Differentiation Potential of Parthenogenetic Embryonic Stem Cells Is Improved by - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 01:01 |只看该作者 |倒序浏览 |打印

作者：Takafusa Hikichia, Sayaka Wakayamaa, Eiji Mizutania, Yasuhiro Takashimab, Satoshi Kishigamia, Nguyen Van Thuana, Hiroshi Ohtaa, Hong Thuy Buia, Shin-Ichi Nishikawab, Teruhiko Wakayamaa ' k' J/ g! U& W! }+ e- G+ c

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      【摘要】. X, S. I' J- P
   Parthenogenesis is the process by which an oocyte develops into an embryo without being fertilized by a spermatozoon. Although such embryos lack the potential to develop to full term, they can be used to establish parthenogenetic embryonic stem (pES) cells for autologous cell therapy in females without needing to destroy normally competent embryos. Unfortunately, the capacity for further differentiation of these pES cells in vivo is very poor. In this study, we succeeded in improving the potential of pES cells using a nuclear transfer (NT) technique. The original pES cell nuclei were transferred into enucleated oocytes, and the resulting NT embryos were used to establish new NT-pES cell lines. We established 84 such lines successfully (78% from blastocysts, 12% from oocytes). All examined cell lines were positive for several ES cell markers and had a normal extent of karyotypes, except for one original pES cell line and its NT-pES cell derivatives, in which all nuclei were triploid. The DNA methylation status of the differentially methylated domain H19 and differentially methylated region IG did not change after NT. However, the in vivo and in vitro differentiation potentials of NT-pES cells were significantly (two to five times) better than the original pES cells, judged by the production of chimeric mice and by in vitro differentiation into neuronal and mesodermal cell lines. Thus, NT could be used to improve the potential of pES cells and may enhance that of otherwise poor-quality ES cells. It also offers a new tool for studying epigenetics. ; p3 I1 |4 C; Z5 {: P) u
      【关键词】 Parthenogenesis Nuclear transfer Reprogramming Embryonic stem Regenerative medicine+ `" ]( U, y9 z3 i6 E2 q9 _" n2 c) w
   INTRODUCTION
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There has been much research into the applications of human embryonic stem (ES) cells in regenerative medicine, where ES cells have been envisioned as potential resources for cell or tissue replacement therapy. However, as with any allogeneic material, ES cells derived from fertilized blastocysts¡ªand the progeny of such cells¡ªinevitably face the risk of immunorejection on transplantation. Therefore, ES cells derived from embryos cloned from the host's own cells by somatic cell nuclear transplantation (NT-ES cells) represent a potential solution to the problem of rejection, as any replacement cells would be genetically identical to the recipient's cells . Such techniques can help reduce ethical concerns, but they could not be applied to humans therapeutically as any cell line would differ genetically from the patient.1 y6 N! H! c9 ]% r& U' A4 f

One other possibility is to establish ES cell lines from parthenogenetically activated embryos (parthenogenetic embryonic stem . Thus, there may be limitations to the use of pES cells in human regenerative medicine.0 A- `" o; Y# a8 B% X3 i

On the other hand, it has been demonstrated that somatic cell nuclear transfer (SCNT) techniques can reprogram donor cell nuclei from a differentiated state to an undifferentiated one . Here, we hypothesized that successive rounds of NT might also reprogram the epigenetic status of parthenogenetically derived ES cell nuclei. Following the reestablishment of NT-pES cells, these might improve the potential for differentiation over that of the original pES cells. To test this hypothesis, we established several second- and third-generation NT-pES cell lines from original pES cell lines by NT. These were examined for their potential to differentiate in vivo and in vitro and were compared with the original pES cell lines. We also examined the DNA methylation status of the differentially methylated domain (DMD) H19 and the differentially methylated region (DMR) IG, regions upstream of Dlk1, which defines a cluster of imprinted genes.

