CN101775368A - Human embryonic stem cell line with homozygous androgen sites on X chromosomes - Google Patents

Abstract

The invention relates to a human embryonic stem cell line with homozygous androgen sites on X chromosomes. The human embryonic stem cell line is characterized in that: the average colony multiplication time of the embryonic stem cell line is about 32 hours; undifferentiated clone AKP of the embryonic stem cell line is detected to be strong positive and has high alkaline phosphatase activity; the embryonic stem cell line can express specific surface antigens SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 and does not express a surface antigen SSEA-1; the embryonic stem cell line can express an Oct-4 transcription factor and keeps undifferentiated property; the embryonic stem cell line is subjected to once karyotype analysis every other 10 generations and has a normal karyotype; the embryonic stem cell line has two X chromosomes; the deactivation analysis of the X chromosomes shows that the androgen sites on the X chromosomes are homozygous; and the embryonic stem cell line has a unique short tandem repeat sequence.

Description

Human embryonic stem cell with homozygous androgen sites on the X chromosome

Technical field

The present invention relates to zygote source human embryo stem cell, specifically relate to a kind of human embryonic stem cell with homozygous androgen sites on the X chromosome.

Background technology

HESC (human embryonic stem cell, hES) be inner cell mass (inner cell mass by blastaea, ICM) separation and Culture and come, cell with totipotential differentiation properties, have self and multidirectional differentiation potential, have the meaning of being sought after in fields such as cell therapy, fetal development, drug screening, gene therapy and heredity, epigenetic Mechanism Study.HESC research starts from 1998, and it has become after the Human Genome Project most active research field in the life science.In recent years, the separation and purification of embryonic stem cell, performance study, cultivation amplification, induce differentiation etc. that breakthrough has been arranged.1998, Univ Wisconsin-Madison USA Thomson laboratory utilizes the fresh or refrigerated blastaea of 36 pieces of infertile Mr. and Mrs' donations of clinical treatment, successfully separate, set up 5 strain hES clone (Thomson JA first, Itskovitz Eldor J, Shapiro SS, et al.Embryonic stem celllines derived from human blastocysts.Science, 1998,282:1145-1147).2000, Australia Monash University cooperates with the genocentric expert of Singapore university, has successfully set up the undifferentiated hES clone of 2 strains (Reubinoff BE et al.Embryonic stem cell lines from human blastocysts:somatic differentiation invitro.Nat Biotechnol.2000 Apr from people's blastaea; 18 (4): 399-404.).Domestic after 2000, medical research units such as Guangzhou, Hunan, Beijing, Shanghai have begun the research of embryonic stem cell in succession.2002, Zhongshan Medical Univ. reported once that they used the blastaea in 5 routine people's zygote sources tentatively to build up 3 embryonic stem cell strain (He Zhixu etc.The preliminary foundation of human embryonic stem cell. Chinese Medical Journal, 2002,82:1314).2004, the scientist of Japan and Britain has also obtained hES clone (Suemori H etal.Establishment of human embryonic stem cell lines and their therapeuticapplication.Rinsho Byori, 2004,52:254; Stojkovic M et al.Derivation ofhuman embryonic stem cells from day-8 blastocysts recovered afterthree-step in vitro culture.Stem Cells.2004; 22 (5): 790-7).Having a laboratory surplus the U.S., Britain, Singapore, Australia, Sweden, Japan, China, the Korea S etc. 20 to have relevant hES cell that 400 strain left and right sides hES cells publish so far in the world builds the treatise that is more than 300 piece of (Guhr A is arranged, Kurtz A, et al.Current state of human embryonic stem cell research; Anoverview of cell lines and their use in experimental work.Stem Cells.2006Oct; 24 (10): 2187-91).The people ES cell strain of NIH registration surpasses strain more than 60 till settled the present.

The hES cell can keep undifferentiated state and unlimited multiplication capacity under external conditions suitable, also can directional induction be divided into the cell of almost all kinds.Because stem cell has above feature, can become the source of human cell or organ transplantation in theory, for human treatment of diseases has been brought new hope.Nearly 10 years development, the hES cell culture technology is increasingly mature, for the Clinical Application of stem cell provides possibility.Utilize the hES cytothesis or substitute that damaged and dysfunction is the epoch-making innovation of human disease treatment pattern because of the human tissue organ that various factors caused.The hES cell that is used for the cell replacement treatment must possess security, validity, immunity exemption property three big characteristics, thereby lot of domestic and international research concentrates on following problem: (1) purifying hES cell culture environment, avoid the pollution of animal derived material; (2) the hES cell induction is divided into various one-tenth somatocyte and precursor cell and transplanting; (3) foundation of body-cell neucleus transplanting and ntES clone; (4) foundation of human parthenogenetic embryo stem cell line; (5) epigenetics of hES cell changes, as the detection of imprinted gene and methylation level, x chromosome inactivation etc.; (6) hES cell and gene therapy.

Chinese people embryonic stem cell-like (hES) is built the source and the cultural method that are and is mainly contained following several:

(1), the main source of hES system is blastaea in vitro fertilization, the hES system of nuclear transplantation embryo and early stage morula of growing and people's parthenogenetic embryo tire separation and Culture.

The blastaea method

(Inner cell m ass ICM) is the most frequently used material that separates hES to the blastaea inner cell mass.To blastaea, separation and Culture goes out ICM with the embryo culture of the embryo of artificial insemination or normal internal fertilization, and further the ICM of separation and Culture propagation obtains the hES cell colony, the propagation that goes down to posterity and detect after promptly obtain hES clone.The hES system that is set up at present obtains (Baharvand H by the artificial ICM separation and Culture that is cultured to blastula stage in vitro fertilization, A sh tiani S K, V alo jerdiM R, et al.Establishment and in v itro differentiation of a new embryonic stem cell linefrom human blastocyst[J] .D ifferentiation, 2004,72 (5): 224-229.; Verlinsky Y, Strelchenko N, Kukharenko V, et al.Human embryonic stemcell lines w ith genetic diso rders[J] .Rep rod Biomed Online, 2005,10 (1): 105-110.).Its concrete technological line is: blastaea → take off zona pellucida, (Tyrode acid or chain protease or mechanical process) → go trophocyte, the ICM of the ICM → discrete propagation that increases on the feeder layer of the ICM of (immunosurgery method or enzyme digestion) → purifying → suitable be minicell agglomerate → renewed vaccination cultivating on the feeder layer → discrete hES cell colony be minicell agglomerate → be seeded in feeder layer upload be commissioned to train foster → a plurality of hES sample colonies → on feeder layer occur or do not have feeder layer enlarged culturing → identify → set up hES system.The separation of ICM is a big difficult point.The method of separating ICM mainly contains two kinds of immunosurgery method and mechanical process.Present most of hES clone all is to adopt the immunosurgery method, with special antibody, and the monoclonal antibody of BeWO cell for example, anti-people's whole serum, perhaps erythrocyte antibody (EA) is isolated the ICM cell from blastaea.(major histocompatibilitycomplex MHC) in conjunction with forming immunocomplex, can cause the trophocyte that immune lysis takes place when complement exists, thereby obtain the ICM cell major histocompatibility antigen on these antibody and trophocyte surface.But there is animal derived pollution problem in the immunosurgery method, exists to a great extent to propagate animal epidemic and produce the heteroimmunity risk of rejection.Reduce animal derived pollution as far as possible, improve the condition of in vitro culture of embryonic stem cell, the method for setting up more effective easier acquisition embryonic stem cell has become the emphasis of various countries' research now.

