CN101914491A - Method for obtaining parthenogenetic embryonic stem cells - Google Patents

Abstract

The invention relates to a method for obtaining parthenogenetic embryonic stem cells. The parthenogenetic embryonic stem cells are isolated from IPN embryos discarded in IVF; and over 99 percent of areas of genomes of the parthenogenetic embryonic stem cells are discovered to be homozygous by the analysis of an SNP chip, and hereditary substances of the parthenogenetic embryonic stem cells are proved by DNA fingerprints to be completely derived from mothers without the participation of the genomes of fathers. Through the method, the IPN embryos discarded in the IVF processes are proved to be a source for the isolated homozygous parthenogenetic embryonic stem cells, so the obtained homozygous parthenogenetic embryonic stem cells have simple genetic background and HLA lotus homozygosis, can meet the organ transplantation requirements of oocyte suppliers and more people with the same HLA types, are important seed cell sources for cell replacement therapy, and in addition, are effective tools for researches on parthenogenetic development and the functions of imprinted genes.

Description

A kind of method that obtains parthenogenetic embryo stem cell line

Technical field

The present invention relates to a kind of method that obtains parthenogenetic embryo stem cell line.

Background technology

Parthenogenesis (Parthenogenesis) is meant under the situation that does not have sperm to participate in, is an embryo's process by ovum direct development.Parthenogenesis is the normal modes of reproduction of nature, comparatively general insect, the also visible parthenogenesis phenomenon of reptiles, fish, birds, if but mammiferous parthenogenetic embryo tire is modified and can't be born without intergenic suppression, usually after embryo plantation soon, because embryo outside organization's dysplasia, can't form normal placenta and die young.Though the gynecogenic embryo of Mammals can't grow normal birth, the chimeric embryo that lonely female cell and normal cell form can life birth.The lonely female mosaic test (Thomson, 1998) of mouse is injected into the embryo of normal fertilization to lonely female cell, can observe lonely female cellular integration in the histoorgan of three germinal layers of embryo, and the growth of participation corresponding organ tissue.Nineteen ninety-five, Nature Genetics has also reported 14 a years old spadger who has lonely female cell chimerism, contains the cell in the lonely female source of part in his skin, peripheral blood leucocyte is then fully by the female cellularity of orphan, caryogram shows as 46, XX (Strain, 1995).These phenomenons have illustrated that lonely female cell can participate in the growth of organ in the body, can also clearly bring into play function at the part organ.

Along with the in vitro fertilization and maturation embryo culture technology, become a reality at manipulation in vitro ovum and embryo.In the IVF-ET process, the following transvaginal puncture of B ultrasonic guiding gets ovum and of short duration culturing process subsequently causes accidentally that also ovum is by the female activation phenomena of orphan (Muechler, 1989; Sultan, 1995; Chen, 2009), thereby form the parthenogenetic embryo tire, and further therefrom separate parthenogenetic embryo stem cell line.(parthenogenetic embryonicstem cells pESCs) promptly comes from 1PN embryo (Lin, 2007) in the IVF process to the homozygous human parthenogenetic embryo stem cell line of isolating first strain in the world in this laboratory in 2007.Except natural situation, utilize artificial activation's method also can obtain the parthenogenetic embryo tire.Have article to be reported in the unfertilized freezing ovum, 86.1% ovum can be by the artificial activation, has 96.8% can develop into the parthenogenetic embryo tire among the activated embryo again, finally has 16.7% can grow the blastaea stage (De Fried, 2008).At present, there are many species to utilize artificial activation's method to obtain lonely female blastaea, and obtain parthenogenetic embryo stem cell line final the separation, as: (Cibelli, 2006) such as mouse, rat, rabbit, pig, goat, ox, monkeys.2007, this laboratory and some other study group also successively utilized artificial lonely female activated method to obtain human parthenogenetic embryo stem cell line (Revazova, 2007,2008; Lin, 2007; Mai, 2007).