MATERIALS AND METHODS

Reconstruction of Hybrid Oocytes and Induction of Parthenogenetic Embryos Expressing Green Fluorescent Protein
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In this study, we established original pES cell lines by combining two genetically different mouse oocytes instead of the usual method of producing parthenogenetic embryos from single oocytes (Fig. 1). Genetically, the resulting constructs are gynogenetic F1 hybrids, which may help avoid any inbred degeneracy or genetic imbalance from an F2 background. Moreover, an F1 genetic background is known to enhance the SCNT cloning success rate  for 1 hour. Reconstructed oocytes that extruded two second polar bodies were cultured 4 days further in CZB medium. When the parthenotes developed to blastocysts, they were used for the establishment of pES cells.2 j5 G" B$ \& n, A

Figure 1. Diagram of hybrid parthenogenetic/gynogenetic mouse embryo. The metaphase II chromosome-spindle complex of a 129/Sv strain mouse oocyte was removed and fused with a C56BL/6-GFP oocyte. Those oocytes become tetraploid and possess two MII spindles. When reconstructed oocytes are activated by SrCl2, those oocytes extrude two second polar bodies and became diploid embryos. These embryonic cells express GFP but also have the potential to enhance nuclear transfer cloning efficiency because of their F1 genetic heterogeneity. Abbreviation: GFP, green fluorescence protein.
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Figure 2. In vitro differentiation of parthenogenetic embryonic stem (pES)-nuclear transfer (NT) and pES cells. (A¨CD): The NT-pES cells showed pluripotency and potential for differentiation. (A): Phase contrast imaging of PG1(III)-1 NT-pES cells. (B): Expression of green fluorescence protein (GFP) under UV light. (C, D): Cells positive for the expression of the pluripotency marker Oct3/4 (C) and embryoid body formation (D). (E¨CG): Chimeric mice derived from PG1(I) pES cells. (H¨CJ): Chimeric mice derived from PG1(III)-1 NT-pES cells. Brain (F, I) and heart (G, J) tissues were extracted from those chimeric mice. The contributions of pES/NT-pES cells were detected from GFP fluorescence signals, which were expressed only in the pES or NT-pES cells (E'¨CJ'). PG1(III)-1 cells contributed more to chimeric mice than PG1 cells. (K, L): Spectral karyotyping with fluorescence in situ hybridization painting of long-passaged pES cells showing abnormal karyotypes. (K): All cells had lost one X chromosome and often showed trisomy of chromosome 8. (L): PG3 and PG3(III)-1, the third generation of cell line PG3(I) showing triploid karyotypes in all cells. (M): Comparison of contribution rate of pES and NT-pES in each organ of the chimeric mice studied. We estimated the contribution rate by fluorescence-activated cell sorting analysis using the GFP signal as a marker. The rates of contribution of some NT-pES cells¡ªPG1(II)-1, PG1(II)-3, and PG2(II)-1¡ªwere higher than those of the pES cells PG1(I) and PG2(I). The contribution rates of PG1(I) long-passaged cell lines were not higher than those of the original PG1 cell line. However, the contribution rates of all NT-pES cell lines were not higher than control embryonic stem cells. Details are shown in Table 3.1 r" m5 r, v/ }8 w0 u3 }9 [, r# x

Establishment of Parthenogenetic ES Cells
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Parthenogenetic or cloned embryos at the morula or blastocyst stages were used to establish ES cell lines as described .
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Nuclear Transfer and Establishment of NT-pES Cell Lines
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Nuclear transfer was performed as described . All established cell lines were examined for the production of GFP to demonstrate the success of nuclear transfer. Even if some parthenogenetic embryos had been produced accidentally, they could have been detected because they would not express the gene for GFP. As a control, 129/Sv oocytes were activated parthenogenetically with cytochalasin B, which prevented second polar body extrusion and allowed the establishment of diploid parthenogenetic ES cell lines.

Immunohistochemistry7 W7 _0 o6 F7 k- L  V6 ?
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We used the appropriate manufacturers' staining procedures throughout. Alkaline phosphatase staining was according to the manufacturer's protocol (Sigma-Aldrich). Immunohistochemistry was performed using the following antibodies: anti-Oct3/4 (monoclonal 1:100; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), anti-Nanog (monoclonal 1:200; ReproCELL Inc., Tokyo, http://www.reprocell.com/en); anti-stage-specific embryonic antigen (AAEA)-1 and -3 (monoclonal 1:100; Chemicon, Temecula, CA, http://www.chemicon.com). Alexa Fluor 568-labeled secondary antibodies (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) were used for detection as appropriate.

