liquid nitrogen container, and 37 ℃ thaw rapidly; At room temperature centrifugal 800 change, and 10 minutes, abandoning supernatant; Cell precipitation is suspended with the PBS of 1ml again, be transferred to the aseptic centrifuge tube of 15-ml, the PBS that adds 9ml is centrifuge washing once (800 change 10 minutes) at room temperature.

5.1-2-2. abandoning supernatant; The stem cell nutrient solution of cell precipitation with 1ml suspended, be transferred to 75-cm2 culturing bottle 5%CO2 in cell culture incubator, cultivate under 37 ℃ of conditions.

5.1-2-3.4-5 after it was cultivated, cell proliferation covered the culturing bottle bottom surface of 70-90%.Nutrient solution is discarded; With the PBS of 10-20ml with twice of cell washing.The 0.25%Trypsin-EDTA that adds 5ml was then hatched 5 minutes for 37 ℃.

5.1-2-4. with the cell transfer that the suspends aseptic centrifuge tube to 15-ml, at room temperature centrifugal 800 change and separated in 10 minutes; And once with the PBS centrifuge washing of 10ml.

5.1-2-5. the extremely low adherent 6-hole of cell transfer culture dish with washing, (the DMEM substratum adds 20% knockout serum with the embryoid body nutrient solution, the L-glutamine of 1mM, 0.1M β-mercaptoethanol and 1% nonessential amino acid) 5%CO2 in cell culture incubator, cultivate to form embryoid body under 37 ℃ of conditions.

5.1-2-6. cultivate after 4-6 days, the big I of embryoid body reaches 300-2000 μ m.Then embryoid body is transferred to the 10-cm2 culture dish, cultivates, changed liquid once in every 2-3 days with differentiation culture liquid (α-MEM adds 10% FCS, the DEX of 10-8M, the β gp of the AA of 50 μ g/ml and 5mM).

5.1-2-7. with being inverted the morphologic variation of observation by light microscope: have or not the zone that mineralizes in the differentiated tissues.

5.1-2-8. histology is identified: adopt von Kossa dyeing to determine the formation (PurpuraKA et al.2004) of bone brief summary.

5-1-2-9. immunohistochemical methods is identified: adopt the OCN specific antibody to press the described cellular immunization group of 1.1-8 method and identify the formation (Aubin JE.1998) of determining osseous tissue.

5.2 result

Adding about the 14th day that differentiation culture liquid is cultivated, and be differentiated to form osteocyte with being inverted the about embryoid body more than 90% of observation by light microscope.Figure 19 demonstration is induced the osteoblastic morphological feature of differentiation from these stem cell directionals.Figure 19 A and B are at the morphological feature of cultivating the 14th day osteocyte, 400 times of Figure 19 A zoom; Figure 19 B amplifies 100 times.Figure 19 C and D are at the morphological feature of cultivating the 22nd day osteocyte; 100 times of zoom.The zone mineralizes in the visible differentiated tissues of microscopically.Break up the 22nd day osseous tissue than the 14th day bigger of differentiation, the zone that mineralizes is also more obvious.

The osteocyte that adopts von kassa dyeing to show that directional induction was broken up the 22nd day has formation (Figure 20,100 times of Figure 20 A zoom of bone brief summary; 200 times of Figure 20 B zoom).Figure 21 shows that then immunohistochemical methods detects the expression that osteocyte that directional induction broke up the 22nd day has OCN.OCN is osteogenetic later stage mark; Consistent with mineralizing of osteocyte.100 times of Figure 21 A image multiplications; 200 times of Figure 21 B image multiplications.

5.3 conclusion

The stem cell with embryonic stem cell characteristic that umbilical cord of collecting from the pregnant period 1 abortion tissue of being pregnent or intestinal tissue separation and purification go out can directional induction be divided into osteocyte in vitro culture.

The foundation in embodiment 6 myeloid-lymphoid stem cell storehouses

1, the fetal placental tissue sample that will build the pregnant period 1 miscarriage of collecting in the storehouse is carried out various communicable diseases and comprise HIV, HBC, HCV, HTLV etc. detect according to a conventional method;

2, according to method separation and purification stem cell from different fetal placental tissues of embodiment 1;

3, the stem cell to separation and purification carries out the karyomit(e) detection according to a conventional method;

4, the method for employing MTT detects the stem cell strain activity of separation and purification;

5, the method that adopts PCR is to the evaluation of classifying of the histocompatibility antigen type of each stem cell strain;

6, set up the database of frozen stem cell with above-mentioned classification appraising datum;

7, according to the stem cell strain of the frozen separation and purification of method of embodiment 1.