Parthenogenetic embryo stem cell line is one of method that makes up patient's autospecific multipotential stem cell, the genetic material of pESCs (comprising nucleus and mitochondrial genome) all comes from ovum, its HLA and those for egg are mated fully, the functioning cell that obtains after the differentiation is transplanted in those for egg's body, be equivalent to autotransplantation, can not evoke immunological rejection, be equivalent to " clone " of female patient at reproduction age.Lonely female artificial activation's method mostly is the discharge that stops second polar body at present, thereby keeps the diploid condition of cell.In this case, owing to there is the exchange of genetic material between joint conference between the homologous chromosomes and the non-sister chromatid, the HLA of 70.9% clone shows as heterozygous state (Kim, 2007), the crowd's service that can only mate fully for those for egg or HLA.Another artificial activation's mode, promptly combined utilization A23187 and puromycin allow the discharge of second polar body, and the monoploid of formation dna replication dna before mitotic division once recovers diploid condition, thus the parthenogenetic embryo stem cell line that obtains isozygotying.If can more than enough storage in embryonic stem cell bank common like this HLA haplotype isozygotys even full genome isozygotys clone, help improving the using value of embryonic stem cell bank, reduce the clone quantity that embryonic stem cell bank needs.And because pESCs comes from and do not have the parthenogenetic embryo of potentiality of development tire, for the hESCs that destroys fetal tissues, ethics is disputed on still less, is the important cells system source of embryonic stem cell bank.

The key reason that causes the parthenogenetic embryo fetal hair to educate defective just is the unusual of genomic imprinting, owing to lack father source property genomic imprinting, the outer tissue of embryo can not normal development, and the parthenogenetic embryo tire can be survived at most to conceived mid-term, embryo itself grows slow, but form is normal.In the experiment of mouse mosaic, observe lonely female cell and can be incorporated into the growth that participates in each histoorgan in the fetal tissues, but in embryo outside organizations such as trophectoderm and extraembryonic endoderm, do not see lonely female cell, lonely male cell then is enriched in the embryo outside organization substantially, participate in embryo's growth itself hardly, prompting marking state can influence the differentiation destiny of cell.Building is the marking state that process does not change parthenogenetic embryo tire stem cell, and its imprinted gene expression pattern is still similar to the parthenogenetic embryo tire, promptly express the Disease in Infants imprinted gene, and father source sexual imprinting gene is not expressed.

The potential safety hazard of the following clinical application maximum of parthenogenetic embryo tire stem cell is its imprinted gene abnormal expression.If but this marking is repaired unusually, just might Secure Application.Korea S scientist's experiment has given good prompting.They grow crucial imprinted gene (H19/Igf2 by the genetic modification small portion; Dlk1-Gtl2) can improve the quality of parthenogenetic embryo tire even obtain mouse (Kono, 2004 of lonely female birth; Wu, 2006).Equally, the hemopoietic stem cell in lonely female source can be rebuild the hemopoietic system of mouse after the lethal exposure, reach the hematopoietic stem cell transplantation (Eckardt that stablizes and function is arranged, 2007), and one rare have lonely female chimeric mankind, its peripheral blood leucocyte comes from lonely female cell fully, and the function of can bringing into normal play (Strain, 1995).Therefore, although can not guarantee that all histoorgans of parthenogenetic embryo tire source of human stem cell can normal development, in some histoorgan, can it work at least.They are safety and effective if can carry out heredity and functional analysis proof to the tissue of parthenogenetic embryo tire source of human stem cell, and parthenogenetic embryo tire stem cell also may be represented a kind of cell source of effective tissue substitute treatment.

Summary of the invention

The object of the present invention is to provide a kind of method that obtains parthenogenetic embryo stem cell line, it is to separate the homozygous parthenogenetic embryo tire of the genome stem cell that obtains from the 1PN embryo.

Different with the hESC of report in the past, the parthenogenetic embryo stem cell line that the present invention obtained derives from the 1PN embryo in the IVF process, and the SNP chip analysis is found that its genome zone more than 99% shows as and isozygotied.