Karyotype Analysis by Giemsa and Spectral Karyotyping with Fluorescence In Situ Hybridization Painting
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Chromosomes from NT-pES cells were stained using Giemsa and spectral karyotyping with fluorescence in situ hybridization (SKY-FISH) chromosome painting techniques (Spectral Imaging Ltd., Vista, CA, http://www.spectral-imaging.com) according to the manufacturer's protocols. All NT-pES cells were used within five passages except for the PG1 long-passaged cells (described below), for which 30 passages were used. More than 50 metaphase nuclei (for Giemsa staining) or 15¨C20 metaphase nuclei (for SKY-FISH staining) were examined for each examined cell line.) ^8 M$ ^( |0 P) j$ C

Production of Chimeric Mice, g, L& C. v( [7 _: G

Blastocyst injections were performed as described . Briefly, ICR females were mated with ICR males, and eight-cell embryos were collected at 3 days post coitum (dpc). In some cases, BDF1 female mice mated with BDF1 male mice were also used to collect embryos. The next day, 15¨C20 of the pES or NT-pES cells were injected into each blastocyst, and those injected embryos were transferred into 2.5-dpc pseudopregnant ICR females. At 18.5¨C19.5 dpc, some of the pregnant females were euthanized by cervical dislocation, and chimeric offspring were obtained by cesarean section. Others were delivered naturally, and the chimeric offspring were examined for coat color contribution and germline transmission.* M4 t8 [: L' U6 ]
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In Vivo Differentiation Analysis
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The rates of contribution of pES or NT-pES cells to each chimeric mouse were analyzed. Six organs (brain, heart, liver, kidney, intestine, and skin) from the chimeric offspring were collected at 18.5 or 19.5 dpc, and small pieces of each tissue were homogenized with 0.25% trypsin. After filtration and washing, single cells were resuspended, and GFP-positive (parthenogenetic origin) or -negative (host embryo origin) cell numbers were counted using fluorescence-activated cell sorting (FACSAria cell sorter; BD Biosciences, San Diego, http://www.bdbiosciences.com), or collected into a separate tube. Nonviable cells were eliminated by 7-amino actinomycin D staining (BD Biosciences). The number of nonviable cells was 3% or less of the total in all internal organs. It is known that the GFP gene promoter is not subject to gene silencing and that GFP-positive cells can contribute to all internal tissues of adult chimeric mice, except for erythrocytes and hair .. Q4 f, m% k* B6 s- s

In Vitro Differentiation
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Induction of ES cell differentiation into neuronal and mesodermal cell lines was performed as described , and the rate of differentiation was analyzed using a FACSCalibur cell sorter (BD Biosciences) at 4 days after induction. Two normally fertilized embryo-derived ES cell lines, E14tg2a (unknown passage number) and 129B6F1 (established in our laboratory and used within five passages), were used as controls.5 G$ v8 a' p/ h/ R' O
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Allele-Specific DNA Methylation Analysis
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Genomic DNA was extracted from each set of cultured ES cells using DNeasy Tissue Kits (Qiagen, Hilden, Germany, http://www1.qiagen.com). The DNA was subjected to bisulfite modification, polymerase chain reaction (PCR) amplification, subcloning, and sequencing as described  with some modifications. The probes used were derived from PCR products, using the following primer sets: H19-bi forward (F), 5'-GGT TGA GGA TTT GTT AAG GTG TTA TTG-3'; H19-bi reverse (R), 5'-TAA TAA CTA ATT TAA ACA CTC CTC ACC-3'; IG-bi-F, 5'-AAG GTA TTT TTT ATT GAT AAA ATA ATG TAG-3'; and IG-bi-R, 5'-CCT ACT CTA TAA TAC CCT ATA TAA TTA TAC-3'.
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RESULTS