Method of the present invention can be more easily from the fetal tissue of pregnant first phase artificial abortion successfully separation and purification go out myeloid-lymphoid stem cell and the stable undifferentiated state that keeps myeloid-lymphoid stem cell, and then set up the myeloid-lymphoid stem cell storehouse, for the damaged and handicapped disease of the various human tissue organs of cellular replacement therapy, provide a kind of new way as Parkinson's disease, diabetes, traumatic spinal cord injury, cytopathy disease, burn, myocardosis and bone injury etc.

[reference]

(1)Anker?PS，Noort?WA，Scherjon?SA，et?al.Mesenchymal?stem?cellsin?human?second-trimester?bone?marrow，liver，lung，and?spleen?exhibita?similar?immunophenotype?but?a?heterogeneous?multilineagedifferentiation?potential.Haematologica?2003；88：845-852.

(2)Aubin?JE.Bone?stem?cells.J?Cell?Biochem?Suppl?1998；30-31：73-82.Bajada?S，Mazakova?I，Richardson?JB，Ashammakhi?N.Updates?on?stem?cellsand?their?applications?in?regenerative?medicine.J?Tissue?Eng?Regen?Med.2008；2：169-83.

(3)Beattie?GM，Lopez?AD，Bucay?N，Hinton?A，Firpo?M，King?CC，HayekA.Activin?A?a?maintains?pluripotency?of?human?embryonic?stem?cellsin?the?absence?og?feeder?layers.Stem?Cells?2005；23：489-95.Bullock?GR，Petrusz?P：Techniques?in?Immunocytochemistry，AcademicPress，New?York，1982.

(4)Chan?S，Harris?J.Adam’s?fibroblast？The(pluri)potential?of?iPCs.J?Med?Ethics.2008；34：64-6.

(5)Durcova?G，yamaguchi?M，takahashi?S，Imai?H.Immunomagneticisolation?of?mouse?embryonic?stem?cells?from?heterogeneous?cellspopulation.J?Reprod?Devel?1998；44：85-9.

(6)Guillot?PV，Cui?W，Fisk?NM，Polak?DJ.Stem?cell?differentiationand?expansion?for?clinical?applications?of?tissue?engineering.J?CellMol?Med.2007；11：935-44.

(7)He?JQ，Ma?Y，Lee?Y，Thomson?JA，Kamp?T.Human?embryonic?stem?cellsdevelop?into?multiple?types?of?cardiac?myocytes：action?potentialcharacterization.Circ?Res?2003；93：32-39.

(8)Javois?LC(edt).Immunocytochemistry?methods?and?protocols.2ndedition.1999；Human?Press.

(9)Karp?JM，Ferreira?LS，Khademhosseinin?A，Kwon?AH，Yeh?J，LangerRS.Cultivation?of?human?embryonic?stem?cells?without?the?embryoid?bodystep?enhances?osteogenesis?in?vitro.Stem?Cells?2006；24：835-43.

(10)Kim?JH，Do?HJ，Choi?SJ，Cho?HJ，Park?KH，Yang?HM，Lee?SH，KimDK，Kwack?KB，Oh?SK，Moon?SY，Cha?KY，Chung?HM.Efficient?gene?deliveryin?differentiated?human?embryonic?stem?cells.Exp?Mol?Med.2005；37：36-44.

(11)Lee?OK，Kuo?TK，Chen?WM，et?al.Isolation?of?multipotentmesenchymal?stem?cells?from?umbilical?cord?blood.Blood，2004；103：1669-1675.

(12)Lumelsky?N，Blondel?O，Laeng?P，Velasco?I，Ravin?R，McKay?R.Differentiation?of?embryonic?stem?cells?to?insulin-secretingstructures?similar?to?pancreatic?islets.Science?2001；1389-94.

(13)McElreavey?KD，Irvine?AI，Ennis?KT?et?al.Isolation，culture?andcharacterization?of?fibroblast-like?cells?derived?from?the?Wharton’sjelly?portion?of?human?umbilical?cord.Biochem?Soc?Trans?1991；19：29S.