The concrete preparation method of the homozygous parthenogenetic embryo stem cell line of this genome can be may further comprise the steps:

1, observes after 12 hours at embryo fertilization, select single protokaryon embryo further to be cultured to the blastaea stage;

2, cut the blastaea inner cell mass, be seeded on the preprepared feeder layer cells, and adopt the stem cell media that suppresses cytodifferentiation to cultivate;

3, undifferentiated cell is further gone down to posterity, amplification, frozen, and carry out the evaluation of lonely female feature and cells and characteristic of stem.

The lonely female source of the parthenogenetic embryo stem cell line that the present invention obtained and complete genomic homozygotic state analyze by HLA somatotype, STR site and the SNP chip analysis proves:

The HLA somatotype shown parthenogenetic embryo tire stem cell HLA-A ,-B and-DR site and those for egg half coupling, with paternal HLA do not match fully (Fig. 1).15 STR sites and Amelogenin gene all show as and isozygoty, and with those for egg's allelotrope half-matched, with paternal allelotrope (Fig. 2) inequality substantially.The SNP chip has been analyzed 500,447 SNP sites of full genome altogether, and the result shows that the site more than 99% shows as isozygotys, and heterogeneous site is dispersed in distribution (Fig. 3) in genome range.

The imprinted gene analysis of the parthenogenetic embryo stem cell line that the present invention obtained comprises father source sexual imprinting gene SNRPN, IPW, KCNQ10T1, PEG3, IGF2, Disease in Infants imprinted gene NES55, H19.Wherein SNRPN, IPW, KCNQ10T1, PEG3 all do not detect, and IGF2 only has faint expression, and the expression ratio fetal tissues stem cell of Disease in Infants imprinted gene NES55, H19 strengthens (Fig. 4) to some extent.These results provide further evidence for the lonely female source of chHES-32.

Evaluation through cells and characteristic of stem, this parthenogenetic embryo stem cell line chHES-32 expresses stem cell specific marker (Fig. 5) and versatility genes involved, keep normal diploid caryogram 46, XX, have strong telomerase activation (Fig. 6), can be in vivo and in vitro to the cytodifferentiation (Fig. 7) of three germinal layers.

This strain provided by the invention separates the homozygous substantially parthenogenetic embryo stem cell line of genome and the derivative thereof that obtain and has great importance for inquiring into father's source property genome and the influence for growth course on genetics and epigenetics of Disease in Infants genome from the 1PN embryo, be the important tool of research imprinted gene biological action in embryo development procedure.In addition, this clone has the ability to three germinal layer cytodifferentiation, and HLA isozygotys in the site, be induced to differentiate into the cell replacement treatment that is used for HLA half-matched patient behind the specific functioning cell, can avoid immunological rejection, be the important seed cell source of stem cell bank, have broad application prospects.

Description of drawings

Fig. 1 is the STR detection case of parthenogenetic embryo stem cell line chHES-32: the STR site of chHES-32 shows as isozygotys, with those for egg's half-matched, and with paternal inconsistent substantially.

Fig. 2 is the snp analysis result of parthenogenetic embryo stem cell line chHES-32.Zone in the genome range more than 99% shows as isozygotys, and the zone less than 1% is a heterozygosis, and is dispersed in distribution and the genome.

Fig. 3 is the imprinted gene expression of parthenogenetic embryo stem cell line chHES-32 and fetal tissues stem cell chHES-35: chHES-35 expresses father source property and Disease in Infants imprinted gene simultaneously, and chHES-32 only expresses Disease in Infants imprinted gene (NES55, H19), and does not express father source sexual imprinting gene (SNRPN, IPW, KCNQ10T1, PEG3, IGF2).

Fig. 4 identifies for the parthenogenetic embryo the form of the foetus attitude and the dyeing of cell characteristic mark in parthenogenetic embryo stem cell line chHES-32 source.A is form under fertilization observation in 12 hours 1PN embryo's the light microscopic; B is 6 days a blastaea form of 1PN fetal development to the; C is the undifferentiated cell form of chHES-32; D is the alkaline phosphatase staining result, shows as strong positive; E-I is respectively stem cell specific marker SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and OCT-4 immunocytochemical stain result, all is shown as the positive.