(2), set up and keep the condition of hES system

Feeder layer

Feeder layer is a prerequisite of setting up and keep ES clone.Major part has been separated the people hES clone that obtains and has all been set up under the feeder layer condition and keep.There are some researches show that (Mouse embryonic f ibroblast is effective and feasible (Daylon J, A riel J L, Daniel B, et al. as the feeder layer of setting up hES system MEF) to mouse embryo fibroblasts Nodal signaling isnecessary fo r themaintenance of p luripo tency in human embryonic stemcells[J] .Development, 2005,132:1273-1282.).Because MEF is the allos cell, cultivates hES with it and have the danger that spreads disease substance and cause foreign protein to pollute.Therefore, people are attempting substituting MEF with other feeder layers, as human embryonic fibroblast (RichardsM, Fong C Y, ChanW K, et al.Human feeders suppo rt p ro longed undifferentiated grow th of humaninner cell masses and embryonic stem cells[J] .N at Bio techno I, 2002,20 (9): 933-936.), foreskin inoblast (Prow se B, M cQ uade L R, BryantK J, et al.A p ro teome analysis of conditioned media from human neonatalfibroblasts used in the maintenance of human embryonic stem cells[J] .Proteom ics, 2005,5 (4): 978-989.) and commercial adult and fetal fibroblast (RichardsM, Tan S, Fong C Y, et al.Comparative evaluation of varioushuman feeders for pro-longed undifferentiated growth of humanembryonic stem cells[J] .Stem Cells, 2003,21:546-556.).People source feeder layer cells can substitute MEF to a certain extent, as the feeder layer cells of hES.The artificial rearing confluent monolayer cells external can continuous passage, not only can save the plenty of time, and the MEF than different batches is stable relatively to make feeder layer with same material than MEF.In addition, there are danger such as foreign protein pollution in the time of also can avoiding with STO or MEF cultivation hES.

Substratum is formed

The substratum of hES system is formed the general high sugared DM EM nutrient solution that adopts, and adds 15%～20% foetal calf serum (FBS) or blood serum substituting product (SR) and some other micro-nutrient composition (as non-essential amino acid, glutamine etc.), antioxidant (as 2 mercaptoethanols (2-M E)) and microbiotic etc.Most researchs are thought, Prostatropin (bFGF), FBS or SR have vital role (Jo s é l in hES cultivates, Karin, M arie G M A, et al.Derivation of human embryonicstem cell lines in serum replacement medium using postnatal humanfibroblasts as feeder cells[J] .Stem Cells, 2005,23:544-549.).Most researchs think, bFGF is a hES self and to keep undifferentiated state necessary.At present, the hES research emphasis is to screen and makes embryonic stem cell remain on the best culture system of undifferentiated state; Whether the embryonic stem cell biological characteristics that check obtains is stable.

The biological characteristics and the authentication method of hES system

The hES volume is little, and nuclear is big and obvious, one or more kernels is arranged, the nuclear-cytoplasmic ratio height.The hES cell is the growth of colony shape when vitro culture, be loose relatively, flats colony, and colony inner cell boundary is may be seen indistinctly.HES has normal stable diploid caryogram and banding pattern.The alkaline phosphatase activities of hES cell is positive, and the cell that has broken up is weak positive or negative.The human body early embryo phasic specificity antigen that hES expresses has: SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81.HESCs also expresses key protein (the Lee J Bs relevant with versatility such as Oct4, Fgf4, H2az, Foxd3, Nanog and Sox2, Kim J M, Kim S J, et al.Comparative characteristics of three human embryonic stem cell lines[J] .Mo I Cells, 2005,19 (1): 31-38.).In addition, hES has high telomerase activation and to the potential of 3 germinal layer cytodifferentiation.The biological characteristics of hES is self and omnidirectional's differentiation.

HES molecular genetic, epigenetics research

Genomic imprinting is some allelic epigenetic modification, and is relevant with abnormal development of fetus and tumor growth.At the ontogeny different times, genomic imprinting state difference.There are some researches show, unusual or unstable (the Kirn JH of mouse embryonic stem cell genomic imprinting, Auerbach JM, Rodriguez2Gomez JAet al.Dopamine neurons derived from embryonic stem cells function in ananimal of Parkinson ' s disease[J] .Nature, 2002,418 (6893): 50256.); But another group investigator then genomic imprinting of finder and mouse EG cell is normal substantially; this may get rid of a big obstacle (Humpherys D for the embryonic stem cell clinical application; Eggan K; Akutsu et al.Epigenetic instability in ES cells and cloned mice[J] .Science; 2001,293 (5527): 95297).Imprinted gene, epigenetics such as methylate are changed at hES cell (Bo WenSun, A.Cong Yang et al.Temporal and parental-specific expression ofimprinted genes in a newly derived Chinese human embryonic stem cell lineand embryoid bodies Human Molecular Genetics, 2006, Vol.15, No.1 65-75) research has become now various countries at present at the focus of embryonic stem cell area research.Molecular genetics is also significant to the research of embryonic stem cell at aspects such as caryogram variation, homozygote variation, homology exchanges.

X chromosome inactivation was an integral part of epigenetic mechanism, and this phenomenon has just had the LYON hypothesis in 1961, that is: have only an X chromosome activity to be arranged, another inactivation and pyknosis in (1) female mammal cell; (2) inactivation occurs in embryo's early stage (blastula stage); (3) inactivation is at random, and promptly the X chromosome of inactivation both can also can be from mother from father, in case a cell bar X chromosome inactivation, the daughter cell that is come by this cells multiply all has the X chromosome of same inactivation.

