Mouse cloning and somatic cell reprogramming using electrofused blastomeres

Mouse cloning from fertilized eggs can assist development of approaches for the production of "genetically tailored" human embryonic stem (ES) cell lines that are not constrained by the limitations of oocyte availability. However, to date only zygotes have been successfully used as recipients of nuclei from terminally differentiated somatic cell donors leading to ES cell lines. In fertility clinics, embryos of advanced embryonic stages are usually stored for future use, but their ability to support the derivation of ES cell lines via somatic nuclear transfer has not yet been proved. Here, we report that two-cell stage electrofused mouse embryos, arrested in mitosis, can support developmental reprogramming of nuclei from donor cells ranging from blastomeres to somatic cells. Live, full-term cloned pups from embryonic donors, as well as pluripotent ES cell lines from embryonic or somatic donors, were successfully generated from these reconstructed embryos. Advanced stage pre-implantation embryos were unable to develop normally to term after electrofusion and transfer of a somatic cell nucleus, indicating that discarded pre-implantation human embryos could be an important resource for research that minimizes the ethical concerns for human therapeutic cloning. Our approach provides an attractive and practical alternative to therapeutic cloning using donated oocytes for the generation of patient-specific human ES cell lines.     

治疗性克隆

治疗性克隆是利用核移植技术将病人的体细胞核移植到去核的卵母细胞中 ,使其重编程并发育成囊胚 ,然后再用胚胎干细胞分离技术从克隆囊胚的ICM分离出多能胚胎干细胞 (ES)。这种干细胞在遗传学上和病人完全一致 ,再定向诱导其分化成病人所需要的体细胞进行移植 ,以取代和修复患者已丧失功能的细胞、组织或器官 ,而达到完全治愈。治疗性克隆不仅解决移植物与受者间的免疫排斥反应问题 ,而且可以解决移植物的来源问题。     

Embryonic stem cells contribute to mouse chimeras in the absence of detectable cell fusion

Embryonic stem (ES) cells are capable of differentiating into all embryonic and adult cell types following mouse chimera production. Although injection of diploid ES cells into tetraploid blastocysts suggests that tetraploid cells have a selective disadvantage in the developing embryo, tetraploid hybrid cells, formed by cell fusion between ES cells and somatic cells, have been reported to contribute to mouse chimeras. In addition, other examples of apparent stem cell plasticity have recently been shown to be the result of cell fusion. Here we investigate whether ES cells contribute to mouse chimeras through a cell fusion mechanism. Fluorescence in situ hybridization (FISH) analysis for X and Y chromosomes was performed on dissociated tissues from embryonic, neonatal, and adult wild-type, and chimeric mice to follow the ploidy distributions of cells from various tissues. FISH analysis showed that the ploidy distributions in dissociated tissues, notably the tetraploid cell number, did not differ between chimeric and wild-type tissues. To address the possibility that early cell fusion events are hidden by subsequent reductive divisions or other changes in cell ploidy, we injected Z/EG (lacZ/EGFP) ES cells into ACTB-cre blastocysts. Recombination can only occur as the result of cell fusion, and the recombined allele should persist through any subsequent changes in cell ploidy. We did not detect evidence of fusion in embryonic chimeras either by direct fluorescence microscopy for GFP or by PCR amplification of the recombined Z/EG locus on genomic DNA from ACTB-cre::Z/EG chimeric embryos. Our results argue strongly against cell fusion as a mechanism by which ES cells contribute to chimeras.     