Fig. 5 is the evaluation of versatility genes involved, Telomerase and the caryogram of parthenogenetic embryo stem cell line chHES-32.A is versatility genes involved OCT-4, NANOG, REX-1, SOX2, LEFTY A, TDGF, FGF4, TERF1, the PCR detected result of THY1 and DPPA2; B is the Telomerase activity result, is shown as high telomerase activation; C is shown as normal diploid caryogram-46, XX.

Fig. 6 is the evaluation of parthenogenetic embryo stem cell line chHES-32 inside and outside differentiation capability.A-C is respectively β-tubulin (A), SMA (B) and AFP (C) for the chHES-32 cells in vitro carries out the immunocytochemical stain result after breaking up for 3 weeks; D-I be the teratoma that in SCID mouse body, forms of chHES-32 cell through section HE coloration result, can find cartilage (D), bone (E), fatty tissue (F), neuro epithelium (G), sebiferous gland (H) and digestive tube epithelium tissues such as (I).

Embodiment

Embodiment one: the acquisition of the homozygous parthenogenetic embryo stem cell line of genome

One, patient's informed consent

This experiment strictly observes the relevant rules (human auxiliary procreation technology standard, 2003) of Ministry of Health of the People's Republic of China.Experiment is through the approval of Ethics Committee of hospital, and whole process is subjected to the supervision of Ethics Committee.Donations residue embryo or gamete are all informed experiment flow in detail for the IVF patient of stem-cell research, comprise that purpose, meaning, gamete or the embryo's of experiment use approach, voluntary, the free principle of donating behavior, investigator's secret, nothing injure principle etc., promptly by complete sufficient explanation and introduction, the contributor is answered and explanation about necessity of inquiry, make the contributor fully understand the Positive and Negative Aspects of donations behavior, under the situation of knowing the inside story fully, independently, voluntary, rational must make responsible decision.Guarantee that all embryos only limit to scientific research and use, forbid to be used for the reproduction purpose.

Two, 1PN embryo's cultivation

Place 1ml to contain the B2 substratum of 10% maternal serum the oocyte corona cumulus complex that takes out, continue ripe the cultivation and waited for insemination in 4～6 hours.Inseminate by the density of 5 * 10 translational motion sperm/ml during insemination, average every 1ml adds 2～3 oocyte corona cumulus complexs.The fertilization situation was observed in 16～18 hours in the insemination back, and the embryo (1PN) who only observes 1 protokaryon is moved in advance and in the 50ul G1.2 substratum droplet of equilibrate overnight, is coated with paraffin oil on it and cultivates in incubator.In the 3rd day morning, 6～8 cell stages of growing are moved on in the 50ul G2.2 substratum droplet be cultured to the separation that the blastaea stage is used for embryonic stem cell.

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

Blastaea by cut mechanically into two, half that contains inner cell mass will be planted in people's embryo fibroblast (the human embryonic fibroblasts through mitomycin C mitotic division deactivation, hEFs, density is 5,000cells/cm2) further cultivate on the feeder layer, substratum is the DFSR substratum, comprising: 75%DMEM/F12,15%KO-SR, the left-handed glutamine of 2mM, 0.1mM beta-mercaptoethanol, 1% non-essential amino acid and 50ng/ml bFGF.Change liquid every other day, after 4～7 days, have the embryonic stem cell-like agglomerate of tiling growth to grow, mechanical picking does not break up part, is divided into to plant behind the fritter to continue to cultivate on new feeder layer, goes down to posterity once in about 7 days.After treating the early stage cell of embryonic stem cell amplification number generation and frozen q.s, transfer to the conventional DFSR substratum long term culture of adding 4ng/ml bFGF.

