334 FACTORS AFFECTING HEMATOPOIETIC ENGRAFTMENT OF MONKEY EMBRYONIC STEM CELLS IN SHEEP FETUSES

2015 ◽  
Vol 27 (1) ◽  
pp. 255
Author(s):  
Y. Nagao ◽  
T. Abe ◽  
A. Hara ◽  
B. Sarentonglaga ◽  
M. Yamaguchi ◽  
...  
Keyword(s):  
Subcutaneous Tissue ◽  
Es Cells ◽  
Embryonic Stem ◽  
Factors Affecting ◽  
Colony Pcr ◽  
Donor Cells ◽  
Haematopoietic Tissue ◽  
Cell Layers ◽  
Differentiation Stage ◽  
Teratoma Formation

Previously, we generated monkey/sheep haematopoietic chimeras by in utero transplantation (IUT) of monkey embryonic stem (ES); however, the factors that control how the ES cells successfully engraft and differentiate into haematopoietic tissue in sheep fetuses remain uncertain. Here, we examined factors that might influence donor cells and recipient sheep and affect successful ES cell engraftment. We transplanted either undifferentiated monkey ES cells or ES-derived cells at an early haematopoietic differentiation stage into sheep fetuses. The latter cells were allowed to differentiate by culturing on OP9 cell layers for 6 days. Cells were transplanted into the liver or subcutaneous tissue of recipient sheep fetuses at 43 to 50 or 51 to 67 days of gestation (full term = 147 days) using ultrasound to identify the site for transplantation. After birth, monkey haematopoietic engraftment in the bone marrow was analysed in 40 lambs using colony-PCR with cells grown in methylcellulose in the presence of defined cytokines; teratoma formation was analysed by biopsy and immunohistochemistry. We found that haematopoietic engraftment was only observed when ES-derived cells at the early differentiation stage were transplanted into fetal livers at 51 to 67 days of gestation (6/9). However, teratoma formation with mature monkey tissue structures was only observed following transplantation of undifferentiated ES cells into fetal subcutaneous tissues at 43 to 50 days of gestation (4/6), but that was not observed when both types of cells were transplanted into the liver (0/18) or at 51 to 67 days of gestation (0/24). These results demonstrate that the differentiation status of the donor cells, the transplantation site, and the age of the fetus at transplantation are important factors in engraftment and differentiation into haematopoietic tissue or teratoma formation in sheep fetuses.

In vitro differentiation of reprogrammed murine somatic cells into hepatic precursor cells

2008 ◽  
Vol 389 (7) ◽  
Author(s):  
Tobias Cantz ◽  
Martina Bleidißel ◽  
Martin Stehling ◽  
Hans R. Schöler
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Es Cells ◽  
Marrow Cell ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Precursor Cells ◽  
Differentiation Potential ◽  
Teratoma Formation ◽  
Transplantation Experiments

Abstract Recently, a new approach to reprogram somatic cells into pluripotent stem cells was shown by fusion of somatic cells with embryonic stem (ES) cells, which results in a tetraploid karyotype. Normal hepatocytes are often polyploid, so we decided to investigate the differentiation potential of fusion hybrids into hepatic cells. We chose toxic milk mice (a model of Wilson's disease) and performed initial transplantation experiments using this potential cell therapy approach. Mononuclear bone marrow cells from Rosa26 mice were fused with OG2 (Oct4-GFP transgenic) ES cells. Unfused ES cells were eliminated by selection with G418 for OG2-Rosa26 hybrids and fusion-derived colonies could be subcloned. Using an endodermal differentiation protocol, hepatic precursor cells could be generated. After FACS depletion of contaminating Oct4-GFP-positive cells, the hepatic precursor cells were transplanted into immunosuppressed toxic milk mice by intrasplenic injection. However, five out of eight mice showed teratoma formation within 3–6 weeks after transplantation in the spleen and liver. In conclusion, a hepatic precursor cell type was achieved from mononuclear bone marrow cell-ES cell hybrids and preliminary transplantation experiments confirmed engraftment, but also showed teratoma formation, which needs to be excluded by using more stringent purification strategies.