Isolation and characterization of embryonic stem-like cells from canine blastocysts

Embryonic stem (ES) cells are pluripotent cells with the capacity to generate any type of cell. Here we describe the isolation of ES-like cells from canine blastocysts. Canine embryos were collected from beagle bitches at day 11-16 of first estrus. A total of 80 normal embryos were obtained from 15 dogs. Of the embryos, 13 were at the morulae stage, 39 at the blastocyst stage, and 28 at the hatched blastocyst stage. The inside of morulae or inner cell masses (ICMs) of blastocysts were isolated mechanically, and cultured onto mouse embryonic fibroblasts (MEF) as feeder layers. Primary cell colonies were formed in 0% (0/13) of morulae, 25.6% (10/39) of blastocysts, and 67.9% (19/28) of hatched blastocysts. These colonies were separated either by enzymatic dissociation or by mechanical disaggregation. Dissociation with collagenase resulted in immediate differentiation, but with mechanical disaggregation these cells remained undifferentiated, and two ES-like cell lines (cES1, cES2) continued to grow in culture after eight passages. These cells had typical stem cell-like morphology and expressed specific markers such as alkaline phosphatase activity, stage specific embryonic antigen-1 and Oct-4. These cells formed embryoid bodies (EBs) in a suspension culture; extended culture of EBs resulted in the formation of cystic EBs. When the simple EBs were cultured on tissue culture plates, they differentiated into several types of cells including neuron-like, epithelium-like, fibroblast-like, melanocyte-like, and myocardium-like cells. These observations indicate that we successfully isolated and characterized canine ES-like cells.     

Comparison of ES cell fate in sandwiched aggregates and co-cultured aggregates during blastocyst formation by monitored GFP expression

Markers and the means to detect them are required to monitor the fate of living cells. However, few suitable markers for living cells were known until a green fluorescent protein (GFP) was discovered. We have established mouse embryonic stem (ES) cell lines that express mutant GFP under the chicken beta-actin (CAG) promoter. Using these cell lines, we were able to follow the migration of ES cells during blastocyst formation both in sandwiching and coculture methods, even if only a single ES cell was used. Furthermore, the contribution of ES cells to the inner cell mass (ICM) was easily estimated at the blastocyst stage. We compared sandwiching with coculture aggregation relative to the contribution of the ES cell in the ICM, and the results indicated that there was no difference in the ratios of chimeric embryos having ICM contributed from cultured ES cells. Furthermore, an aggregated single ES cell was able to contribute three or four cells to the ICM at the blastocyst stage. Thus we conclude that one, instead of two, embryos is enough to make aggregation with ES cells, and a single ES cell attached to an embryo is enough to produce germline chimeras. Moreover, we could clearly observe single cell fate during blastocyst formation. This suggests that our established cell line can be used for monitoring single cell fate in vivo. In addition, we have shown that up to five doses of 30 sec of UV irradiation using GFP filters have no effect on the embryonic development.     

Development of reconstituted embryos derived from transgenic embryonic stem cell nuclei

This study was carried out to transform embryonic stem (ES) cells and to produce the reconstituted embryos derived from transgenic ES cell nuclei. Then, in vitro/vivo developmental potency of transgenic ES cells were compared to that of control ES cells (non-transgenic ES cells) in the reconstituted embryos. Unfertilized B6D2F1 ooplasm at metaphase II (M II) and two kinds of ES cell lines, 129SV and transgenic (tg) 129SV transformed by EGFP gene, were used as nuclear recipients and nuclear donors, respectively. The M II chromosome-spindle complex was aspirated into the pipette with a minimal volume of ooplasm. After enucleation, the ES cell nuclei was injected into the enucleated ooplasm directly. Then, reconstituted embryos were activated in SrCl2, and they were cultured in HTF medium. There was no difference of developmental rate between reconstituted embryos derived from the control (non-transgenic) and the tg ES cells. From this result, we indicated that transgenic ES cells might not change the property of peculiarity of the ES cell by gene transfer. The expression of GFP gene in the embryos was observed by fluorescence microscope at the 4-cell and more stage. As comparison with development of the embryos derived from the control and tg ES cells, the difference of the development could not be confirmed between the two cell groups. When the reconstituted embryos derived from the control and tg ES cells were transferred into oviduct or uterus of pseudopregnant females, fetuses were observed 13.5 days post coitum. However, in all fetuses, developmental arrest and regression were seen 19.5 days post coitum.     