The selection and the startup of x chromosome inactivation occur in blastula stage, this process is controlled by X inactivation centre (XIC), XIC is a cis acting site, comprise information and the XIST gene of distinguishing the X chromosome number, the former can guarantee only to have a karyomit(e) that activity is arranged, but mechanism is not clear, and XIST genetically deficient will cause the x chromosome inactivation failure.The process of x chromosome inactivation is: XIST genes encoding XISTRNA, after transcribing, it is wrapped on the X chromosome that synthesizes it, XISTRNA accumulates on X chromosome and constantly expansion subsequently, and following a pair of gene silencing has the factor of critical function to be recruited again, and comprises PCG albumen EED and ENX1.These albumen are formed temporary transient site on the X chromosome of inactivation, inducing DNA methylates and the generation of histone modification immediately, and this is to the foundation of x chromosome inactivation and keep important effect.The karyomit(e) of inactivation still continues synthetic XISTRNA, keeps the inactivated state of itself.

The human genome DNA has 3 * 10 ⁹Bp, wherein 10% is tandem repetitive sequence, is called satellite DNA.Press the length of repeated fragment, can be divided into large satellite, middle satellite, moonlet and microsatellite sequence again.Wherein repeated fragment is little satellite that is called of 2-7bp composition, be called STR (Short TandemRepeat again, be called for short STR), the number of different human body genome satellite DNA repeating unit is variable, therefore forms extremely complicated allelotrope fragment length polymorphism.It is generally acknowledged that the average per 6～10kb of Human genome just has 1 str locus seat, the abundant source of high information gene seat is provided for legal medical expert individual identification and paternity test.In practice, unite and use a plurality of STR can produce hundreds of millions of genotype combinations, and that each is combined in the frequency that occurs in the colony is all very low, can on high probability level very, assert the suspect and carry out sibship and identify.Therefore, the PCR somatotype of str locus seat is because of its highly sensitive, and high efficiency practical characteristics have become domestic and international legal medical expert's individual recognition and the topmost technology of paternity test at present.

Summary of the invention

The object of the present invention is to provide a kind of human embryonic stem cell with homozygous androgen sites on the X chromosome, this human embryonic stem cell has two X chromosomes, and androgen sites is a homozygosity on its X chromosome, and have unique STR (STR), this type of cell and derivative thereof have the feature of unique identification in potential is used from now on.

Human embryonic stem cell with homozygous androgen sites on the X chromosome of the present invention is characterized in that:

(a) the average population doubling time of described embryonic stem cell line is about 32 hours;

(b) the not differentiation of described embryonic stem cell line clone alkaline phosphatase is that AKP detects to strong positive, has high alkaline phosphatase activity;

(c) described embryonic stem cell line can be expressed one of following specific surfaces antigen: phasic specificity antigen-3 is SSEA-3, phasic specificity antigen-4 is SSEA-4, tumor response antigen-1-60 is TRA-1-60, and tumor response antigen-1-81 is TRA-1-81, and expression phase specific antigens-1 is not SSEA-1;

(d) to express eight aggressiveness be the Oct-4 transcription factor in conjunction with transcription factor 4 to described embryonic stem cell line, keeps not differentiation characteristic;

(e) described embryonic stem cell line carries out a karyotyping every 10 generations, has normal karyotype;

(f) described embryonic stem cell line has two X chromosomes, and the x chromosome inactivation analysis shows that androgen sites is a homozygosity on its X chromosome;

(g) described embryonic stem cell line has unique STR.

Among the present invention, chosen the state that human androgen receptor's gene (Human androgen receptor gene) comes the Analysis of X x chromosome inactivation.Human androgen receptor's gene first exon comprises high polymorphism trinucleotide repeats sequence.Studies show that relevant with x chromosome inactivation from the about 100bp of these trinucleotide repeat sequences local methylated Hpall and Hhal site far away.Select for use methylation status of PTEN promoter to come the state of Analysis of X x chromosome inactivation in this experiment.(Takeo?Kubota?et?al(1999)A?newassay?for?the?analysis?of?X-chromosome?inactivation?based?onmethylation-specific?PCR.Hum?Genet?104：49-55)。In normal women and have in the human embryo stem cell of women's caryogram, the inactivation of X chromosome should take place at random, and what the present invention obtained is that androgen sites is a strain human embryonic stem cell line of homozygosity on the X chromosome.

Known in the art, normal women has two X chromosomes (be paternal source, another is maternal source), and the male sex has only one.For two X chromosome genetic expressions of balance, inactivation can take place in women's a X chromosome, and the gene on it is not all expressed.It is early stage that this deactivation phenomenom occurs in the embryo, is at random, promptly both can be the x chromosome inactivation that father originates, and also can be the x chromosome inactivation that mother originates.At each cell, this selection is independently, and as a whole, ratio paternal and maternal x chromosome inactivation is 50% pair 50%.In the chain disease of some asymptomatic X, nonrandom x chromosome inactivation can take place, promptly in all cells, be from the x chromosome inactivation of paternal line or be x chromosome inactivation from maternal side, perhaps these inactivation ratios are extremely uneven, at least reach 90% to 10%, claim that in the industry this inactivation is nonrandom x chromosome inactivation.Having in theory under the embryonic stem cell normal circumstances of XX caryogram should be inactivation at random, and the methylation status of PTEN promoter detected result should show: active X karyomit(e) and inactivation X chromosome have two fragment are different but peak that area approximation equates, but same peak all appears in its active X karyomit(e) of embryonic stem cell of the present invention and inactivation X chromosome, discloses that androgen sites is the homozygosity (see figure 6) on this strain human embryo stem cell X chromosome.

Among the present invention, what carry out that the STR somatotype adopted is the fluorescent mark 16 locus composite amplification systems of Promega company, and identification capacity has reached 10 ^-17, be used for the evaluation of individuality or sibship and all be enough to make that conclusion.These 16 sites comprise that U.S. FBI recommends to be used for building national DNA database (Combined DNA index System, CODIS) 13 locus, be D3S1385, TH01, D21S11, D18S511, VWA, D8S1179, TPOX, FGA, D5S818, D31S317, D7S820, D16S539, CSF1PO (these 13 sites have become the internationally recognized locus that is used for medical jurisprudence STR somatotype), and Penta E, Penta D and a sex site Amelogenin.

It is to utilize to exist the different STR pulsating characteristic that increases to identify between individuality that STR (STR) is analyzed, every 6-10KBb just has a STR site in the Human genome genomic dna, and its polymorphism becomes the abundant source of forensic and paternity test.Embryonic stem cell is owing to be to set up from different zygote sources, its genetic background is also different, the STR site is analyzed and can clearly different embryonic stem cell strain differences be come, we have different STR site features by this strain embryonic stem cell of invention, can be differentiated (see figure 4) with other people embryonic stem cell line.

