CN101525592A - Human parthenogenetic embryo stem cell line with two active X chromosomes and derivatives thereof - Google Patents

Abstract

The invention relates to a human parthenogenetic embryo stem cell line with two active X chromosomes and derivatives thereof. A karyotype of the human parthenogenetic embryo stem cell line is 46, XX before 10 generations; the karyotype of the cell line becomes a chimera from the 20th generation; all cells of the cell line lose an active X chromosome at the 35th generation; and in the inactivation detection of the X chromosome, only the state of the active X chromosome is maintained all along, but the presence of the inactivated X chromosome is not discovered. The parthenogenetic activation can be achieved by adopting an artificial activation condition causing a second polar body to be discharged; therefore, the human parthenogenetic embryo stem cell line and the derivatives thereof can be obtained. The cell line and the derivatives thereof have important significance on researching the characteristics of the cell on genetics and epigenetics and further applying the cell to clinically treating certain diseases; and a cell source for cell replacement therapy can be obtained by transforming genes of the cell or modifying the genes on the epigenetics.

Description

Human parthenogenetic embryo stem cell line and derivative thereof with two active X chromosomes

Technical field

The present invention relates to the versatility embryonic stem cell, especially relate to a kind of human parthenogenetic embryo stem cell line and derivative thereof.

Background technology

As everyone knows, so-called parthenogenesis is to need not under the condition of sperm, and is spontaneous or through physics, chemical stimulation, makes egg cell development become an embryo's process.Promptly there is not any effect of male gamete, by the embryo of female gamete generation.No matter whether it develops into individuality, all be called parthenogenesis.The nature parthenogenesis is a kind of reproductive patterns, also exists certain parthenogenesis phenomenon reptile, fish animal, birds.

Though Mammals can not breed in this way, can be by external artificial stimulation simulation fertilization process, ovocyte is activated under the sperm condition need not, thereby obtains the parthenogenetic embryo tire.This is an important biotechnology, can be used for assessing indirectly ovocyte quality, analyze that paternal maternal gamete interacts in the early embryonic development, the initial mechanism of research fetal development.The external Activiation method of while mature oocyte still carries out animal cloning by nuclear transplantation prerequisite (Gasparrini et al., 2004; Sengoku et al., 2004; Zhu et al., 2003).Lonely female activated ovocyte provides alternative resource (Sengoku et al., 2004) to the activation process of research human oocytes and protokaryon embryo's formation.

The parthenogenesis technology also is to set up embryonic stem cell (Embryonic Stem Cell, an important technology that ESC) is (Mizutani et al., 2004).ESC is from body early embryo inner cell mass (inner cellmass, ICM) or archeocyte (primordial germ cells, PGCs) multipotent stem cells of separating, the latter is called embryonic genital cell (embryonic germ cells, EG cells) again.These cells can keep not differentiation state in external long-term propagation, has normal diploid karyotype, express various multipotential stem cell specific markers, and can be divided into various cells and the types of organization's (comprising sexual gland and sexual cell) that represents three germinal layers in vivo and in vitro.Though development along with medical technology, increasing disease is captured, but because irreversible necrosis of self cell or disease that functional defect caused, effective methods of treatment is not arranged so far yet, and wherein representational disease has diabetes, Parkinson's disease, Alzheimer syndromes, Spinal injury, leukemia, myocardial infarction etc.Treat the effective means of this class disease and carry out cell or organ transplantation exactly, regrettably, be subjected to number of patients that these diseases torment considerably beyond supplying transplanted organ quantity.HESC's appearance makes these treatment of diseases become possibility, though the research of present stage still fails to reach the level of clinical application, but the research that utilizes embryonic stem cell to carry out vitro culture, propagation, differentiation has obtained very big progress, thereby for further cell, tissue or organ transplantation are laid a good foundation, can be used in the future countless treatment of diseases, its application relates to the every field of medical science, is the seed cell of the tool potentiality of following regenerative medicine.The major obstacle of ESC clinical application is that the transplanting of its derivative may cause allogeneic rejection (Drukker and Benvenisty, 2004).The hazard level of this rejection is presented proteic inconsistent degree with the cell-surface antigens between the confession acceptor and is become positive correlation.Optimal transplanting state is, and the main histocompatibility complex of donor tissue (Major HistocompatibilityComplex, MHC) in full accord with acceptor.Human MHC is named as human leucocyte antigen (HLA) (HumanLeukocyte Antigen, HLA) system.Can reduce the reaction of recipient cell poison T cell for the HLA antigen coupling of acceptor, thereby increase the probability of graft survival.In order to solve the immunological rejection problem after the transplanting, must use anti-rejection drugs or set up a very big ES cell bank and join type.The former uses anti-rejection drugs harmful, and the latter is a huge engineering, can not realize at no distant date.Because the appearance of somatic cell clone technique, people are recognized set up the imagination that has with the embryonic stem cell line of patient homologue consistency mixture to become possibility.So, people can obtain not having the embryonic stem cell line of immunological rejection, induce then to be divided into required cell and to carry out surrogate therapeutic.Shortcomings such as it is big that but this method exists, and cloning efficiency is low, the ovum source is limited and ethics is disputed on.