(14)Mitchell?KE，Weiss?ML，Mitchell?BM.Matrix?cells?from?Wharton’sjelly?form?neurons?and?glia.Stem?Cells?2003；21：50-60.

(15)Mummery?C，Oostwaard?DW，Doevendans?P，Spijker?R，van?den?BrinkS，Hassink?R，van?der?Heyden?M，Opthof?T，Pera?M，de?la?Riviere?AB，Passier?R，Tertoolen?L.Differentiation?of?human?embryonic?stem?cellto?cardiomyocytes：role?of?coculture?with?visceral?endoderm-like?cells.Circulation?2002；107：2733-40.

(16)Passier?R，Oostwaard?DW，Snapper?J，Kloots?J，Hassink?RJ，KuijkE，Roelen?B，de?la?Riviere?AB，Mummery?C.Increase?cardiomycytedifferentiation?from?human?embryonic?stem?cells?in?serum-freecultures.Stem?Cells?2005；23：772-780.

(17)Pera?MF，Trounson?AO.Human?embryonic?stem?cells：prospects?fordevelopment.Development?2004；131：5515-25.

(18)Purpura?KA，Aubin?JE，Zandstra?PW.Sustained?in?vitro?expansionof?bone?progenitors?is?cell?density?dependent.Stem?Cells?2004；22：39-50.

(19)Romanov?YA，Svintsitskaya?VA，Smirnov?VN.Searching?foralternative?sources?of?postnatal?human?mesenchymal?stem?cells：candidate?MSC-Like?cells?from?umbilical?cord.Stem?Cells?2003；21：105-110.

(20)Rogers?I，Casper?RF.Stem?cells：you?can’t?tell?a?cell?by?its?cover.Hum?Reprod?Update.2003；9：25-33.

(21)Segev?H，Kenyagin-Karsenti?D，Fishman?B，Gerecht-Nir?S，ZiskindA，Amit?M，Coleman?R，Itskovitz-Eldor?J.Molecular?analysis?ofcardiomyocytes?derived?from?human?embryonic?stem?cells.Develop?GrowthDiffer?2005；47：295-306.

(22)Takahashi?K，Yamanaka?S.Induction?of?pluripotent?stem?cells?frommouse?embryonic?and?adult?fibroblast?cultures?by?defined?factors.Cell.2006；126：663-76.

(23)Wang?HS，Hung?SC，Peng?ST，et?al.Mesenchymal?stem?cells?in?theWharton’s?jelly?of?the?human?umbilical?cord.Stem?Cells?2004；22：1330-1337.

(24)Yan?YP，Yang?DL，Zarnowska?ED，Du?ZW，Werbel?B，Valliere?C，PearceRA，Thomson?JA，Zhang?SC.Directed?differentiation?of?dopaminergicneuronal?subtypes?from?human?embryonic?cells.Stem?Cells2005；23：781-90.

(25)Young?HE，Black?AC?Jr.Adult?stem?cells.Anat?Rec?A?Discov?MolCell?Evol?Biol.2004；276：75-102.

(26)Yu?J，Vodyanik?MA，Smuga-Otto?K，Antosiewicz-Bourget?J，FraneJL，Tian?S，Nie?J，Jonsdottir?GA，Ruotti?V，Stewart?R，Slukvin?II，Thomson?JA.Induced?pluripotent?stem?cell?lines?derived?from?humansomatic?cells.Science.2007；318(5858)：1917-20.

Claims (19)

1, a kind of separation and purification comprises from the method for the myeloid-lymphoid stem cell of people's early abortion fetal tissue: the fetal placental tissue sample of a, the period 1 miscarriage of collection gestation; B, from the sample of step a, separate to obtain fetal cord or intestinal tissue; C, from the fetal cord of step b or intestinal tissue, obtain fetal cord or intestinal tissue cell; Embryonic stem cell mark OCT-4, SSEA-3, SSEA-4, the stem cell of TRA-1-60 and TRA-1-81 are expressed in separation and purification d, the fetal cord that obtains from step c or the intestinal tissue cell.

2, method according to claim 1 also comprises: the purifying stem cell that e, frozen steps d obtain; F, the frozen purifying stem cell of recovery.