The present invention obtains to have the human embryonic stem cell of homozygous androgen sites on the X chromosome, this type of cell and derivative thereof are in the future possible epigenetics Mechanism Study, hESC's clinical application, to the foundation of women's X chromosome relative disease model, and the feasibility assessment of noble cells in clinical application that is produced by this class cell induction provides the ideal cell model.

Description of drawings

What Fig. 1 showed is clone's developmental process of FY-hES7 clone, is clone's size of the 1st day, the 2nd day, the 3rd day and the 4th day successively, and the doubling time that calculates this clone is 31.25 hours (100X scales=100um).

Fig. 2 is the stem cell biological characterization result figure of human embryo stem cell FY-hES7, distinguishes the biological nature of AKP level, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 from left to right;

Fig. 3 shows that FY-hES7 and other two strains clone all can express Oct-4 transcription factor (B-D), keeps not differentiation characteristic, and FY-hES-1 also expresses Oct-4 transcription factor (E) in contrast, and F is the expression of house-keeping gene β-actin.Oct-4 is the important gene that the ES cell is kept self, and undifferentiated ES clone can high expression level.Among Fig. 3, A: molecular weight standard Marker; B:FY-hES-5 OCT-4 expresses; C:FY-hES-7 OCT-4 expresses; D:FY-hES-8 OCT-4 expresses; E:FY-hES-1 OCT-4 expresses; F: positive with reference to β-actin.

Fig. 4 shows the STR result of human embryo stem cell FY-hES-7; Show among the figure that human embryo stem cell FY-hES-8 has unique STR site, is respectively to express the peak on the D3S1358 site 15, No. 17, expresses the peak on the TH01 site 8, No. 9, other are followed successively by D21S11:29, and 30; D18S51:12,14; PENTA E:12,14; D5S818:7,13; D13S317:10,11; D7S820:9,10; D16S539:11,11; CSF1PO:9,11; PENTA D:12,12; VWA:14,14; D8S1179:11,16; TPOX:8,9; FGA:22,23.

Fig. 5 is that its caryogram of karyogram of human embryo stem cell FY-hES-7 is 46, XX.

Fig. 6 is the interior differentiated result figure of the body of human embryo stem cell FY-hES, shows respectively from top to bottom to be divided into angling squamous epithelial tissue (ectoderm), nervous tissue (ectoderm), cartilaginous tissue (mesoderm), fatty tissue (mesoderm), body of gland epithelium (entoderm).

Fig. 7 a is the x chromosome inactivation analysis chart of human embryo stem cell FY-hES-7, and last figure is x chromosome inactivation product figure, and figure below is an active X karyomit(e) product; Show among the figure: the X chromosome of its active condition and the X chromosome of inactivated state are all expressed same peak 197bp.

Fig. 7 b is the x chromosome inactivation analysis chart of normal women's blood DNA, and the left side is x chromosome inactivation product figure, and the right side is active X karyomit(e) product figure, and the X chromosome that shows its inactivated state is at random.The X chromosome of inactivation has 194bp and 207 two peaks, and active X chromosome also has 194bp and two peaks of 207bp, and two peak area ratios were near 1: 1.

Embodiment

One, the foundation of human embryonic stem cell

Material

1. embryonic origin

The embryonic cell storehouse that hospital's reproductive center provides is in vitro fertilization (IVF) or single-semen injection (ICSI) back the 3rd day, is not suitable for carrying out embryo transfer or refrigerated inferior quality embryo, is used for this experiment after donations Mr. and Mrs informed consent.

2. key instrument

CO ₂Incubator (Forma), dissecting microscope (Olympus), common inverted microscope (Nikon), whizzer (KUBOTA 21000), thermostat water bath (H.H.S-11.2, Shanghai medicine equipment three factories), electronic analytical balance (Startorius), general refrigerator (Haier),-80 ℃ of cryogenic refrigerators (Forma 725), liquid nitrogen container (MVE, the U.S.) Millipore pure water instrument (the Purelab maxima. U.S.), liquid-transfering gun (Eppendorf, Germany), ophthalmic instruments (East China, Zhejiang Province pharmaceuticals), Bechtop, tissue culturing plate, culture dish (Falcon), divide tubulature, taper centrifuge tube (NUNC).

3. main agents:

4. solution preparation: PBS damping fluid, DMEM liquid, 0.1% gelatin, Mitomycin-C stock solution, collagenase IV stock solution, bFGF stock solution, MEF nutrient solution, blastaea are optimized nutrient solution, phES nutrient solution, phES frozen storing liquid etc., with reference to " stem cell handbook=Handbook of stem cells " (Lanza, volumes such as R, compilings such as professor Pei Xuetao.-Beijing: Science Press, 2006).

Method

1. the separation and Culture of mouse embryo fibroblasts (PMEF)

1.1 get pregnant 13.5-14.5 days the pregnant mouse of Kunming white, adopt the cervical vertebra dislocation method to put to death, cut open the belly under the aseptic condition and take out the uterus, place PBS liquid to wash repeatedly, remove blood stains.

Take out the embryo 1.2 cut off Uterus wall, after removing head, four limbs, internal organ, shred mouse embryo tissue, move in the centrifuge tube of 3～4 15ml, 0.25% trypsinase/0.02%EDTA the Digestive system that adds 2 times of volumes, 37 ℃ of water-bath digestion 8min blow and beat repeatedly with suction pipe, discard the supernatant liquor of digestion for the first time.

1.3 add the 0.25% trypsinase/0.02%EDTA Digestive system of 2 times of volumes again, 37 ℃ of water-bath digestion 5～8min blow and beat repeatedly with suction pipe, leave standstill 2min and draw supernatant liquor, move into and stop digestion, the centrifugal 5min of 1000rpm in the centrifuge tube that fills nutrient solution, abandon supernatant, add new nutrient solution.

1.4 repeat 1.3 steps 5～6 time, get off until the most tissues cell dissociation.

1.5 collect all cell precipitations after centrifugal in same centrifuge tube, recentrifuge is abandoned supernatant, adds new nutrient solution, adjusting cell density is 4 * 10 ⁵/ ml is inoculated in the culture dish, at 37 ℃, 5%CO ₂, cultivate in the saturated humidity incubator, changed liquid 1 time in 2～3 days; Treat that cell reaches 90% when converging, the cultivation of can going down to posterity, frozen or make feeder layer.

2. the preparation of feeder layer cells

2.1 when the growth of the 1st～3 generation MEF cell is joined together, add Mitomycin-C in culture supernatant, making final concentration is 10 μ g/ml, puts back to incubator effect 2.5hr.