500. THE MANIPULATION OF THE EPIGENETIC MARK HISTONE 3 LYSINE 9 TRIMETHYLATION IN DONOR CELLS AND ITS EFFECTS ON THE DEVELOPMENT OF CLONED MOUSE EMBRYOS

2009 ◽  
Vol 21 (9) ◽  
pp. 101
Author(s):  
J. Antony ◽  
F. Oback ◽  
R. Broadhurst ◽  
S. Cole ◽  
C. Graham ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Expression Profiles ◽  
Es Cells ◽  
Embryonic Stem ◽  
Histone Demethylase ◽  
Specific Gene ◽  
Epigenetic Mark ◽  
Epigenetic Marks ◽  
Donor Cells

To produce live cloned mammals from adult somatic cells the nuclei of these cells must be first reprogrammed from a very restricted, cell lineage-specific gene expression profile to an embryo-like expression pattern, compatible with embryonic development. Although this has been achieved in a number of species the efficiency of cloning remains very low. Inadequate reprogramming of epigenetic marks in the donor cells correlated with aberrant embryonic gene expression profiles has been identified as a key cause of this inefficiency. Some of the most common epigenetic marks are chemical modifications of histones, the main structural proteins of chromatin. A range of different histone modifications, including acetylation and methylation, exists and can be attributed to either repression or activation of genes. One epigenetic mark which is known to be very stable and difficult to remove during reprogramming is the trimethylation of lysine 9 in histone H3 (H3K9Me3). To test the hypothesis that H3K9Me3 marks are a major stumbling block for successful cloning we are attempting to remove these marks by overexpression of the H3K9Me3 specific histone demethylase, jmjd2b, in donor cells, prior to their use for nuclear transfer. We have engineered mouse embryonic stem (ES) cells for the tet inducible expression of a fusion protein with a functional jmjd2b or non-functional mutant jmjd2b histone demethylase. Approximately 94% and 88% of the cells can be induced for the expression of functional and mutant jmjd2b-EGFP in the respective ES cell lines. Immunofluorescence analyses have shown that induction of functional jmjd2b-EGFP results in an approximately 50% reduction of H3K9Me3 levels compared to non-induced cells and induced mutant jmjd2b-EGFP cells. The comparison of the in-vitro embryo development following nuclear transfer with induced and non-induced donor cells show significantly better overall development to blastocysts and morulae from induced donor cells with reduced H3K9Me3 levels.

Prevention of Tumor Formation after Allogeneic In Utero Transplantation of Cynomolgus Monkey Embryonic Stem Cell-Derived Hematopoietic Precursors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 189-189 ◽  
Author(s):  
Yutaka Hanazono ◽  
Hiroaki Shibata ◽  
Naohide Ageyama ◽  
Yujiro Tanaka ◽  
Yukiko Kishi ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Allogeneic Transplantation ◽  
In Utero ◽  
Tumor Formation ◽  
Full Term ◽  
Fetal Sheep ◽  
Hematopoietic Organ ◽  
Teratoma Formation ◽  
Time Point