A novel retrovirally induced embryonic lethal mutation in the mouse: assessment of the developmental fate of embryonic stem cells homozygous for the 413.d proviral integration

A genetic screen of transgenic mouse strains, carrying multiple copies of an MPSV neo retroviral vector, has led to the identification of a recessive embryonic lethal mutation, termed 413.d. This mutation is associated with a single proviral insertion and when homozygous, results in the failure of the early postimplantation embryo at the gastrulation stage of development. Embryonic stem cell lines (ES cells) were derived from 413.d intercross embryos. Genotyping, with respect to the 413.d integration site, identified wild-type, heterozygous and homozygous ES cell lines. The differentiation abilities and developmental potential of the ES cell lines were assessed using a number of in vitro and in vivo assays. Results indicate that the ES cell lines, regardless of genotype, are pluripotent and can give rise to tissue and cell types derived from all three germ layers. Furthermore, analysis of midgestation conceptuses (10.5 p.c.) and adult chimeras generated by injecting mutant ES cells into host blastocysts, provides strong evidence that the mutant cells can contribute to all extraembryonic tissues and somatic tissues, as well as to functional germ cells. These results indicate that the homozygous mutant cells can be effectively 'rescued' by the presence of wild-type cells in a carrier embryo.     

Green fluorescent protein gene-transfected peafowl somatic cells participate in the development of chicken embryos

This study was performed to investigate whether the embryonic somatic cells are capable of reconstituting and participating in the embryonic development of chickens to produce chimeras. In order to track the migration behavior of the donor cells, a cell line, originally isolated from an Indian peafowl embryo, was fluorescent-labeled by transfection of the cells with enhanced Green Fluorescent Protein (GFP) and Neomycin resistant (Neo) genes prior to injection into the stage X blastoderm of White Leghorn chickens. The injection was performed with a medium in the presence of 1-5% polyethylene glycol. The development of putative chimeric embryos between the stages three and 24 was examined for GFP expression under fluorescent light. To trace the peafowl cells in the developing chicken embryos, both a species-specific genetic marker originating from the mitochondrial DNA cytochrome b (cyt b) gene and a DNA fragment of GFP gene were used. Of the 185 fertile eggs manipulated, 173 developed into embryos. Fifty-five of them showed positive GFP patches in extra-embryonic tissues, and 15 expressed GFP in intra-embryonic tissues such as those of the head, heart, and gonad. PCR analysis revealed that PCR fragments for the peafowl mitochondrial DNA cyt b and GFP genes were detected in the samples of the GFP positive extra- and intra-embryonic tissues of the chimeras. The present results provide evidence that fluorescent-labeled peafowl embryonic cells carrying GFP and Neo genes are able to participate in the development of chicken embryos to generate chimeras.     

Evaluation of developmental competence of vitrified-warmed early cleavage stage embryos and their application for chimeric mouse production.

The developmental competence of in vitro cultured embryos vitrified-warmed at an early cleavage stage (2- or 4, 8-cell stage) was examined by both direct transfer into recipient animals and after in vitro manipulation for chimeric mice production using embryonic stem (ES) cells. Vitrified-warmed embryos transferred at the morulae and blastocyst stages showed fetus development comparable to control embryos, although blastocyst development of vitrified-warmed embryos was significantly slower than that of controls. When vitrified-warmed early cleavage stage embryos were used for chimeric mouse production using ES cells, 1 to 10% of the injected or aggregated embryos developed into chimeric neonates and germ-line chimeric mice were obtained from all ES cell lines. This study indicates that embryos developed in vitro from vitrified-warmed embryos have equivalent competence with unvitrified embryos irrespective of stage of vitrification and that these vitrified-warmed embryos maintain adequate viability even after in vitro manipulation such as aggregation and microinjection with ES cells.     