Establishment of pES and NT-pES Cells
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To establish original pES cell lines, 20 reconstructed oocytes were activated parthenogenetically, and six of these developed into blastocysts. When these were plated onto ES cell establishment medium, four first-generation (I) pES cell lines were established and labeled PG1(I) to PG4(I) (Table 1). Next, newly established pES cell nuclei were transferred into enucleated B6D2F1 oocytes, and the resulting NT blastocysts were used for new ES cell derivation . As a result, totals of 18, 5, 7, and 6 second-generation NT-pES cell lines were obtained from the PG1(I), PG2(I), PG3(I), and PG4(I) pES cell lines (75%¨C88%), respectively (Table 1). We labeled the second-generation (II) NT-pES cell lines PG1(II)-1 to PG1(II)-18, derived from PG1(I) pES cell nuclei (Table 1). We repeated this process and obtained totals of 9, 15, 12, and 12 third-generation (III) NT-pES cell lines (71%¨C83%), respectively, which were labeled PG1(III)-1 to PG1(III)-9 and so forth (Table 1).

Table 1. Establishment of pES and NT-pES cell lines
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All of the established pES/NT-pES cell lines were positive for alkaline phosphatase, Oct3/4 (Fig. 2C), Nanog, and SSEA-1 (ES cell marker) and formed embryoid bodies (evidence of totipotency; Fig. 2D). By microscopy (x20), no difference could be observed between pES and NT-pES cells.. j" @# {) h  j5 E3 g( B
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Karyotyping& v2 k$ s/ y7 P' j5 E' m
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Seventeen cell lines (four original pES cell lines, one PG1(I) long-passaged cell line, four second-generation NT-pES cell lines, and eight third-generation NT-pES cell lines) were karyotyped by Giemsa staining and SKY-FISH painting. These showed that a normal extent of karyotypes was maintained, even to the third generation (58%¨C88% of cells). The only exception was the PG1(I) long-passaged cell, PG3, and its later generations. PG3(I), one of the original pES cell lines, showed trisomy in most cells (87%), and this phenotype was inherited to the third generation as shown in PG3(III)-1 (Fig. 2L).
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In Vivo Differentiation Potential of NT-pES Cells
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Using these four lines of pES and 10 lines of NT-pES cell lines, we compared the potential for in vivo differentiation in chimeric mice produced by cell injection into the blastocoels of ICR or B6D2F2 strain blastocysts. Because these cells were GFP-positive, we could distinguish the injected from the host cells. Chimeric offspring were collected at 19.5 dpc, and the rate of contribution of pES/NT-pES cells was determined by GFP expression under fluorescent microscopy (Fig. 2E¨C2H). The birth rates of chimeric offspring increased with successive generations (48%¨C78%; Table 2), but offspring often died just after birth, irrespective of the generation. We found that the contribution rate of the pES cells was very low; only two pups (18%) were born with high GFP expression in the skin, but they were cannibalized the next day. However, when NT-pES cell lines were used, more than 60% of the chimeric mice showed high contribution rates, as determined by GFP expression throughout the body, irrespective of the cell line. Note that in this experiment, some of the chimeric mice showed growth retardation when either the pES or the NT-pES cell contribution rate was high. This was also reported previously when chimeric mice were constructed by aggregation between parthenogenetic embryos and normally fertilized embryos . As there were several differences between these published studies and those reported here, such as the genetic background of the pES cell lines used, it is difficult to know the reason, but it is possible that our established pES cell lines retained the lethal genetic defects of parthenogenetic embryos, even after NT.
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Table 2. Contribution of parthenogenetic embryonic stem (pES)/nuclear transfer-pES cells to chimeras following cell injection into normal fertilization-derived blastocysts

To further examine the contribution rates of pES and NT-pES cells, three to six offspring were collected randomly from each chimeric line, and six organs (brain, kidney, liver, heart, intestine, and skin) were examined by fluorescence-activated cell sorting (FACS) analysis (Fig. 2F, 2G, 2I; Table 3). The average contribution rate of pES cells in the chimeric mice was only 2%¨C8% in all tissues, as in previous reports , and thus these results did not confirm germline transmission, the most important criterion for genetic normality. However, when chimeric mice were produced from the second generation of NT-pES cells, germline transmission was confirmed in the PG1(II)-1 cell line by natural mating. Moreover, in some organs, especially in the brain and intestine, 7 of 10 examined NT-pES cell lines showed that contribution rates were increased up to fivefold compared with pES cell lines (Fig. 2I, 2I'; Table 3). However, the contribution rates were lower than in control ES cells, and in the heart and liver, the contribution rate did not increase even in the third-generation NT-pES cell line (3% in NT-pES vs. 2% in pES; Fig. 2J, 2J'). On the other hand, such significant improvement in the potential for differentiation in vivo was not observed in long-passaged PG1 cell lines (Fig. 2M). The contribution rate was not improved in successive NT generations, as shown in the PG(III)-1 cell line (Table 3).