Abstract Although human ES cell-based transplantation therapies would hold a great potential in the treatment of a variety of diseases and injuries, there is a concern for teratoma formation after transplantation. In this study, we assessed the risk of tumor formation during the hematopoietic engraftment derived from cynomolgus ES cells (cyESCs) in an allogeneic transplantation model. cyESCs expressing GFP were cultured on OP9 stromal cells and induced to differentiate into the putative hematopoietic precursors. According to the flow cytometric analysis, CD34 was up-regulated on day 6 but decreased thereafter. Notably, CD31, CD144 (VE-cadherin) and VEGFR-2 (Flk-1) were all up-regulated on day 6. These are key markers of hemangioblasts (which generate endothelial and hematopoietic lineages). Despite the hemangioblast marker expression on day 6, the SCL gene was up-regulated at this time point as assessed by RNA-PCR, implying that the hematopoietic commitment might have already occurred on day 6. CD45, however, was not detected until day 12. The day-6 cells were transplanted in utero into allogeneic (cynomolgus) preimmune fetuses (n = 3) in the liver under ultrasound guidance around the end of first trimester (49–66 days/full term 165 days). We transplanted day-6 cells because the CD34 expression was the highest at this time point. We transplanted the cells into the liver because the liver is the major hematopoietic organ at these gestation days. Fetuses were delivered at 3 months after transplantation (almost at full term). The transplanted cell-derived, GFP-positive hematopoietic colony-forming cells were successfully detected in the newborns (4–5%). Hematopoietic engraftment from cyESCs was thus achieved albeit at low levels. However, teratomas formed in all the three newborns. They were derived from transplanted cells, because they expressed GFP. The risk of tumor formation was unexpectedly high, given that we had seldom observed tumor formation in immunodeficient mice or fetal sheep that had been transplanted with the same day-6 cyESC-derivatives. Innate immune responses against cynomolgus-derived tumors might be more rigorous in xeno-transplanted mice and sheep than in allo-transplanted monkeys, resulting in failure to detect tumorigenesis in the xeno-transplantation models. Our monkey allogeneic transplantation setting would therefore allow the strict evaluation of in vivo safety of transplantation therapies using ES cells. It turned out that day-6 cyESC-derivatives included residual SSEA-4-positive pluripotent cells (38.2 ± 10.3%) despite the rigorous differentiation culture. Presumably those cells were responsible for the teratoma formation. We purged an SSEA-4-positive fraction of day-6 cyESC-derivatives using a cell sorter and transplanted the negative fraction into the fetal liver (n = 6). At delivery, tumors were no longer observed in all the six animals, while the cyESC-derived hematopoietic engraftment was unperturbed (2–5%). SSEA-4 is therefore a clinically-relevant pluripotency marker of primate ES cells. Purging cells with this surface marker would be a promising method for clinical progenitor cell preparations using human ES cells.

scholarly journals Selective apoptosis of pluripotent mouse and human stem cells by novel ceramide analogues prevents teratoma formation and enriches for neural precursors in ES cell–derived neural transplants

2004 ◽  
Vol 167 (4) ◽  
pp. 723-734 ◽  
Author(s):  
Erhard Bieberich ◽  
Jeane Silva ◽  
Guanghu Wang ◽  
Kannan Krishnamurthy ◽  
Brian G. Condie
Keyword(s):  
Stem Cell ◽  
Neural Differentiation ◽  
Embryoid Body ◽  
Es Cells ◽  
Embryonic Stem ◽  
Human Stem Cells ◽  
Pluripotency Marker ◽  
Lineage Differentiation ◽  
Induced Apoptosis ◽  
Teratoma Formation

The formation of stem cell–derived tumors (teratomas) is observed when engrafting undifferentiated embryonic stem (ES) cells, embryoid body–derived cells (EBCs), or mammalian embryos and is a significant obstacle to stem cell therapy. We show that in tumors formed after engraftment of EBCs into mouse brain, expression of the pluripotency marker Oct-4 colocalized with that of prostate apoptosis response-4 (PAR-4), a protein mediating ceramide-induced apoptosis during neural differentiation of ES cells. We tested the ability of the novel ceramide analogue N-oleoyl serinol (S18) to eliminate mouse and human Oct-4(+)/PAR-4(+) cells and to increase the proportion of nestin(+) neuroprogenitors in EBC-derived cell cultures and grafts. S18-treated EBCs persisted in the hippocampal area and showed neuronal lineage differentiation as indicated by the expression of β-tubulin III. However, untreated cells formed numerous teratomas that contained derivatives of endoderm, mesoderm, and ectoderm. Our results show for the first time that ceramide-induced apoptosis eliminates residual, pluripotent EBCs, prevents teratoma formation, and enriches the EBCs for cells that undergo neural differentiation after transplantation.