2.2 0.1% aqueous gelatin solution is sucked in the culture dish, get final product can cover the culture dish surface, put back to more than the incubator effect 0.5hr; Before the use, inhale and abandon unnecessary aqueous gelatin solution, super clean bench is air-dry standby.

2.3 abandon the MEF cell culture supernatant that contains Mitomycin-C, with PBS washing three times.

2.40.25% behind trypsinase/0.02%EDTA liquid digestion 1min, add nutrient solution piping and druming wash-out cell, become 4 * 10 with inoculum preparation ⁶The cell suspension of individual/ml density is planted in advance in the culture dish of handling with gelatin.

2.5 at 37 ℃, 5%CO ₂, cultivate in the saturated humidity incubator.

2.6 the feeder layer ware for preparing is placed on standby in the incubator (can use) in 1～3d, uses the preceding 4～24h of prerequisite to be replaced by the embryonic stem cell nutrient solution.

3. blastaea is cultivated

3.1 the sequential culture of blastaea:

3.1.1 preparation G2.3 nutrient solution: in the G2.3 of 4.75ml substratum, add the HSA (concentration is 5%) of 0.25ml, in the culture dish of 35mm, do the droplet of 35 μ l, covering mineral oil, 37 ℃, 5%CO with liquid-transfering gun ₂, saturated humidity incubator inner equilibrium spends the night.

3.1.2 with reproductive center D3 depleted embryo by change in the G1.3 droplet in the G2.3 nutrient solution clean 3 times after, forward to again in the fresh G2.3 droplet and cultivate; Observe the fetal development situation every day until D7, and the blastaea that forms is carried out classification.

3.2 the optimization of blastaea is cultivated

3.2.1 the preparation blastaea is optimized nutrient solution: get above-mentioned preparation G2.3 nutrient solution 1ml and add the hLIF of 2000ul and the bFGF of 10ng, in the culture dish of 35mm, do the droplet of 35 μ l, covering mineral oil, 37 ℃, 5%CO with liquid-transfering gun ₂, saturated humidity incubator inner equilibrium spends the night.

3.2.2 reproductive center D3 depleted embryo forwards in the G2.3 droplet and is cultured to D5, changes in the blastaea optimization cultivation drop to cultivate again; Observe the fetal development situation every day until D7, and the blastaea that forms is carried out classification (with reference to Gardner blastaea staging), blastaea can be divided into three grades of A, B, C according to what of inner cell mass number: the A level, the ICM cell number is many and it is fine and close to arrange, and size obviously as seen; The B level, the ICM cell number is few and arrange to evacuate, and size as seen; C level, ICM cell number seldom almost lose.

4.ICM separation

4.1 mechanical cutting method:

4.1.1 making cutting needle: the middle part of the Glass tubing of external diameter 90 μ m, internal diameter 70 μ m is pulled into two sections on the outer flame of spirit lamp.Again that the thin portion of glass needle that pulls out is top-notch at the interior flame of spirit lamp, thin portion is evenly dwindled, pull out the very thin pin cutting edge of a knife or a sword that is about 1cm, the about 30 μ m of middle part diameter.

4.1.2 removal zona pellucida: blastaea is put into pronase (0.5mg/ml), wash 2 times after, change among another pronase, under dissecting microscope, observe the variation of blastaea zona pellucida, behind about 1～2min, see that zona pellucida begins to forward to immediately in the hES cultivation drop after the dissolving, wash 2-3 time.Hatch then directly to cultivate in the drop as blastaea and wash at hES.

4.1.3 mechanical separation ICM: go the blastaea of zona pellucida to change hES cultivation drop over to, the offside that left hand is pushed down the blastaea inner cell mass with 1ml syringe needle (29G) under anatomical lens is fixed, and the right hand cuts trophoderm outside the removal with homemade glass fine needle (30 μ m thickness) in the both sides of ICM.

4.1.4 the ICM after separating with a mouthful suction pipe change over to hES cultivate clean three times in the drop after, be inoculated on the MEF feeder layer of evening making in advance.

4.2 immunosurgery method

4.2.1 removal zona pellucida: same 4.1.2

4.2.2 antibodies: the blastaea that will remove behind the zona pellucida moves among the anti-human whole serum (1: 50), hatches 30min, changes hES immediately over to and cultivates the drop thorough washing 3 times.

4.2.3 complement combination: antibody effect blastaea later is transferred in the fresh guinea pig serum (complement), hatch 30min, at any time observe in the treating processes, expand when growing the germinal layer cell, being transparent cavity shape is termination, change hES over to and cultivate in the drop, blow and beat repeatedly with thin suction pipe and remove outer trophocyte.

4.2.4 the ICM after separating is inoculated on the MEF feeder layer of advanced processing making after hES cultivation drop wash-in is washed 3 times.

5. former generation clone's cultivation

5.1hES the preparation of nutrient solution

K-DMEM+15%SR+5%FBS+ other.

5.2. former generation clone's observation

ICM inoculation back was left standstill motionless in preceding 5 days, in order to avoid influence the adherent of ICM; Gave the growing state that half amount is changed liquid and observe ICM under anatomical lens on the 5th day, when being cultured to the 10th day, how much be divided into A, B, C three classes according to nido clone's size and periphery trophocyte:

6.hES the propagating method of cell relatively

Former be commissioned to train support 10-12d after, will clone in former ware with the glass fine needle and mechanically be divided into the 3-4 piece, small cell cluster is changed on the new MEF feeder layer with a mouth suction pipe go down to posterity first then.Go down to posterity first and cultivate 4～5 days again, after the clone increases to 1～2mm size, go down to posterity according to 1: 2～1: 3 ratio.Available mechanical process, collagenase IV digestion and the trysinization method that goes down to posterity that goes down to posterity, specific as follows:

6.1 machinery goes down to posterity

6.1.1 under dissecting microscope,, successively carry out vertically and horizontal cutting apart, be the cell mass of 100 * 100 μ m sizes with clone and separate on ES clone surface with making the glass fine needle by oneself.

6.1.2 the cell mass after will separating with the Bath mouth suction pipe that draws is transferred on the feeder layer of new system, and the culture dish that rolls makes clone's uniform distribution; Observe, write down ES cell growing state every day.

6.2 collagenase IV had digestive transfer culture

6.2.1 abandon original fluid, with DPBS washing 1 time, adding concentration is 1mg/ml collagenase IV, consumption is can cover cell degree of being, at 37 ℃, 5%CO ₂, cultivate 15～20min in the saturated humidity incubator.

6.2.2 abandon Digestive system, rap culture dish bottom, add an amount of nutrient solution and stop digestion, blow and beat gently with suction pipe most of cell is broken away from the bottom of ware.