3, method according to claim 1 is characterized in that: the described gestation of step a period 1 aborted fetus is organized as the fetal placental tissue of gestation 8 thoughtful 12 all artificial abortion or spontaneous abortion.

4, method according to claim 1 is characterized in that: among the step c, earlier fetal cord or intestinal tissue are washed with the physiological saline of sterilizing, be cut into small pieces again, with collagenase digesting to cellular constituent behind state, centrifugal acquisition cell precipitation, standby with physiological saline washing.

5, method according to claim 1, it is characterized in that: in the steps d, to express OCT-4 with the immunomagnetic beads isolation technique earlier, SSEA-3, SSEA-4, the stem cell purifying of TRA-1-60 and/or TRA-1-81, the stem cell with wash-out is transferred to the cell cultures flask culture again, obtains the stem cell of purifying.

6, method according to claim 1 is characterized in that: in the steps d, fetal cord or intestinal tissue cell that step c is obtained suspend with the stem cell nutrient solution, shift to cultivate and go down to posterity, and culture condition is, at 37 ℃ and 5%CO ₂Condition under cultivated 3-7 days or cell proliferation accounts for culturing bottle or more than 70% of culture dish floorage, shift to go down to posterity at least 5 times, obtain the stem cell of purifying.

7, method according to claim 6, it is characterized in that: described stem cell nutrient solution is by the α-MEM substratum of volumn concentration 88%, the bFGF of 10% foetal calf serum or 0.1 μ g/ml, 100X green grass or young crops/Streptomycin sulphate of 1%, the plain B of 1% 100X two property enzymes forms.

8, method according to claim 6 is characterized in that: shift when going down to posterity, cell culture fluid is discarded, use the phosphoric acid buffer washed cell; Stick on the cell suspension of culturing bottle bottom again in the time of will growing with trypsinase-EDTA solution, centrifugation obtains cell under the room temperature; , and about 10% cell transfer to another Tissue Culture Flask continued to cultivate with cell suspension with the stem cell nutrient solution.

9, according to claim 5 or 6 described methods, it is characterized in that: described purifying stem cell can form embryoid body.

10, method according to claim 2 is characterized in that: during frozen stem cell, earlier with the suspend cell of centrifugation in the process that goes down to posterity of stem cell stock solution, being transferred to keep in cold storage pipe and placement then and spending the night for-70 ℃, transferring to the liquid nitrogen container prolonged preservation; Described stem cell stock solution is by the foetal calf serum of volumn concentration 40%, and 50% α-MEM substratum and 10% DMSO form.

11, method according to claim 2 is characterized in that: during the recovery stem cell, the pipe that keeps in cold storage that stem cell is housed is taken out from liquid nitrogen container, be placed on 37 ℃ and thaw; Centrifugation then, and with phosphoric acid buffer washing, the cell transfer that will thaw is to culturing bottle again, with the stem cell nutrient solution at 5% CO ₂With cultivated 24 hours under 37 ℃ the condition, replace fresh nutrient solution continuation at 5% CO ₂With cultivate under 37 ℃ the condition, and every 3-5 days renew bright nutrient solution once.

12, a kind of myeloid-lymphoid stem cell from people's early abortion fetal tissue is obtained by the described method separation and purification of claim 1.

13, stem cell according to claim 12, the embryonic stem cell mark of its expression is OCT-4, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81.

14, stem cell according to claim 12, it expresses one or more embryonic stem cell idiosyncratic transcription factors.

15, stem cell according to claim 14, the embryonic stem cell idiosyncratic transcription factor of its expression is OCT-4, Nanog or Telemerase.

16, stem cell according to claim 12, it can form embryoid body.

17, the described stem cell of claim 12, it is induced to differentiate into the purposes of pancreatic, myocardial cell, neurocyte, osteocyte.

18, a kind of method of setting up the myeloid-lymphoid stem cell storehouse comprises:

A, the fetal placental tissue sample of the early abortion of collecting is done necessary loimology inspection;

B, separation and purification stem cell from different fetal placental tissues in accordance with the method for claim 1;

The evaluation of classifying of the histocompatibility antigen of c, stem cell strain that step b is obtained;

D, set up stem cell strain catalogue according to the classification qualification result of step c;

E, the stem cell strain that step b is obtained keep in cold storage by the described method of claim 10.

19, a kind of myeloid-lymphoid stem cell storehouse of setting up by the method for claim 18.

Images