And, utilizing its ovocyte can obtain these characteristics of blastaea by artificial activation's method for jenny or women, people have proposed to utilize lonely female activation embryo to set up the idea of embryonic stem cell.This idea has at first obtained realization (Szabo and Mann, 1994 on mouse; Kaufman et al., 1983), people have set up the lonely female and lonely male embryonic stem cell line of mouse, and have analyzed its methylation state.Afterwards, scientists finds that the embryonic stem cell in lonely female activation source has and the identical main histocompatibility complex of the female mouse of donor.This also provides experiment basis for this technology is applied to the mankind.At present, the success from mouse (Kaufman et al., 1983), rabbit (Wang et al., 2007), macaque (Cibelliet al., 2002; Vrana et al., 2003) and people (Lin et al., 2007; Mai et al., 2007; Revazova et al., 2007a; Revazova et al., 2007b) etc. the female activated blastaea of mammiferous orphan obtains ESC system.The advantage of utilizing the parthenogenetic embryo tire to set up ESC is that to build be the efficient height, and the ethics dispute is few.

The human genome DNA has 3 * 109bp, and 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) (Edwards et al., 1991), the number of different human body genome satellite DNA repeating unit is variable, therefore form extremely complicated allelotrope fragment length polymorphism (Schaferand Hawkins, 1998; Carramolino et al., 1997).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 (Hallenberg and Morling, 2001) at present.

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, research No.165-75) 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 is an integral part of epigenetic mechanism, this phenomenon has just had the LYON hypothesis in 1961, that is: have only an X chromosome that activity is arranged in (1) female mammal cell, it is at random that another inactivation and pyknosis (2) inactivation occurs in early stage (blastula stage) (3) of embryo inactivation, the X chromosome that is 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.

Summary of the invention

The object of the present invention is to provide a kind of human parthenogenetic embryo stem cell line and derivative thereof with two active X chromosomes.Different with the lonely female stem cell of versatility people of the generation of former report, though described versatility people's parthenogenetic embryo tire stem cell also is to produce by the female activated method of orphan, but the lonely female stem cell line that the present invention obtained has two active X chromosomes, and after culturing process in lose an active X chromosomes gradually, this does not find in similar clone before being.

A kind of human parthenogenetic embryo stem cell line and derivative thereof with two active X chromosomes of the present invention is characterized in that:

(a) caryogram of this human parthenogenetic embryo stem cell line and derivative thereof was " 46, XX " in the past in 10 generations; Since the 20th generation, the caryogram of this clone has become mosaic " 46, XX/45, X0 ", and the part cell has taken place by losing of one active X chromosomes; Along with the increase of generation, 45, the division phase ratio of X0 increases; To the 35th generation, caryogram is " 45, X0 ", and all cells has all taken place by losing of one active X chromosomes;

(b) in the 15th generation caryogram " 46, XX " and the 30th generation caryogram " 46, XX/45; X0 ", there is not the X chromosome of inactivation to exist, in the detection of x chromosome inactivation, keep always and have only the chromosomal state of active X, and do not have discovery that the existence of the X chromosome of inactivation is arranged;

(c) this human parthenogenetic embryo stem cell line and derivative thereof prepare by following steps:

A. obtain the sophisticated ovocyte that is in MII MII;

B. activate the second polar body of ovum and discharge, impel its female pronucleus to double to form zygoid, cultivate this activated cell, induce this cell to become the embryo of containing recognizable inner cell mass and trophectoderm;

C. separate described inner cell mass, change in the substratum of differentiation capable of inhibiting cell and cultivate, make these cells keep undifferentiated multipotency state, and go down to posterity, freezing, identify through the whole bag of tricks, confirm as parthenogenetic embryo stem cell line and derivative thereof.

Preferably, the step a of described preparation comprises: collect corolla mound mixture, cultivated 0.5-1 hour in the nutrient solution that contains 5-10% blood serum substituting product; This corolla mound mixture is exposed in the Unidasa of 80IU/ml 20 seconds～1 minute, removes granulosa cell, washing for several times in containing the nutrient solution of Hepes; And the microscopically observation, the MII phase ovocyte of selective maturation carries out next step lonely female activation.

Preferably, the step b of described preparation comprises: this ovocyte is acted on 3-5 minute in the body early embryo nutrient solution that contains 5 μ M-10mM Calcium ionophores; Change over to contain in the body early embryo nutrient solution that 1-5mM 6-Dimethylamino pyridine is 6-DMAP and 1nM-1mM NSC 630176 and act on 4-6 hour; After test under microscope confirms to discharge second polar body, continue in containing the body early embryo nutrient solution of 1nM-1mM NSC 630176, to activate 8-12 hour; The flush away NSC 630176 changes ovocyte over to cultivate in the body early embryo nutrient solution 2-3 days, and the blastaea nutrient solution was cultivated 2-3 days more then, formed early stage blastaea; The blastaea that early stage blastaea is moved into the rh-bFGF that contains 1000-2000IU/ml people's recombinant leukemia inhibitor factor and 2-10ng/ml is optimized substratum continuation cultivation 2-3 days, obtains described human parthenogenetic embryo stem cell line with two active X chromosomes.

The different new Activiation method of Activiation method of people's parthenogenetic embryo tire of provided by the present invention and aforementioned documents report, promptly adopt to cause that artificial activation's condition that second polar body is discharged realizes lonely female activation, and obtained a kind of two active X chromosome human parthenogenetic embryo stem cell lines that have thus.

People's parthenogenetic embryo tire stem cell (parthenogenetic human stem cell that the present invention obtained, phES) caryogram was 46 in 10 generations in the past, the XX (see figure 2), MLPA (Multiplexligation-dependent Probe amplification, multiple ligand dependence probe amplification) analyzing the lonely female cell of demonstration has and the normal contour X peak type of XX caryogram cell, what FISH (Fluorescence in situhybridization, fluorescence in situ hybridization) also proved this cell has two X chromosomes really.Since the 20th generation, find that the caryogram of this clone has become mosaic, 46, XX/45, X0 (division phase ratio 64: 36); Along with the increase of generation, 45, the division phase ratio of X0 increases; To the 35th generation, can not in analyzing, the G band find 46, and XX divides phase, and cell has become 45 fully, the caryogram (see figure 2) of X0.MLPA analyzes and shows that lonely female cell has only half high X peak type of normal XX caryogram cell, analyzes and has only an X chromosome.Constantly go down to posterity external, the (see figure 2) of losing of an X chromosome taken place in lonely female cell.