6.2.3 the collecting cell suspension in the centrifuge tube of a 10ml, adds the hES washing lotion to 5ml, 1000rpm, centrifugal 4 minutes.

6.2.4 abandon supernatant, add an amount of nutrient solution, with the careful piping and druming of cell precipitation evenly, be transferred to enlarged culturing on the feeder layer of new system, observe every day, record ES cell growing state.

6.3 trysinization is gone down to posterity

6.3.1 abandon original fluid,, add an amount of concentration and be 0.05% pancreatin, at 37 ℃, 5%CO with DPBS washing 1 time ₂, act on 5min in the saturated humidity incubator.

6.3.2 surplus step is with 6.2.

7.hES the cryopreservation resuscitation of cell (a frozen at a slow speed rapid rewarming)

7.1 frozen hES cell

Adding concentration is 1mg/ml collagenase IV, behind 37 ℃ of effect 15～20min, blows and beats into small cell cluster gently, and centrifugal 1000rpm * 5min abandons supernatant, adds the frozen storing liquid mixing; Adjusting cell concn is 10 ⁶Individual/ml, divide the frozen tubule of packing into, every pipe 0.5ml, 4 ℃ of 30min ,-80 ℃ are spent the night, and drop into the medium-term and long-term preservation of liquid nitrogen next day.

7.2 recovery hES cell

From liquid nitrogen container, take out frozen pipe, drop into immediately in 37 ℃ the warm water and rock fast, until having only a fritter frozen storing liquid not melt.Change the cell cryopreservation suspension over to fill the 5ml nutrient solution aseptic centrifuge tube, 1000rpm is centrifugal, and 5min. abandons supernatant, adds nutrient solution, blows and beats mixing gently, and cell inoculation is cultivated on previously prepared feeder layer.

The result

1. by the blastaea in useless embryo source, be used to be cultured in the culture system the 7th day (D7) in preface, mostly be rudimentary blastaea greatly, it is little to show as blister cavities, and inner cell mass is little or do not have, and is unfavorable for that hES builds to be.Be cultured to D7 days discovery blister cavities and obviously expand in blastaea optimization culture system, the ICM cell number increases and is fine and close; But continue to be cultured to the shrinkage of D8 blister cavities, occur a large amount of fragments in the chamber, the more preceding increase of ICM but color and luster dimness, being inoculated into behind the feeder layer can not adherent growth, and the prompting blastaea should be above 7 days in the vitro culture time.

2. the foundation of useless embryo source hES clone

This experiment is adopted among 11 pieces of ICM that K-DMEM+20%SR+5%FBS group nutrient solution cultivates, and has 9 pieces of ICM adherent, obtains 5 pieces of A, the former generation clone of B level, after going down to posterity wherein 4 pieces reach 2-6 generation just dull gradually, dissolve until death; Finally set up 1 strain hES clone.Newly-established hES clone all forms flat circular colony on feeder layer, compact structure, and clear-cut margin, high power lens down visible clone's individual cells gap is clear, and nuclear-cytoplasmic ratio height, kernel are clear.This strain hES clone all can freezing repeatedly recovery, keeps undifferentiated state, and anabiosis rate is approximately 25%～40%.

The hES biological characteristics detects

Material

The cell source

1. the hES clone set up of laboratory, inventor place.

2. laboratory animal

6～8w age male SCID mouse, available from Shanghai Slac Experimental Animal Co., Ltd..

3. instrument

Fluorescence inverted microscope (Nikon), 3100 Genetic analyzer (American AB I company), PE9700PCR amplification instrument (American AB I company), UVP ultraviolet imagery system, electrophoresis apparatus (Biometra)

4. reagent

Trizonl reagent (Shanghai dodge brilliant molecular biosciences Science and Technology Ltd.), Qiagen One Step forRT-PCR Kit (Qiagen); ES Cell Characterization Kit (SCROO1, Chemicon); Human Embryonic Germ Layer Characterization Kit (SCRO30, Chemicon); GOAT ANTI MOUSE IgG-FITC, GOAT ANTI MOUSE IgM-FITC (Santacruz); Promega PowerPlex 16 System kit (Promega, USA).

Method

1.hES the cell colony doubling time (population doubling time, PD time) is detected

The hES cell adopts the collagenase digesting of 1mg/ml to go down to posterity and the low density inoculation,, begins in postvaccinal the 1st day about 5 clones of the bottom of each culture dish mark with marker pen, every the same ES clone of photographic measurement in 24 hours size, until the 4th day.Calculate its doubling time according to formula PD (hour)=Δ txLg2/ (LgNt-LgN0), calculate three PD values at least, average then.

2.hES the cell-specific mark is identified

2.1 alkaline phosphatase detects (AKP dyeing)

When human embryo stem cell was cultured to for the 20th generation, carry out alkaline phosphatase and detect (AKP dyeing): after going nutrient solution to fix 15 minutes with 4% Paraformaldehyde 96, TBST liquid flushing 2 times, add the alkaline phosphatase Incubating Solution, hatch under the room temperature when extremely dyeing was satisfied in 30 minutes, the flushing of TBST liquid, observations under the light microscopic.

2.2 hESC's surface antigen is identified

2.2.1hES passage is cultured to the 4th day, grows to stop when vigorous cultivating, 4% Paraformaldehyde 96 is 20min fixedly.

2.2.2TBST wash each 5-10min 2 times

2.2.30.1%Triton X-100/PBS effect 10min

2.2.4TBST wash each 5-10min 2 times.

2.2.5 add 4% lowlenthal serum blocking-up sealing heterogenetic antigen, hatched under the room temperature 30 minutes.

2.2.6 add an anti-SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 (with the dilution in 1: 50 of 4% lowlenthal serum), incubated at room 1 hour.

2.2.7TBST wash each 5-10min 3 times.

2.2.8 resist incubated at room 30min with two of 4% lowlenthal serum dilution in 1: 100 FITC mark.

2.2.9TBST wash each 5-10min 3 times.

2.2.10PBS cover the fluorescence microscope coloration result.

3.RT-PCR detect the expression of Oct-4 transcription factor in the hES cell

3.1Trizol test kit separates total RNA that purifies

3.1.1 respectively get 10 hES clone cell groups, add the TriZOL of 0.5ml, abundant mixing, room temperature leaves standstill 5min.

3.1.2 add the chloroform of 100 μ l again, abundant mixing, room temperature leaves standstill 5min.

3.1.34 ℃, the centrifugal 15min of 10000rpm.

3.1.4 get supernatant liquor, add the Virahol of 250 μ l, fully the mixing room temperature leaves standstill 5min.