Table 3. Contribution of embryonic stem, pES, and NT-pES cells to chimeric offspring following cell injection into normal fertilization-derived blastocysts; ?2 [  B. ?9 ?5 c6 ^

In Vitro Differentiation Potential of NT-pES Cells9 j9 l* p. m$ z- p- K
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We also examined the in vitro differentiation potential of pES and NT-pES cells into neuronal and mesodermal cells. Anti-nestin, anti-TuJ, and anti-tyrosine hydroxylase antibodies were used as markers for primary, intermediate, and terminal differentiation of neuronal cells, respectively , and the rate of differentiation of PG1(I) and PG1(III)-1 cell lines was analyzed by FACS at 4 days (Fig. 3E¨C3G). The rate of differentiation into mesoderm was higher in the NT-pES cells than the pES cells, but it was still lower than in the control ES cells and was the same as that seen for neuronal differentiation. Thus, although parthenogenetic ES cells have a lower potential for differentiation in vivo and in vitro than ES cells, this potential can be improved by NT and by the reestablishment of pES cells as NT-pES cells.
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Figure 3. Differentiation of parthenogenetic embryonic stem (pES)/nuclear transfer (NT)-pES cells. (A¨CD): In vitro potential to differentiate into dopaminergic neural cells. We cultured pES and NT-pES cells on PA6 feeder cells and induced neural differentiation using the SDAI method. Percentages of nestin-positive (A), TuJ-positive (B), and TH-positive (C) colonies, indicating primary, intermediate, and terminal differentiation, respectively, are shown at 5, 7, and 10 days after differentiation. The rate was calculated on the basis of the numbers of 4,6-diamidino-2-phenylindole-positive colonies. (D): The potential for terminal differentiation to neural cells from TuJ-positive cells. Although neither pES nor NT-pES cells showed lower potential for differentiation into neuronal cells, the potential to differentiate into terminal stages from intermediate stages was increased in NT-pES cells, almost to the level of fertilized ES cells. (E): In vitro potential of pES, NT-pES, and E14 cells to differentiate into mesodermal cells. PDGFR- and FLK1/KDR were used as mesodermal markers, and the rate of differentiation was analyzed by fluorescence-activated cell sorting 4 days after differentiation. In differentiated mesodermal cells, the surface markers FLK1 and PDGFR- were detected as higher signals. The rates of differentiation into mesoderm were higher in PG1(III)-1 NT-pES cells (F) than in PG1 pES cells (E) but still lower than control E14tg2a ES cells (G), as with the case of neuronal differentiation. Abbreviations: FLK-1, fms-related tyrosine kinase 1; PDGFRa, platelet-derived growth factor receptor-; SDIA, stromal cell-derived-inducing activity; TH, tyrosine hydrozylase.% c) M3 {+ l9 {, _& P7 w
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DNA Methylation Status of H19-DMD and IG-DMR

To investigate whether nuclear transfer affects the typical epigenetic errors of parthenogenetic cells, we checked the DNA methylation status of H19-DMD and IG-DMR, which should show only hypomethylated alleles in parthenogenetic cells . As shown in Figure 4, we confirmed that the original pES cell lines (PG1(I) and PG2(I)) showed such a hypomethylated status. Interestingly, although the in vivo differentiation potential of NT-pES cell was improved in chimeric mice, the hypomethylation status of all alleles was maintained.& ?; }7 _# p# X: o6 E! V

Figure 4. Differential methylation status of the H19-DMD and IG-DMR alleles. The methylation patterns of the DMRs of H19 (A) and IG (B), which are upstream of Dlk1, were detected using bisulfite sequencing (filled circles, methylated; open circles, unmethylated). Cytosines are shown for a number of independently sequenced templates (horizontal lines). These DMRs are methylated in paternal gametes only. In the normally fertilized cell, approximately half the sequenced templates were highly methylated, presumably the paternal allele. By contrast, in the parthenogenetic cells, all sequenced templates were unmethylated because they possessed only maternal alleles. In the NT-pES cells, almost all sequenced templates were unmethylated, and significant changes were not detected in H19 or IG DMRs, even after nuclear transfer. Abbreviations: DMD, differentially methylated domain; DMR, differentially methylated region; ES, embryonic stem.