The lonely female source of the phES clone that the present invention obtained adopts HLA somatotype and individual recognition (analysis of STR site) to prove.

The HLA somatotype has shown the HLA-A of lonely female stem cell ,-B and DRB site and ovum donor half coupling, and the different loci on 12 different karyomit(e)s that 32 STR sites are dispersed in also shows this clone and ovum donor's affinity.All these results show that this clone is to be derived from lonely female activated (see figure 2).

The imprinted genes analysis of the phES clone that the present invention obtained comprises paternal imprinted genes H19, maternal imprinted genes IGF2 and SNRPN, and autosomal gene GNAS.Wherein, IGF2 and SNRPN all do not detect, and H19 and GNAS express the positive.And above-mentioned all genes are at the people ES of normal fertilization clone FY-HES-1, all can detect expression in-7.This result provides further evidence (Fig. 5) for the lonely female source of FY-phES 018.

To the gene chip analysis of the phES clone that the present invention obtained, comprising 21522 Human genome probes, 34 known imprinted genes are used to this research.The result shows that most of maternal imprinted genes all are to be the downward modulation state, and most of paternal imprinted genes all is to be the rise state.

The dna methylation analysis of the phES clone that the present invention obtained is found have only the maternal side SNRPN allelotrope site (174bp) that methylates in FY-phES 018, can be detected, do not had unmethylated paternal allelotrope (100bp) (Fig. 5).This is consistent with not expressing of SNRPN gene in the detection of front imprinted gene.The lonely female source of the complete maternal gene really of the cell that this also confirms from another side.In contrast, methylate and all there be (Fig. 5) in two the allelotrope sites that do not methylate in FY-HES-7.

MSP Analysis of X ist gene promoter position point shows, the FY-Hes-7 of XX caryogram exists two the xist gene promoter area allelotrope that do not methylate and methylate, and have in early days among the FY-phES 018 of two active X chromosomes, only found methylated allelic xist gene promoter site (Fig. 5).

The x chromosome inactivation state analysis of the phES clone that the present invention obtained is found, 15 generations (46, XX) and 30 generations (46, XX/45, X0) the X chromosome of inactivation does not exist, explanation, in the early stage generation cell of FY-phES 018, the X chromosome dna methylation is that globality ground reduces.In the external process that constantly goes down to posterity, because the unstable of X chromosome has been lost an X chromosome gradually, and in the detection of x chromosome inactivation, keep always and have only the chromosomal state of active X, and do not have to find to have the existing of X chromosome (Fig. 6) of inactivation.

This versatility human parthenogenetic embryo stem cell line with unique property provided by the present invention and derivative thereof are significant in the characteristic of genetics and epigenetics for inquiring into the phES cell.This phES clone in vivo with the external ability that all has to the differentiation of three germinal layers, and it has the HLA type that mates with those for egg half, transplant after being induced to differentiate into the cell or tissue of particular type, can avoid immunological rejection, some disease is significant for further being applied to treat clinically in the future.Can also modify by genetic modification or epigenetics this phES clone, further provide fundamental basis and practical experience for setting up the phES clone that really can be applied to clinical treatment in the future, cell source as the cell replacement treatment has broad application prospects.

Description of drawings

The behave form of parthenogenetic embryo tire and embryonic stem cell line of Fig. 1; A is the 7th day a blastaea; B is the original colony form of phES cell; C-F is respectively the colony form (100 * microscopically photo) in the 10th generation of phES cell, the 20th generation, the 30th generation, the 40th generation

Fig. 2 the behave karyotyping and the STR detection case of lonely female ES cell; A is the phES caryogram in the 10th generation, 46, and XX; B is the phES caryogram in the 35th generation, 45, and X0; C is the phES fluorescence in situ hybridization in the 35th generation (FISH) picture, shows to have only an X chromosome (shown in the arrow), and red, green, sky blue is represented karyomit(e) respectively 13,21 and No. 18; D and F are the result of the somatic STR of those for egg (STR); E and G are the result of the STR (STR) of phES cell.

Behave evaluation---the detection of alkaline phosphatase, cell surface marker and inside and outside differentiation capability of lonely female ES cell of Fig. 3; A is the alkaline phosphatase staining result of phES cell, is shown as strong positive (100 * microscopically photo); B-D is respectively stem cell surface mark SSEA-4, TRA-1-60, TRA-1-81 immunocytochemical stain result, is shown as the positive; The EB that E is differentiated to form for the phES cells in vitro (100 * microscopically photo); F-H is the cellular immunization dyeing picture of the spontaneous differentiation of phES cells in vitro, respectively alpha-fetoprotein (F, entoderm), the exciting albumen (G, mesoderm) of unstriated muscle, vimentin (H, ectoderm); I-K is the teratoma histological stain result who is differentiated to form in the body, and I is that mesoblastic cartilaginous tissue, J are ectodermic neuroganglion; K is mesoblastic fatty tissue and endoblastic body of gland.