3.1.54 ℃, the centrifugal 10min of 10000rpm.

3.1.6 abandon supernatant liquor, add 75% alcohol 500 μ l, 4 ℃, the centrifugal 10min of 10000rpm.

3.1.7 abandon supernatant liquor, drying at room temperature adds the DEPC water of 25 μ l, mixing again.

3.1.8 ultraviolet spectrophotometer is measured OD260/280, ratio gets final product greater than 1.8.

3.2 single stage method RT-PCR detects the expression of Oct-4

3.2.1 design of primers: OCT-4Primer A:5 '-GTGTTCAGCCAAAAGACCATC-3 ', Primer B:5 '-CCCTGAGAAAGGAGACCCA-3 '

3.2.2RT-PCR reaction system

50 μ l reaction systems, specifically composed as follows

RNase-free?water????????????26.0μl

RT-PCR?Buffer???????????????10.0μl

dNTP?Mix????????????????????2.0μl

Primer?A????????????????3.0μl

Primer?B????????????????3.0μl

RT-PCR?Enzyme?Mix???????2.0μl

Template?RNA????????????4.0μl

3.2.3RT-PCR reaction amplification condition

50 ℃ of reverse transcription reaction 30min

95 ℃ of PCR initial step: 15min

Following steps circulation 36 times

94 ℃ of sex change: 1min

Annealing: 56 ℃ of 1min

Extend: 72 ℃ of 1min

Extend at last: 72 ℃ of 10min

3.2.4 prepare 1.5% sepharose, electrophoresis 30min observes amplified band, the record result.

4. chromosome karyotype analysis

4.1 select to be cultured to after the 10th generation, the hES cell that is in exponential phase of growth carries out karyotyping; Add colchicine (final concentration is 0.25 μ g/ml) and stop at metaphase, put incubator and continue to cultivate 4 hours with inhibition division stage cell.

4.2 blow and beat with suction pipe, most of circular cell metaphase is come off at the bottom of ware, nutrient solution is changed in the centrifuge tube, after cleaning one time with PBS again, add an amount of 0.05% trypsin solution effect 5min, add nutrient solution and blow and beat centrifugal 5 minutes of collecting cell suspension 1000rpm repeatedly, abandon supernatant liquor, stay sedimentation cell.

4.3 add 0.4% Sodium Citrate of 37 ℃ of pre-temperature and the hypotonic medium of 0.4%KCl (1: 1) toward cell precipitation, with suction pipe piping and druming evenly, in the water temperature case, left standstill 5 minutes.

4.4 add freshly prepared methyl alcohol and Glacial acetic acid (3: 1) stationary liquid 0.5ml in the cell suspension after hypotonic processing, blow and beat gently to mix well with suction pipe and pre-fix centrifugal 5 minutes of 1000rpm.

Add stationary liquid 4ml 4.5 abandon supernatant liquor, piping and druming is gently sealed the back room temperature and was left standstill centrifugal 5 minutes of 1000rpm 40 minutes.

Add stationary liquid 2ml 4.6 abandon supernatant liquor, piping and druming is gently sealed the back room temperature and was left standstill centrifugal 5 minutes of 1000rpm 20 minutes.

Discard most of supernatant liquor 4.7 inhale, stay 0.5ml left and right sides supernatant and cell precipitation, get 1-2 behind the mixing and drip sheet, put 65 ℃ of roasting sheets and spend the night.

4.8 the chromosome sectioning for preparing after 0.25% trysinization G shows band, dyes 10min with 10% Giemsa stain of fresh configuration, flowing water is rinsed well, in dry.

4.9 high power lens is the karyotype of analysis cell 20～40 metaphases down, adopts Leica chromosome analysis software observes result, the announcement of transmitting messages.

5. individual recognition (analysis of STR site)

Adopt the Qiagen method to extract genomic dna, carry out pcr amplification, product carries out capillary electrophoresis on ABI 3100 genetic analyzers, analyzes 16 short series connection repetition sites and does individual recognition.16 sites are: D3S1358, TH01, D21S11, D18S51, PentaE, D5S818, D13S317, D7S820, D16S539, CSF1PO, PentaD, amelogenin, vWA, D8S1179, TPOX, FGA.

6. the inside and outside differentiation capability detects

6.1 the vitro differentiation ability detects

6.1.1 embryoid body forms: eugonic embryonic stem cell adds the embryoid body nutrient solution with 0.05% pancreatin effect 3 minutes, blows and beats into small cell cluster gently, changes the Micro-Organism Culture Dish inner suspension over to and cultivates, and changes after 3 days to new Micro-Organism Culture Dish; After this changed liquid 1 time in per 2 days, and formed to embryoid body.

6.1.2 spontaneous differentiation: cultivate 2 weeks of adherent culture in the culture dish that after 7 days the embryoid body that forms is changed over to 0.1% gelatin bag quilt, observe its differentiating phenomenon.

6.1.3 spontaneous differentiation culture is after 4 days, adopt triploblastica cell-specific antibody to detect noble cells antigen presentation situation, these antibody are: the human smooth muscle is exciting, and albumen (is used to detect bone and muscle cell related antigen, mesoderm), alpha-fetoprotein (is used to detect related antigens such as liver cell, entoderm) and vimentin (being used to detect the neurocyte related antigen, ectoderm).

6.2 differentiation capability detects in the body

6.2.1 teratoma forms: after embryonic stem cell digestion is centrifugal, make 1 * 10 ⁶The cell suspension of/ml, be inoculated into 6 the week ages male SCID mouse the subcutaneous and intraperitoneal of inguinal region.Whether after 4 weeks, observing has tumor growth; After 10～12 weeks, put to death mouse, extract lump and carry out following analysis.

6.2.2 histological examination: lump is through 10% neutral formalin fixedly behind the 24h; The gradient ethanol dehydration; Dimethylbenzene is transparent; Paraffin embedding; 5pm section continuously is through 65 ℃ of constant temperature roaster 6h oven dry; Step by step the dewaxing, aquation; Haematoxylin dyeing 5min, washing; Hydrochloride alcohol color separation 30sec, washing; Yihong dyeing 5min, washing; Dehydration, transparent step by step, neutral gum mounting, tissues observed cellular form under the mirror.

The result

1.hES the mean P D time of clone

This strain hES clone is monitored 3 clones' the speed of growth, and calculates its mean doubling time, write down as following table:

Table 1: three clone's mean P D times of this strain hES clone

The clone	??1	??2	??3
				The PD time (hour)	??32.75	??32.25	??31.50

As can be seen from the above table, the PD time of this strain hES clone is identical substantially, and all about 32 hours, prompting hES cell just can increase one times every 32 hours.