DISCUSSION( [0 d* y2 Q/ B% \2 h! x+ {7 n

The established NT-pES cells showed greatly enhanced differentiation potential in vivo and in vitro. When we examined the chimeric mice, the rate of contribution of NT-pES cell varied between organs: for example, brain and intestine showed contribution rates two to five times higher than the original pES cells. This remarkable change occurred in the first round of NT, and successive nuclear transfers produced no significant change in the cell line characteristics. This suggests that the most important genes for embryonic development are reprogrammed by the first NT. This is supported by our previous results, showing that the cloning success rate did not increase when NT was repeated successively several times . It is possible that the effects of NT differ between internal organs or that paternal gene expression is required in particular organs, and this could not be reversed by the NT procedure.- M/ }- n1 p4 B# O, v; l5 u

It is known that there is epigenetic instability between ES cells and their subclones , all the NT-pES cell lines examined here maintained the same methylation status as the original pES cell line for H19. Thus, although we cannot exclude the possibility of NT-based selection, it appears likely that NT techniques could be applied to improve some particular epigenetic disorders of cell lines.# k( ^+ F+ N% B2 ?3 w
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On the other hand, one pES cell line (PG3) and its following NT-pES cell lines were triploid. Probably, when the original reconstructed oocyte was activated, it extruded only a single second polar body. We may have mistaken the first polar body as a second one. Interestingly, this triploid pES cell line could still contribute to chimeric mice, and the karyotypes were maintained after NT, but the rate of contribution of this abnormal cell line was not improved after NT. This suggests that NT can improve only epigenetic disorders, not genetic abnormalities per se.
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In this study, we established NT-pES cell lines from all original pES cell lines, with a very high success rate. Previously, NT-ES cells have been established from somatic cells or ES cells, and the rate of establishment is approximately 30%¨C50% from cloned blastocysts . It seems unlikely to be associated with parthenogenesis per se, because the control ES and pES cells were almost identical except for the imprinted alleles.1 @6 `: t3 @) |7 n6 h
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Embryonic stem cell derivation from parthenogenetically activated oocytes has been envisioned for autologous cell therapy without the need to destroy normally competent embryos or to require donation of another woman's oocytes , their capacity for differentiation is clearly improved by successive nuclear transfer procedures. So far, NT techniques required additional oocytes; however, in this study, only eight oocytes per line were required for establishment (a mean of 12.4% from oocytes; Table 1) because of the high success rate of growing NT-pES cell lines. Thus, we have shown that incompetent ES cells can be improved through in vitro manipulation. Although these nuclei maintained parthenogenetic phenotypes (Fig. 4), those cloned embryos are still destined to die, so ES cell derivation could be done without destroying potentially live individuals. This procedure would help to circumvent the ethical objections to the sacrifice of normal embryos. We have also established a useful tool for investigating the effect of epigenetic modifications, because these NT-pES cells permit the possibility of investigating the roles of imprinted genes.: w' t" n& V9 w

DISCLOSURES$ @/ q8 @! n6 Y" m

The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS# ?. n& C, W1 P. ^7 {8 ^) a

We thank Dr. Sasai, Dr. Watanabe, Dr. J. Cummins, and D. Sipp for discussion and critical reading of the manuscript. We are grateful to the Laboratory for Animal Resources and Genetic Engineering for the housing of mice. T.W. was supported by Grant 15080211, and T.W. and S. N. were supported by a Project for the Realization of Regenerative Medicine from the Ministry of Education, Science, Sports, Culture and Technology of Japan.7 v7 \% V! @0 h$ c2 c8 G" G
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