The behave versatility transcription factor Oct4 of ES cell of lonely female ES cell and normal fertilization of Fig. 4, Nanog, the PCR detected result of Sox2;

The behave imprinted genes expression of ES cell of lonely female ES cell and normal fertilization of Fig. 5; Among the figure, A represent normal fertilization female ES cell (46, XX) express the X chromosome that the explanation of XIST gene has inactivation, and the male ES cell of normal fertilization (46, XY) and the lonely female ES cell of people do not express the X chromosome that the XIST explanation does not have inactivation; Maternal imprinted genes (IGF2 and SNRPN) of the ES cell expressing of normal fertilization and paternal imprinted genes (H19 and GNAS); The people is lonely, and female ES cell is not expressed maternal imprinted genes (IGF2 and SNRPN), but expresses paternal imprinted genes (H19 and GNAS).B is the methylation state of maternal imprinted genes SNRPN: the people is lonely, and female ES cell has only methylated SNRPN gene (174bp), and the ES cell of normal fertilization not only has methylated SNRPN gene, also has unmethylated SNRPN gene.

The behave active detection of X chromosome (red peak is shown as Marker, and blue peak is an X chromosome) of ES cell of lonely female ES cell and normal fertilization of Fig. 6.Left hand view is the activatory X chromosome, and right part of flg is the X chromosome of inactivation.A, B are the phES cell in the 10th generation; C, D are the phES cell in the 30th generation; E, F be normal fertilization male ES cell (46, XY); G, and the female ES cell of H normal fertilization (46, XX); I, and the normal women's hemocyte contrast of J (46, XX).

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment, and described percentage concentration is mass percent concentration if no special instructions.

Embodiment one: the acquisition of people's parthenogenetic embryo tire stem cell

One, volunteer's collection and relevant informed consent

Strict (No.460,2003) the relevant rules of following the The People's Republic of China Ministry of Health of this experiment.

All volunteers are all at length informed experiment flow, comprise the purposes of ovum and the purpose of research etc., and sign a series of Informed Consent Forms voluntarily, do not obtain any economic interests.Guarantee that all ovums all will be used for fundamental research, and be not used in the purpose of reproduction.

Two, volunteer's the short super ovulation and the results of ovum

Each volunteer must comprise communicable diseases such as acquired immune deficiency syndrome (AIDS), hepatitis B, hepatitis C, venereal disease through strict physical examination before entering short super ovulatory cycle.Eligible adopts rectangular case or short scheme to urge super ovulation.Rectangular case is in the analogue (enantone of injecting gonadotropin releasing hormone (GnRH) on the 21st day of menstruation, leuprorelin acetate) 1.8mg falling tone, inject follicular stimulating hormone (fruit receive sweet smell, Switzerland Xue Lannuo company) 150-300IU every day in beginning in the 3rd day of menstruation next time and urge super the ovulation.Short scheme is injected follicular stimulating hormone (fruit receive sweet smell, Switzerland Xue Lannuo company) 150-300IU every day in beginning in the 3rd day of menstruation and urge super ovulation, the analogue (Wy 18481) of while start injection gonadotropin releasing hormone (GnRH) 0.15mg/ days.When treating that dominant follicle reaches 16-18mm, injected the chorionic-gonadotropin hormone HCG of 10000IU that night.Inject and under the guiding of vagina B ultrasonic, got ovum in back about 36 hours.

Three, the preparation of main agents and solution

1, main agents

2, 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).

Four, lonely female activation

During normal fertilization, ovocyte just contacted with sperm and was activated immediately meiosis metaphase (M II phase), experienced a process, and this process comprises the separation of sister chromatid, the discharge of second polar body, produces the chromosomal ovocyte that contains monoploid quantity.And during lonely female activation, because the difference of the osmotic pressure of stimulus, stimulus intensity, nutrient solution etc., second polar body can not be discharged, also inconsistent (the Chen Dayuan etc. of caryogram of the lonely female ovum that obtains thus (parthenogenetic embryo tire), fertilization biology-fertilization mechanism and reproduction engineering, Beijing: Science Press, 2000:252-254).Activate in the present invention and be meant the method for inducing M II phase ovocyte to become the embryo of containing recognizable inner cell mass and trophectoderm, this embryo can be used for producing pluripotent cell, but itself can not grow the individuality of survival.Different with other the production method of people's parthenogenetic embryo tire stem cell is in the present invention, preferably to adopt to cause that artificial activation's condition that second polar body is discharged realizes lonely female activation.Activate the second polar body of ovum in the present invention and discharge, its female pronucleus doubles to form and isozygotys two times.

(Oocyte-Cumulus Complexes OCCs), cultivated 0.5-1 hour in the nutrient solution that contains 5-10% blood serum substituting product to collect corolla mound mixture.OCCs is exposed in the Unidasa (20 seconds～1 minute) of 80IU/ml, and mechanical process is removed granulosa cell, and washing for several times in containing the nutrient solution of Hepes.Microscopically is observed, and the MII phase ovocyte of selective maturation carries out lonely female activation.All cultivations maintain 37 ℃, 5%CO ₂Moist environment in.

The MII phase ovocyte of selective maturation carries out lonely female activation.Ovocyte acts on 5 minutes in the G1.3 body early embryo nutrient solution (Switzerland vitrolife company) of the calcium ion carrier A 23187 that contains 5 μ M (Sigma company), containing 1mM 6-dimethylamino-purine (6-Dimethylamino pyridine subsequently, 6-DMAP, Sigma) and 5nM Trichostatin A (TSA, Sigma) effect is 4 hours among the G1.3, after mirror is checked down and is confirmed to discharge second polar body, continuation activates 10 hours in the G1.3 that contains 5nM TSA, ovocyte with flush away TSA changes 72 hours that cultivate among the G1.3 over to afterwards, and G2.3 blastaea nutrient solution (Switzerland vitrolife company) was cultivated 48 hours more then.The 6th day, early stage blastaea moves into the rh-bFGF that contains 2000IU/ml people's recombinant leukemia inhibitor factor (hLIF, Chemicon company) and 10ng/ml, and (bFGF, G2.3 blastaea Sigma) optimized substratum and continue to cultivate 2 days.Observe fetal development situation and record every day between incubation period.