Embodiment two: the evaluation of parthenogenetic embryo stem cell line

One, the conventional stem cell CHARACTERISTICS IDENTIFICATION of parthenogenetic embryo stem cell line

1. the detection of alkaline phosphatase (AKP)

The Fast Red Substrate Pack test kit that adopts Invitrogen company to provide detects undifferentiated hESCs clone, operation steps is undertaken by the method that test kit provides, ultimate principle is with Gomori naphthyl alcohol phosphoric acid method, be that alkaline phosphatase solves naphthyl alcohol with the Alpha-Naphthyl phosphoric acid water under alkaline condition, form the water-fast salt of red-brown with fast red B salt in the zymophore coupling then.The positive staining result is a bright red.The MEF of periphery is not painted, negative contrast.

2 cell-specific detection of antigens

The multipotent stem cells specific antigens comprises: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, OCT-4; The triploblastica specific marker comprises: AFP (entoderm), β-tubulin (ectoderm), SMA (mesoderm).All adopt indirect immunofluorescence to detect, step is as follows: the ES clone of cultivation fixes 20 minutes with 4% Paraformaldehyde 96; The saturating film of 0.1%Triton-X-100 10 minutes (this step is at nuclear endoantigen OCT-4, and all the other do not need for membrane antigens); Normal goats serum room temperature sealing 30 minutes; Corresponding one anti-4 ℃ of overnight incubation; It is anti-to add Alexa Flour 488-connection two, and the room temperature lucifuge was hatched 1 hour; DAPI redyes nuclear; Fluorescent microscope is observed detected result down and is taken a picture.

The G of 3 hESCs shows the band karyotyping

HESCs is transferred to the pollution of spreading under the no feeder layer system that ware, hEFs-conditioned medium cultivate further amplification and removing people's feeder layer cells with matrigel.Cultivate after 3 days, in substratum, add colchicine (final concentration is 80ng/ml), handled 3.5 hours for 37 ℃.Digest collecting cell with 0.05% trypsinase/0.53mM EDTA.Prepare the karyomit(e) smear by this laboratory ordinary method, Giemsa solution-dyed, gummy mounting, observations under oily mirror.Each sample analysis 5-10 metacinesis phase.

Table 1

Table 1 is the HLA detection case of parthenogenetic embryo stem cell line chHES-32: the HLA site of chHES-32 shows as isozygotys, with those for egg's half-matched, and with paternal inconsistent fully.

The detection of 4hESCs versatility genes involved

Collect undifferentiated hESCs agglomerate, use the Trizol extracted total RNA, the reverse transcription test kit that provides with Fermentas company is cDNA with 1ug RNA reverse transcription, be that template is carried out RT-PCR again with cDNA, to detect the wherein expression of versatility genes involved OCT3/4, Nanog, REX-1, SOX2, THY1, TDGF1, TERF1, LEFTB, DPPA2 and FGF4, β-actin is the reaction system positive control.The PCR reaction conditions: 95 ℃ 1 minute 30 seconds; 30 circulations (94 ℃ 40 seconds, 54 ℃-64 ℃ 40 seconds, 72 ℃ 40 seconds); 72 ℃ were extended 7 minutes.

The detection of 5hESCs differentiation potential

5.1 experiment-teratomatous preparation in the body:

Collect and do not break up the hESCs clone, about 1-2 * 106cells is injected in the hindlimb muscle of male SCID mouse in 6-8 age in week, observes teratomatous formation situation.8-12 takes out tumour after week, after the section of conventional paraffin embedding, and the HE observation of dyeing.

5.2 the preparation of experiment in vitro-embryoid body:

With the mechanical agglomerate that cuts into big or small basically identical of hESCs clone, collect hESCs agglomerate suspension culture in the 60mm Micro-Organism Culture Dish.Substratum is the EB substratum, does not promptly contain the DFSR substratum of bFGF, changes liquid every day, and suspension culture is after 7 days, is inoculated in advance in six orifice plates of handling with 0.1% gelatin adherent culture 14 days, carries out the immunohistochemical staining of triploblastica mark then.