Mouse embryonic stem (ES) cell lines established from neuronal cell-derived cloned blastocysts

We have established mouse embryonic stem (ES) cell lines from blastocysts derived by transfer of nuclei of fetal neuronal cells. These neuronal cell-derived embryonic cell lines had properties that characterize them as ES cells, including typical cell markers and alkaline phosphatase activity. Moreover, the cells had a normal karyotype and were pluripotent, as they were capable of differentiating into all three germ layers. Although they were derived from neuronal donor nuclei, the cells no longer expressed neuronal markers; however, they were capable of differentiating into cells with neuronal characteristics. These results suggest that the clone-derived cells have fully acquired an ES cell character. Thus, ES cells can be derived from embryos resulting from nuclear transfer, which results in reprogramming of the genetic information and acquisition of pluripotency. ES cells established from somatic cell-derived blastocysts could be useful not only as research tools for studying reprogramming but also as models for cell-based transplantation therapy.     

Chemical enhancement in embryo development and stem cell derivation from single blastomeres

Several chemicals targeting the mitogen-activated protein (MAP) kinase signaling pathway, which play an important role in regulating cell growth and differentiation, have shown enhancing effects on the development of the inner cell mass (ICM) and the derivation of ES cells. However, investigation of such chemicals on early embryonic development and the establishment of ES cell lines has not been elucidated. This study was aimed to determine if ACTH, MAP2K1 inhibitor [MAP2K1 (I)], and MAPK14 inhibitor [MAPK14 (I)] could enhance the development of the ICM in preimplantation mouse embryos and blastocyst outgrowths, and the establishment of ES cell lines from blastomeres of early embryos. We have demonstrated that both MAP2K1 (I) and MAPK14 (I) delay early embryo development and inhibit the development of embryos from early blastomeres. On the other hand, ACTH had a positive effect on embryos derived from early blastomeres. As a result, 17 ES cell lines were established. Among these ES cell lines, nine and five ES cell lines were established from single blastomeres of two-cell embryos with and without the supplement of ACTH, respectively. In addition to two-cell isolated blastomeres, three ES cell lines were established from blastomeres of four-cell embryos only with the supplement of ACTH. Our results suggest that ACTH can enhance the derivation of ES cells from single blastomere-derived embryos.     

Irradiation of fish embryos prior to blastomere transfer boosts the colonisation of their gonads by donor‐derived gametes

Blastomere transplantation into fish blastula embryos results in somatic chimeras, which generally provide null or a small proportion of gametes derived from the donor. This may partly explain why none of the ES‐like cell lines established from fish embryos has contributed to the germline of chimeras when transplanted at the blastula stage. Here, we report that a moderate gamma‐irradiation of recipient embryos, followed by transplantation of dispersed blastomeres, considerably enhances the proportion of donor‐derived gametes (53% versus 5% in average). In fish, the resulting protocol should maximise the pluripotency level measured in vivo for embryonic cell lines and for cultured germ cells. Mol. Reprod. Dev. 53:394–397, 1999. © 1999 Wiley‐Liss, Inc.     

Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei

Pluripotent human stem cells isolated from early embryos represent a potentially unlimited source of many different cell types for cell-based gene and tissue therapies [1-3]. Nevertheless, if the full potential of cell lines derived from donor embryos is to be realised, the problem of donor-recipient tissue matching needs to be overcome. One approach, which avoids the problem of transplant rejection, would be to establish stem cell lines from the patient's own cells through therapeutic cloning [3,4]. Recent studies have shown that it is possible to transfer the nucleus from an adult somatic cell to an unfertilised oocyte that is devoid of maternal chromosomes, and achieve embryonic development under the control of the transferred nucleus [5-7]. Stem cells isolated from such a cloned embryo would be genetically identical to the patient and pose no risk of immune rejection. Here, we report the isolation of pluripotent murine stem cells from reprogrammed adult somatic cell nuclei. Embryos were generated by direct injection of mechanically isolated cumulus cell nuclei into mature oocytes. Embryonic stem (ES) cells isolated from cumulus-cell-derived blastocysts displayed the characteristic morphology and marker expression of conventional ES cells and underwent extensive differentiation into all three embryonic germ layers (endoderm, mesoderm and ectoderm) in tumours and in chimaeric foetuses and pups. The ES cells were also shown to differentiate readily into neurons and muscle in culture. This study shows that pluripotent stem cells can be derived from nuclei of terminally differentiated adult somatic cells and offers a model system for the development of therapies that rely on autologous, human pluripotent stem cells.     