In the foregoing description, also available other Calcium ionophores of calcium ion carrier A 23187 or divalent cation replace as ionomycin; 6-DMAP can replace with other protein phosphorylation inhibitor, for example 6-DMAP, okadaic acid (okadaic acid, OA) etc.; TSA can substitute with other NSC 630176, for example system is dripped rhzomorph (Trichostatin A, be called for short TSA, belong to a kind of hydroxamic acid), butyric acid (a kind of short chain fatty acid, SCFA), SAHA (Suberoylanilide hydroxamic acid), MS2272275 (are also referred to as MS2275, be a kind of benzamide derivatives of synthetic), Apicidin (sickle-like bacteria meta-bolites, belong to cyclic tetrapeptide quasi-molecule) etc.; Also available other body early embryo nutrient solutions of G1.3 body early embryo nutrient solution replace; Also available other blastaea nutrient solutions of G2.3 blastaea nutrient solution replace.

Five, the foundation of parthenogenetic embryo stem cell line and cultivation

1, the preparation of feeder layer

Mitomycin-C of doses (mitomycin-C) and gamma-radiation can make cell stop division, but are unlikely at once dead, can be in external survival for some time, and keep secreting the function of the various factors.So use Mitomycin-C or gamma-radiation to handle feeder cell, make it the termination division, still secretion promotes the embryonic stem cell proliferation and the factor that suppresses its autonomous differentiation simultaneously, keeps it and does not break up diploid condition.

(1) when the 1st～3 generation, the mouse embryo fibroblasts growth was joined together, add Mitomycin-C in culture supernatant, making final concentration is 10 μ g/ml, puts back to the incubator effect 2～3 hours.

(2) anticipate culture dish with gelatin.Treatment process is: 0.1% aqueous gelatin solution is sucked in the culture dish, get final product can cover the culture dish surface, at room temperature leave standstill more than 2 hours.Before the use, inhale and abandon unnecessary aqueous gelatin solution, standby.

(3) discard the feeder cell culture supernatant that contains Mitomycin-C.With PBS washing three times.

Behind (4) 0.25% trypsinase/0.02%EDTA liquid digestion feeder cell 1min, add nutrient solution piping and druming wash-out cell, become 3 * 10 with inoculum preparation ⁶The cell suspension of individual cell/ml density is planted in advance in the culture dish of handling with gelatin.The cell count of plantation is: MEF 2 * 10 ⁴～5 * 10 ⁴Individual/cm ²

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

The raising individual layer that (6) will prepare is placed in the incubator standby.In 5d, use, will change the embryonic stem cell nutrient solution before using.

2, blastaea is cultivated

(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.

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

(3) with the parthenogenetic embryo tire of the 3rd day (Day3) by change in the G1.3 droplet clean 3 times in the G2.3 nutrient solution after, forward to again in the fresh G2.3 droplet and cultivate, until the 5th day (D5), change over to again in the blastaea optimization cultivation drop and cultivate, observe the fetal development situation every day, and the blastaea that forms is carried out classification, and 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.

3, immunosurgery method separate inner cell mass

(1) remove zona pellucida: blastaea is put into 0.5% proteolytic enzyme (pronase, Sigma) in, after washing 2 times, 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 phES cultivation drop after the dissolving, wash 2-3 time.

Hatch then directly to cultivate in the drop as blastaea and wash at phES.

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

(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 phES over to and cultivate in the drop, blow and beat repeatedly with thin suction pipe and remove outer trophocyte.

(4) ICM after separating is after phES cultivates the drop wash-in and washs 3 times, is inoculated on the MEF feeder layer that advanced processing makes (ICR mouse embryo fibroblasts) and does further to cultivate.

4, former generation clone's cultivation reaches and goes down to posterity in early days

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, cultivate after 8-10 days, observe the form Outgrowths the same with common embryonic stem cell.How much being divided into A, B, C three classes according to nido clone's size and periphery trophocyte: category-A. former generation clone is the nest like convex growth, and the trophocyte is few; Category-B. former generation clone is small circular, and periphery has more trophocyte to hold; The C class. the dimness of former generation clone color and luster, cessation of growth cessation, or only see that the trophocyte breeds.

Once went down to posterity with mechanical process under dissecting microscope in the every 5-6 of the cell mass of this stem-like cell days in going down to posterity in early days, former generation outgrowths is cut into two to three minicell agglomerates, moves on the fresh feeder layer to cultivate.After going down to posterity through 5 times, can use collagenase 4 instead and carry out enzymatic digest and go down to posterity.HESC's media components is 80%knockout-DMEM (Gibco Invitrogen Corporation), 15% serum substitute (SR, Gibco), 5% high-quality foetal calf serum (FBS, hyclone), 0.1mM2-mercaptoethanol (β-mercaptolethanol, Gibco), 2mM glutamine (Gibco), 0.1mM non-essential amino acid (Gibco), the 8ng/ml Prostatropin (bFGF, Chemicon) and 2000IU/ml human leukemia supressor (hLIF, chemicon).After passing for 5 generations, hLIF can add in substratum, and Prostatropin also can be reduced to 4ng/ml.

5, collagenase IV had digestive transfer culture

(1) abandon original fluid, with PBS washing 1 time, adding concentration is 1mg/ml collagenase IV, and consumption is advisable can cover cell, at 37 ℃, 5%CO ₂, cultivate 15～20min in the saturated humidity incubator.

(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.