Two, the specificity of parthenogenetic embryo stem cell line of the present invention and derivative thereof is identified

1 complete genomic single nucleotide polymorphism (SNP) is analyzed

Embryonic stem cell is transferred to further amplification under the no feeder layer system that Matrigel shop ware, hEFs-conditioned medium cultivate, the pollution of removing people's feeder layer cells simultaneously.0.05% trysinization collecting cell is with DNeasy Blood ﹠amp; Tissue kit extracting complete genome DNA.We adopt 5.0 pairs of 2 normal fertilization hESCs systems of GeneChip Human Mapping SNP Array and 2 pESCs systems to carry out full genome SNP phenotypic analysis, help judge the source of clone according to the gene type in full genome SNP site.Analytical procedure is: all karyomit(e)s beyond the Y chromosome are from centromere position, and every 1Mb to karyomit(e) two ends subregion, calculates the heterozygosis frequency (the SNP number of sites of heterozygosis/total SNP number of loci) of each unit interval SNP as a unit then.Our regulation: if the heterozygosis frequency of SNP is lower than 5% in the single interval, judge this zone so for isozygotying, otherwise, if the heterozygosis frequency is higher than 5%, judge that then this zone is a heterozygosis.Because the chHES-8 caryogram is 46, XY, its X chromosome should show as and isozygoty, and chip results shows that it is heterozygosis that this X chromosome has 0.5% zone, our heterozygosis frequency that this karyomit(e) is total is controlled the word error probability that whole gene type is analyzed as the systematic error of SNP chip with this.

The RT-PCR of 2 imprinted genes detects

Use the Trizol extracted total RNA, the reverse transcription test kit that provides with Fermentas company is cDNA with 1ug RNA reverse transcription, is that template is carried out RT-PCR again with cDNA, detects the expression of imprinted gene, and β-actin is the reaction system positive control.The PCR reaction conditions: 95 ℃ 1 minute 30 seconds; 25-30 circulation (95 ℃ 40 seconds, 54 ℃-64 ℃ 40 seconds, 72 ℃ 40 seconds); 72 ℃ were extended 7 minutes.

The 3DNA fingerprinting

Embryonic stem cell is transferred to further amplification under the no feeder layer system that Matrigel shop ware, hEFs-conditioned medium cultivate, the pollution of removing people's feeder layer cells simultaneously.0.05% trysinization collecting cell is used DNeasyBlood ﹠amp; Tissue kit extracting complete genome DNA, 3130 genetic analysis instrument carry out the dna fingerprint analysis.Adopt

16system analyzes 15 STR sites and Amelogenin altogether, and operation steps is referring to the test kit specification sheets.

The detection of 4HLA somatotype

To embryonic stem cell all carry out human leucocyte antigen HLA-A ,-B ,-dna typing in DR site.Embryonic stem cell is transferred to further amplification under the no feeder layer system that Matrigel shop ware, hEFs-conditioned medium cultivate, the pollution of removing people's feeder layer cells simultaneously.0.05% trysinization collecting cell is with DNeasy Blood ﹠amp; Tissue kit extracting complete genome DNA.The AB/DR SSP UniTray that adopts Invitrogen company to provide carries out the detection of HLA somatotype, the method for utilization PCR-SSP, and concrete steps are referring to the test kit specification sheets.

Claims (2)

1. method that obtains parthenogenetic embryo stem cell line is characterized in that this parthenogenetic embryo tire stem cell separates to obtain from the 1PN embryo.

2. the method for acquisition parthenogenetic embryo stem cell line according to claim 1 is characterized in that it may further comprise the steps:

(1) observes after 12 hours at embryo fertilization, select single protokaryon embryo further to be cultured to the blastaea stage;

(2) cut the blastaea inner cell mass, be seeded on the preprepared feeder layer cells, and adopt the stem cell media that suppresses cytodifferentiation to cultivate;

(3) undifferentiated cell is further gone down to posterity, amplification, frozen, and carry out the evaluation of lonely female feature and cells and characteristic of stem.

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