Development of reconstituted embryos derived from somatic cell nuclei in the rabbit

Production of cloned laboratory animals is helpful in the establishment of medical models. In this study, we examined to produce reconstituted embryos derived from somatic cell nuclei, and to establish embryonic stem (ES) cell lines from the embryo in rabbits. Metaphase II (M-II) oocytes from superovulated rabbit were used as nuclear recipients. Nuclear donor cells were fibroblasts collected from a Dutch Beleted rabbit. The M-II chromosome and the 1st polar body were aspirated, and a fibroblast was inserted into the perivitelline space of the enucleated oocyte. The pairs were electrofused for cell membrane fusion using a cell fusion apparatus, and reconstituted embryos were produced. The embryos were activated and cultured in modified HTF medium and DMEM. The embryos developed to the blastocyst stage were removed their zona pellucida, and they were cultured on the feeder cell layer. As a result of having observed development of reconstituted embryos, 21.2% of the embryos were developed to the blastocyst stage. In the embryos cultured on the feeder cells, the adhesion on feeder cells was observed. We obtained inner cell mass (ICM) colony derived from reconstituted embryos. At present, we are investigating to establish the ES cell lines derived from the embryos reconstituted by nuclear transfer.     

Development of reconstituted embryos derived from somatic cell nuclei in the rabbit

Production of cloned laboratory animals is helpful in the establishment of medical models. In this study, we examined to produce reconstituted embryos derived from somatic cell nuclei, and to establish embryonic stem (ES) cell lines from the embryo in rabbits. Metaphase II (M-II) oocytes from superovulated rabbit were used as nuclear recipients. Nuclear donor cells were fibroblasts collected from a Dutch Beleted rabbit. The M-II chromosome and the 1st polar body were aspirated, and a fibroblast was inserted into the perivitelline space of the enucleated oocyte. The pairs were electrofused for cell membrane fusion using a cell fusion apparatus and reconstituted embryos were produced. The embryos were activated and cultured in modified HTF medium and DMEM. The embryos developed to the blastocyst stage were removed their zona pellucida, and they were cultured on the feeder cell layer. As a result of having observed development of reconstituted embryos, 21.2% of the embryos were developed to the blastocyst stage. In the embryos cultured on the feeder cells, the adhesion on feeder cells was observed. We obtained inner cell mass (ICM) colony derived from reconstituted embryos At present, we are investigating to establish the ES cell lines derived from the embryos reconstituted by nuclear transfer.     

Experimental embryology of mammals at the Jastrzebiec Institute of Genetics and Animal Breeding

Our Department of Experimental Embryology originated from The Laboratory of Embryo Biotechnology, which was organized and directed by Dr. Maria Czlonkowska until her premature death in 1991. Proving successful international transfer of frozen equine embryos and generation of an embryonic sheep-goat chimaera surviving ten years were outstanding achievements of her term. In the 1990s, we produced advanced fetuses of mice after reconstructing enucleated oocytes with embryonic stem (ES) cells, as well as mice originating entirely from ES cells by substitution of the inner cell mass with ES cells. Attempts at obtaining ES cells in sheep resulted in the establishment of embryo-derived epithelioid cell lines from Polish Heatherhead and Polish Merino breeds, producing overt chimaeras upon blastocyst injection. Successful re-cloning was achieved from 8-cell rabbit embryos, and healthy animals were born from the third generation of cloned embryos. Recently mice were born after transfer of 8-cell embryonic nuclei into selectively enucleated zygotes, and mouse blastocysts were produced from selectively enucleated germinal vesicle oocytes surrounded by follicular cells, upon their reconstruction with 2-cell nuclei and subsequent activation. Embryonic-somatic chimaeras were born after transfer of foetal fibroblasts into 8-cell embryos (mouse) and into morulae and blastocysts (sheep). We also regularly perform the following applications: in vitro production of bovine embryos from slaughterhouse oocytes or those recovered by ovum pick up; cryopreservation of oocytes and embryos (freezing: mouse, rabbit, sheep, goat; vitrification: rabbit, cow); and banking of somatic cells from endangered wild mammalian species (mainly Cervidae).     