(3) the collecting cell suspension adds the phES washing lotion to 5ml in the centrifuge tube of 10ml, 1000rpm, centrifugal 4 minutes.

(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, frozen phES cell

Adding concentration is 1mg/ml collagenase IV, behind 37 ℃ of effect 15～20min, blows and beats into small cell cluster gently, centrifugal 1000rpm, and 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, recovery phES 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, the centrifugal 5min of 1000rpm.Abandon supernatant, add nutrient solution, blow and beat mixing gently, cell inoculation is cultivated on previously prepared feeder layer.

The result

Grow blastaea for 4 after the lonely female activation in the 7th day 18 zygote.Isolate 3 inner cell masses with the immunosurgery method, move on the MEF feeder layer and cultivate after 8-9 days, inner cell mass presents dome-shaped colony form.Successfully set up 1 strain human parthenogenetic embryo stem cell line (FY-phES 018) (Fig. 1).018 doubling time of FY-phES is 72 hours, and normal control FY-hES-1 is about 36 hours.

The evaluation of embodiment 2 phES cells

One, the routine of phES cell is identified

1, alkaline phosphatase detects (AKP dyeing)

PhES cell cultures to the is during 20 generations, carries out alkaline phosphatase and detects (AKP dyeing): remove nutrient solution, fix 15 minutes with 4% Paraformaldehyde 96 after, PBS liquid flushing 2 times adds the alkaline phosphatase Incubating Solution, hatches under the room temperature when extremely dyeing was fit in 30 minutes, the flushing of PBS liquid, observations under the light microscopic.

2, the human embryo stem cell surface antigen is identified

(1) the phES passage is cultured to the 4th day, grows to stop when vigorous cultivating, and 4% Paraformaldehyde 96 is 20min fixedly.

(2) the PBS flushing is 2 times, each 5-10min

(3) 0.1%Triton X-100/PBS effect 10min

(4) the PBS flushing is 2 times, each 5-10min.

(5) PBS that adds 4% lowlenthal serum seals heterogenetic antigen, hatches under the room temperature 30 minutes.

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

(6) the PBS flushing is 3 times, each 5-10min.

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

(8) the PBS flushing is 3 times, each 5-10min.

(9) PBS covers, the fluorescence microscope coloration result.

3, RT-PCR detects the expression of hES transit cell record factor Oct-4, Nanog, Sox2

(1) the Trizol test kit separates total RNA that purifies

(2) respectively get 10 phES clone cell groups, add the Trizol of 0.5ml, abundant mixing, room temperature leaves standstill 5min.

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

(4) 4 ℃, the centrifugal 15min of 10000rpm.

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

(6) 4 ℃, the centrifugal 10min of 10000rpm.

(7) abandon supernatant liquor, add 75% alcohol 500 μ l, 4 ℃, centrifugal 10 minutes of 10000rpm.

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

(9) ultraviolet spectrophotometer is measured OD 260/280, and ratio gets final product greater than 1.8.

(10) design of primers: adopt Primer Premier 5 softwares, respectively with people's H19, IGF2, SNRPN, GNAS, GAPDH, OCT4, SOX2, NANOG, the XIST gene order designs corresponding primer for contrast.

(11) RT-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

(12) RT-PCR reaction: 50-60 ℃, 30-40 circulation

(13) preparation 1.5% sepharose, electrophoresis 30min observes amplified band, the record result.

4, differentiation capability detects

(1) the vitro differentiation ability detects

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 once in per 2 days, and formed to embryoid body.

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.

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).

(2) differentiation capability detects in the body

Teratoma forms: after embryonic stem cell digestion is centrifugal, make 1 * 10 ⁶The cell suspension of/ml, be inoculated into 6 week male Reconstruction in Sever Combined Immunodeciency in age (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.

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 shows that FY-hpES 018 cell shows typical embryonic stem cell form, presents the alkaline phosphatase activities strong positive, expresses TRA-1-60, TRA-1-81, and SSEA-4 (Fig. 3), but do not express SSEA-1.Important totipotency genes involved Oct-4, Sox2, Nanog also express in FY-phES 018, and the ES clone FY-hES-1 of normal fertilization in contrast also obtains identical result (Fig. 4).

The presentation of results of EB differentiation, the cell of its differentiation shows human smooth muscle Actin muscle (mesoderm), alpha-fetoprotein (entoderm) and Nestin (ectoderm) the antibody staining positive.The result shows that FY-hpES018 has the ability that is divided into three germinal layers.

Vitro differentiation result shows, can form teratoma, it has the feature of all three germinal layers section statining inspection explanation, comprises entoderm (intestinal epithelial cells), mesoderm (cartilage and muscle) and ectoderm (tesselated epithelium, neuroderm and neuroganglion) are (Fig. 2).

Two, inventor's parthenogenetic embryo tire stem cell and derivative thereof determines

1, chromosome karyotype analysis

(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.

(2) with suction pipe piping and druming, most of circular cell metaphase is come off at the bottom of ware, nutrient solution is changed in the centrifuge tube, with after the PBS cleaning one time, add an amount of 0.05% trypsin solution effect 5min again, add nutrient solution and blow and beat the collecting cell suspension repeatedly; Centrifugal 5 minutes of 1000rpm abandons supernatant liquor, stays sedimentation cell.

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

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

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

(7) suction discards most of supernatant liquor, stays 0.5ml left and right sides supernatant liquor and cell precipitation, gets 1-2 behind the mixing and drips sheet, puts 65 ℃ of roasting sheets and spends the night.

(8) chromosome sectioning for preparing dyes 10min with 10% Giemsa stain of fresh configuration after 0.25% trysinization G shows band, flowing water is rinsed well, drying.