2. stem cell specificity marker thing is identified

The not differentiation clone AKP of this strain hES clone detects and is strong positive, and black-and-blue particle deposition is arranged in the kytoplasm, and a little less than the clone of part differentiation was painted, feeder layer cells was not painted, illustrates that the hES cell has high alkaline phosphatase activity.ES surface antigen S SEA-1, SSEA-4, TRA-1-60 and TRA-1-81 detected result show, SSEA-4, TRA-1-60 and the TRA-1-81 Immunofluorescence Reactions of this strain hES clone present strong green, SSEA-1 antigen-antibody Immunofluorescence Reactions negative (referring to Fig. 2), this strain hES clone energy expression specificity surface antigen S SEA-3, SSEA-4, TRA-1-60 and TRA-1-81 are described, do not express surface antigen S SEA-1.

3.Oct-4 the expression of transcription factor

Oct-4 is the important gene that the ES cell is kept self, and undifferentiated ES clone can high expression level.This experiment adopts the method for RT-PCR to detect this factor, and the newly-established hES clone of results suggest can be expressed the Oct-4 transcription factor, keeps not differentiation characteristic.(referring to Fig. 3)

4. individual recognition:

This strain hES clone specific peak occurs and itself and other hES clone can be made a distinction on 16 STR sites, referring to Fig. 4.

5, chromosome karyotype analysis:

This strain hES clone is carried out a karyotyping every 10 generations, and analytical results points out this strain clone to have normal karyotype, referring to Fig. 5.

6. the inside and outside differentiation capability detects:

6.1 the cell of vitro differentiation potential: FY-hES7 is at D7 days formation capsule embryoid bodies (EB) of suspension culture; Forward in the culture dish that is covered with gelatin after forming capsule EB, adherent culture to the four days is carried out triploblastica specific marker quality testing and is surveyed, and the result shows that the hES cell can spontaneously be divided into the cell of three germinal layers such as neurocyte, epithelial cell, smooth muscle cell.

6.2 the cell of differentiation potential: FY-hES7 begins to occur lump being inoculated into SCID mouse inguinal region about subcutaneous 6 weeks in the body; Lump increases about 30mm3 size during 12 weeks, puts to death mouse, extracts lump, does the tissue of visible three germinal layers of pathological section, comprises keratinized stratified squamous epithelium, neurocyte, pigment epithelium (ectoderm); Unstriated muscle, cartilage (mesoderm) and enteraden epithelium and cilium cylindrical epithelium (entoderm).Referring to Fig. 6.

Methylation status of PTEN promoter detects the x chromosome inactivation state

The extraction of embryonic stem cell DNA

The DNA that extracts uses the methylating reagent box to carry out bisulfite and handles, and makes the unmethylated cytosine(Cyt) among the DNA be converted into uridylic, and methylated cytosine(Cyt) then remains unchanged

Prepare two cover PCR reaction systems, one is methylated X allelotrope, another set of be unmethylated X chromosome reaction system with.

Add cell DNA sample that 1 μ l handled in the PCR reaction tubes, reaction system is as follows

10x?FX?buffer????????1.5

10mM?dNTP????????????0.75

Primer?ARM-F?????????0.2

Primer?ARM-R?????????0.2

AmpliTaq?Gold????????0.1

H ₂O 11.25 (μ l) (system methylates)

10x?FX?buffer????????1.5

10mM?dNTP????????????0.75

Primer?ARU-F?????????0.2

Primer?ARU-R?????????0.2

AmpliTaq?Gold???????0.1

H ₂O 11.25 (μ l) (system does not methylate)

Reaction conditions is as follows

95℃for?12’

94℃for?30”????????←

58℃for?30”????????←

72 ℃ of for 30 " ← 28 circulations

72℃for?7’

4 ℃ of placements

The purifying of product

Genetic Analyzer 310 carries out product analysis, and human androgen receptor's gene product size is between 170-230bp.

The analysis of x chromosome inactivation state

During normal women's x chromosome inactivation is analyzed, the peak of its inactivation should have two, and active peak also should be two, can calculate two x chromosome inactivation ratios from the ratio of two peak areas (or revising the back area), normal ratio is near 1: 1, but also can fluctuate to 8: 2.If this ratio is higher than 9: 1 or has only a peak, then this state is nonrandom inactivation.(reference: T Kubota et al 1999, A newassay for the analysis of X-chromosome inactivation based onmethylation-specific PCR, Hum Gent 104:49-55).

The result

The x chromosome inactivation analysis chart of embryonic stem cell FY-hES-7 shows that the X chromosome of its active condition and the X chromosome of inactivated state all express same peak 197bp.Referring to Fig. 7 a.

Normal women's DNA analysis x chromosome inactivation shows that the X chromosome of its inactivated state is at random.The X chromosome of inactivation has 194bp and 207 two peaks, and active X chromosome also has 194bp and two peaks of 207bp, and two peak area ratios were near 1: 1.Referring to Fig. 7 b.

Claims (1)

1. have the human embryonic stem cell of homozygous androgen sites on the X chromosome, it is characterized in that:

(a) the average population doubling time of described embryonic stem cell line is about 32 hours;

(b) the not differentiation of described embryonic stem cell line clone alkaline phosphatase is that AKP detects to strong positive, has high alkaline phosphatase activity;

(c) described embryonic stem cell line can be expressed one of following specific surfaces antigen: phasic specificity antigen-3 is SSEA-3, phasic specificity antigen-4 is SSEA-4, tumor response antigen-1-60 is TRA-1-60, and tumor response antigen-1-81 is TRA-1-81, and expression phase specific antigens-1 is not SSEA-1;

(d) to express eight aggressiveness be the Oct-4 transcription factor in conjunction with transcription factor 4 to described embryonic stem cell line, keeps not differentiation characteristic;

(e) described embryonic stem cell line carries out a karyotyping every 10 generations, has normal karyotype;

(f) described embryonic stem cell line has two X chromosomes, the x chromosome inactivation analysis chart shows that the X chromosome of active condition of this embryonic stem cell line and the X chromosome of inactivated state all express same peak 197bp, shows that androgen sites is a homozygosity on its X chromosome;

(g) described embryonic stem cell line has unique STR, is respectively to express the peak on the D3S1358 site 15, No. 17, expresses the peak on the TH01 site 8, No. 9, and other are followed successively by D21S11:29, and 30; D18S51:12,14; PENTA E:12,14; D5S818:7,13; D13S317:10,11; D7S820:9,10; D16S539:11,11; CSF1PO:9,11; PENTAD:12,12; VWA:14,14; D8S1179:11,16; TPOX:8,9; FGA:22,23.

Images