Increasing the cell number of host tetraploid embryos can improve the production of mice derived from embryonic stem cells

Tetraploid (4n) embryo complementation assay has shown that embryonic stem (ES) cells alone are capable of supporting embryonic development (ES mouse), allowing the recovery of mouse lines directly from cultured ES cell lines. Although the advantages of this technique are well recognized, it remains inefficient for generating ES mice. In the present study, we investigated the effects of cell number of host 4n embryos on the production of ES mice. Four independent ES cell lines (two general ES cell lines and two nuclear transfer-derived ES cell lines) were used, and each cell line was aggregated with single (1x) to triple (3x) host 4n embryos. We found that birth rate of ES mice using 1x 4n embryos was quite low (0-2%) regardless of cell line, whereas except for one cell line, approximately 6-14% of embryos developed to full term in the case of 3x 4n embryos. Contamination of host 4n cells in ES mice was quite rare, being comparable to that generated using general methods even if they were delivered from 3x 4n host embryos. These results demonstrate that the use of 3x 4n embryos is effective for generating ES mice. Our technique described here will be applicable to any ES cell line, including general ES cell lines used for gene targeting.     

Developmental potential of human oocytes reconstructed by transferring somatic cell nuclei into polyspermic zygote cytoplasm

The generation of patient-specific nuclear transfer embryonic stem cells holds huge promise in modern regenerative medicine and cell-based drug discovery. Since human in vivo matured oocytes are not readily available, human therapeutic cloning is developing slowly. Here, we investigated for the first time whether human polyspermic zygotes could support preimplantation development of cloned embryos. Our results showed that polyspermic zygotes could be used as recipients for human somatic cell nuclear transfer (SCNT). The preimplantation developmental potential of SCNT embryos from polyspermic zygotes was limited to the 8-cell stage. Since ES cell lines can be derived from single blastomeres, these results may have important significance for human ES cells derived by SCNT. In addition, confocal images demonstrated that all of the SCNT embryos that failed to cleave showed abnormal microtubule organization. The results of the present study suggest that polyspermic human zygotes could be used as a potential source of recipient cytoplasm for SCNT.     

Contributions to somatic and germline lineages of chicken blastodermal cells maintained in culture

Chicken blastodermal cells were cultured for 48 hr as explanted intact embryos, as dispersed cells in a monolayer, or with a confluent layer of mouse fibroblasts. The cells were then dispersed and injected into stage X (E-G&K) recipient embryos that were exposed to 600 rads of irradiation from a ⁶⁰Co source. Regardless of the conditions in which the cells were cultured, chimeras with contributions to both somatic tissues and the germline were observed. When blastodermal cells were co-cultured with mouse embryonic fibroblasts, significantly more somatic chimeras were observed and the proportion of feather follicles derived from donor cells was increased relative to that observed following the injection of cells derived from explanted embryos or monolayer cultures. Culture of blastodermal cells in any of the systems, however, yielded fewer chimeras that exhibited reduced contributions to somatic tissues in comparison to the frequency and extent of somatic chimerism observed following injection of freshly prepared cells. Contributions to the germline were observed at an equal frequency regardless of the conditions of culture, but were significantly reduced in comparison to the frequency and rate of germ-line transmission following injection of cells obtained directly from stage X (E-G&K) embryos. These data demonstrate that some cells retain the ability to contribute to germline and somatic tissues after 48 hr in culture and that the ability to contribute to the somatic and germline lineages is not retained equally. © 1996 Wiley-Liss, Inc.