(9) karyotype of analysis cell 20～40 metaphases under the high power lens adopts Leica chromosome analysis software observes result.

2, fluorescence in situ hybridization fish analysis

The ES cell suspending liquid sheet that wets is fixed, 63 ℃ of dried overnight, and 70%, 85% and 100% sequential concentration dehydrated alcohol hybridization down then.FISH uses Vysis

PGT polychromatic probe (Vysis company, numbering 32-131080), (LSI 13, SpectrumRed comprising 5 probes; CEP 18, SpectrumAqua; LSI 21, SpectrumGreen; CEP X, SpectrumBlue; CEP Y SpectrumGold).Fluorescently-labeled probe is with damping fluid dilution in 1: 3,73 ℃ of sex change 5 minutes, 37 ℃ of hybridization 4 hours.PBS washes 3 times then, and fluorescent microscope is observed down.

The result shows: FY-phES 018 is 46 in the caryogram in the 10th generation, and XX (Fig. 2) MLPA analyze to show that lonely female cell has and the normal contour X peak type of XX caryogram cell,, what FISH also proved this cell has two X chromosomes really.But since the 20th generation, find that the caryogram of this cell has become mosaic, 46, XX/45, X0 (division phase ratio 64: 36).Along with the increase of generation, 45, the division phase ratio of X0 increases, and to the 35th generation, can not find 46 in the G band is analyzed, the division phase of XX, cell has become 45 fully, the caryogram of X0 (Fig. 2).MLPA analyzes and shows that lonely female cell has only half high X peak type of normal XX caryogram cell, analyzes and has only an X chromosome.Constantly go down to posterity external, lose (Fig. 2) of an X chromosome taken place in lonely female cell.

3, detect the lonely female source of phES clone

(1) HLA somatotype

The sequence pcr Auele Specific Primer is used for HLA somatotype (biotest landsteinerstr.dreieich Germany, biotest human leucocyte antigen test kit).The PCR product carries out agarose gel electrophoresis, and utilization Biotest SSP somatotype software is determined the DNA band under UV-light, with to human leucocyte antigen-A, human leucocyte antigen-B and HLA-DR site are analyzed. the strict contrast agents specification sheets of all manipulations.

(2) individual recognition (analysis of STR site)

Adopt Qiagen to organize test kit (Qiagen) to extract cell genomic dna.The DNA that extracts carries out pcr amplification with the Promega PowerPlex 16 System kit (Promega) that contain 16 different gene locuss, goes up at full-automatic ABI 3100 genetic analysis instrument (Applied Biosystem) then and analyzes with capillary electrophoresis.16 STR (STR) site is respectively D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, Amelogenin, vWA, D8S1179, TPOX and FGA.

The HLA somatotype has shown the HLA-A of lonely female stem cell ,-B and DRB site and ovum donor half coupling.Different loci on 12 different karyomit(e)s that 32 STR sites are dispersed in also shows this clone and ovum donor's affinity. all these results show that this clone is to be derived from lonely female activation (Fig. 2).

4, the lonely female stem cell imprinted genes under the undifferentiated state is analyzed

In the female ES cell of orphan, owing to lack the gene in paternal source, so paternal expressing gene (being maternal imprinted genes) is expression not to be arranged.In this experiment, chosen paternal imprinted genes H19, maternal imprinted genes IGF2 and SNRPN, and autosomal gene GNAS is at FY-phES 018 and FY-HES-1 compare analysis in-7.

Qiagene one one step RT-PCR kit for testing is used in lonely female stem cell imprinted genes analysis under the undifferentiated state.Selected imprinted genes comprises H19, IGF2, and SNRPN and GNAS. polymerase chain reaction condition are 55 ℃, 45 circulations.2% polyacrylamide gel electrophoresis is analyzed the PCR product, and GAPDH is as a positive control.Negative control confirms that no genomic dna pollutes.

The result shows that in FY-hpES 018 cell, IGF2 and SNRPN all do not detect, and H19 and GNAS express the positive.Above-mentioned all genes are at FY-HES-1, all can detect expression in-7.This result provides further evidence (Fig. 5) for the lonely female source of FY-phES 018.

5, the gene chip analysis of imprinted genes

Human Genome Oligo Set Version 2.1 Genechip, comprising 21522 Human genome probes, 34 known imprinted genes are used to this research.Use Cy5 and Cy3 dye marker respectively from total RNA of female stem cell of orphan and FY-hES-4 cell extraction; microarray data is analyzed with AgilentG2567AA FeatureExtraction Software; in pairs the comparing in twos of mRNA level (40 generations, 3 independently experiments) has and do not have different expression patterns to detect in the stem cell in female stem cell of orphan and normal fertilization ovum source imprinted genes.

The result shows that most of maternal imprinted genes all are to be the downward modulation state, and most of paternal imprinted genes all is to be the rise state.Also found the expression disorder of indivedual maternal imprinted genes, as PEG10.

6, dna methylation analysis

To undifferentiated lonely female stem cell (46, XX/45, X0, the 30th generation) and FY-hES-7 (46, the research of epigenetics steady state and methylation level has been carried out with the Xist promoter region in marking control center (IC) zone of the methylation patterns of human SNRPN gene XX, the 30th generation).Measure with methylation status of PTEN promoter (MSP).PWS and patient AS and normal people DNA sample are in contrast.7% polyacrylamide gel electrophoresis carries out the PCR product analysis.

The result shows, has only the maternal side SNRPN allelotrope site (174bp) that methylates can be detected in FY-phES 018, do not have unmethylated paternal allelotrope (100bp) (Fig. 5).This is consistent with not expressing of SNRPN gene in the detection of front imprinted gene.The lonely female source of the complete maternal gene really of the cell that this also confirms from another side.In contrast, methylate and all there be (Fig. 5) in two the allelotrope sites that do not methylate in FY-HES-7.

MSP Analysis of X ist gene promoter position point shows, the FY-HES-7 of XX caryogram exists two the xist gene promoter area allelotrope that do not methylate and methylate, and have in early days among the FY-phES 018 of two active X chromosomes, only found methylated allelic xist gene promoter site (Fig. 5).

7, x chromosome inactivation state

Human androgen receptor's gene first exon contains a height polymorphism trinucleotide repeats sequence.Found to be no less than Hpalland/or Hhal site methylated relevant of 100bp distance at present with the X inactivation from this polymorphism STR (STR).The lonely female stem cell methylation state of different generations is analyzed with MSP.The genomic dna that extracts is respectively applied for two cover PCR and detects.Wherein a cover is a methyl X allelotrope, and another set of is to be unmethylated allelotrope.The MSP primer is: PrimerARM-F 5 '-GCG AGC GTA GTA TTT TTC GGC-3 ', Primer ARM-R 5 '-AACCAA ATA ACC TAT AAA ACC TCT ACG-3 '; Primer ARU-F 5 '-GTT GTG AGTGTA GTA TTT TTT GGT-3 ', Primer ARU-R 5 '-CAA ATA ACC TAT AAA ACCTCT ACA-3 '.The explanation of contrast agents box is increased and the gel interpretation of result.Male sex's hemocyte DNA of the general methylated DNA of the CpGenome of sodium bisulfite-conversion (Chemicon, Temecula, CA, http://www.chemicon.com) and sodium bisulfite-conversion is as positive control.Genetic analyzer 310 is analyzed clip size.The PCR product of androgen receptor gene is between 177 to 221bp.

The result shows that in FY-HES-7 (46, XX, 30 generations), existing activity has the X chromosome of inactivation again.What is interesting is, find FY-phES 018 15 generations (46, XX) and 30 generations (46, XX/45, X0) the X chromosome of inactivation does not exist, and illustrate, in the early stage generation cell of FY-phES 018, the X chromosome dna methylation is the minimizing of globality ground.In the external process that constantly goes down to posterity, because the unstable of X chromosome has been lost an X chromosome gradually, and in the detection of x chromosome inactivation, keep always and have only the chromosomal state of active X, and do not have to find to have inactivation X chromosome have a (see figure 6).

Claims (3)

1, a kind of human parthenogenetic embryo stem cell line and derivative thereof with two active X chromosomes is characterized in that:

(a) caryogram of this human parthenogenetic embryo stem cell line and derivative thereof was " 46, XX " in the past in 10 generations; Since the 20th generation, losing of an active X chromosomes taken place in the part cell, and the caryogram of this clone has become mosaic " 46, XX/45, X0 "; Along with the increase of generation, 45, the division phase ratio of X0 increases; To the 35th generation, caryogram is " 45, X0 ", and all cells of this clone has all taken place by losing of one active X chromosomes;

(b) in the 15th generation caryogram " 46, XX " and the 30th generation caryogram " 46, XX/45; X0 ", there is not the X chromosome of inactivation to exist, in the detection of x chromosome inactivation, keep always and have only the chromosomal state of active X, and do not have discovery that the existence of the X chromosome of inactivation is arranged;

(c) this human parthenogenetic embryo stem cell line and derivative thereof prepare by following steps:

A. obtain the sophisticated ovocyte that is in MII;

B. activate the second polar body of ovum and discharge, impel its female pronucleus to double to form zygoid, cultivate this activated cell, induce this cell to become the embryo of containing recognizable inner cell mass and trophectoderm;

C. separate described inner cell mass, change in the substratum of differentiation capable of inhibiting cell and cultivate, make these cells keep undifferentiated multipotency state, and go down to posterity, freezing, identify through the whole bag of tricks, confirm as parthenogenetic embryo stem cell line and derivative thereof.

2, human parthenogenetic embryo stem cell line according to claim 1 and derivative thereof is characterized in that, the step a of described preparation comprises:

Collect corolla mound mixture, in the nutrient solution that contains 5-10% blood serum substituting product, cultivated 0.5-1 hour;

This corolla mound mixture is exposed in the Unidasa of 80IU/ml 20 seconds～1 minute, removes granulosa cell, washing for several times in containing the nutrient solution of Hepes; And

Microscopically is observed, and the MII phase ovocyte of selective maturation carries out next step lonely female activation.

3, human parthenogenetic embryo stem cell line according to claim 1 and derivative thereof is characterized in that, the step b of described preparation comprises:

This ovocyte is acted on 3-5 minute in the body early embryo nutrient solution that contains 5 μ M-10mM Calcium ionophores;

Change over to contain in the body early embryo nutrient solution that 1-5mM protein phosphorylation inhibitor 6-Dimethylamino pyridine is 6-DMAP and 1nM-1mM NSC 630176 and act on 4-6 hour;

After test under microscope confirms to discharge second polar body, continue in containing the body early embryo nutrient solution of 1nM-1mM NSC 630176, to activate 8-12 hour;

The flush away NSC 630176 changes ovocyte over to cultivate in the body early embryo nutrient solution 2-3 days, and the blastaea nutrient solution was cultivated 2-3 days more then, formed early stage blastaea;

The blastaea that early stage blastaea is moved into the rh-bFGF that contains 1000-2000IU/ml people's recombinant leukemia inhibitor factor and 2-10ng/ml is optimized substratum continuation cultivation 2-3 days, obtains described human parthenogenetic embryo stem cell line with two active X chromosomes.

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