225 TERATOMA FORMATION BY BOVINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 229
Author(s):  
M. L. Lim ◽  
I. Vassiliev ◽  
P. J. Verma
Keyword(s):  
Growth Factor ◽  
Es Cells ◽  
Calf Serum ◽  
Embryonic Stem ◽  
Scid Mice ◽  
Bovine Embryos ◽  
Differentiation Potential ◽  
Teratoma Formation

Teratoma formation is commonly used as a model for examining the in vivo differentiation potential of embryonic stem cells. We wanted to investigate the teratoma-forming ability of bovine ES cells; however, there are no reports of teratoma-forming ability of bovine pluripotent cells including pre-implantation embryos. In vivo-produced bovine embryos at stages earlier than Day 14 failed to develop teratomas when transplanted into one of the kidneys of immuno-deficient mice (Anderson et al. 1996 Anim. Reprod. Sci. 45, 231–240), and this prompted questions about the ability of bovine embryos to form teratomas. Bovine oocytes were cultured for 20 to 22 h after aspiration at 39�C (5% CO2/95% air) in TCM-199-bicarbonate medium supplemented with GlutaMax6" (Invitrogen Australia Pty Ltd., Mount Waverley, Victoria, Australia), penicillin/streptomycin, β-mercaptoethanol, 17β-estradiol, fetal calf serum, LH, follicle stimulating hormone, basic fibroblast growth factor, epidermal growth factor, glycine, and l-cysteine. Oocytes were fertilized with IVF media (Cook Australia, Brisbane, Queensland, Australia) and kept for 7 days at 39�C in 5% CO2/95% air to generate blastocysts. The zona pellucida of Day 7 blastocysts was enzymatically removed, and one or two zona-free embryos were injected into each testis of 5-week-old immunodeficient (SCID) mice (CB-17/ICR-Prkdcscid strain; Walter and Eliza Hall Institute, Melbourne, Australia). Eight weeks post-injection, teratomas partially expelled from testes were identified. Histological analysis has confirmed the derivatives of all 3 germ layers in teratomas. In conclusion, we report that Day 7 in vitro-produced embryos can form teratomas when injected into testes of SCID mice.

41 UPDATING THE ZONA-FREE METHOD FOR MOUSE CLONING USING HM1 EMBRYONIC STEM CELLS

2015 ◽  
Vol 27 (1) ◽  
pp. 113
Author(s):  
I. Lagutina ◽  
M. Lizier ◽  
M. Paulis ◽  
F. Lucchini ◽  
A. Castelli ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Cumulus Cells ◽  
Farm Animals ◽  
Blastocyst Development ◽  
Donor Cells ◽  
M Phase ◽  
Fetal Bovine ◽  
Spherical Cells ◽  
Old Mice

The zona-free method of SCNT designed for bovine and pig cloning (Booth et al. 2001; Vajta et al. 2001; Oback et al. 2003) was successfully used for horse (Galli et al. 2003). Although simple and efficient in farm animals, its application in the mouse met several problems (Ribas et al. 2005, 2006). The aim of our work was to produce cloned mice using HM1 embryonic stem (ES)cells adapting a zona-free method. Seven- to 24-week-old superovulated B6D2F1 female mice were used as oocytes donors. Cumulus cells were removed by 0.3% hyaluronidase and the zona pellucida by 0.5% pronase in KSOM-HEPES (KSOM-H) 1 h later (Ribas et al. 2006) or immediately after hyaluronidase treatment at 37°C. The HM1 ES cells were cultured in KnockOut DMEM supplemented with leukemia inhibitory factor and 15% fetal bovine serum with or without 2i (Ying et al. 2008) and were synchronized at M phase by 3 ng mL–1 nocodazole for 3 h before fusion. Only spherical cells were selected for NT. Metaphase II chromosome spindle complexes were removed by micromanipulation in KSOM-H medium with 5 μg mL–1 cytochalasin B. Lectin-treated enucleated oocytes were attached to the donor cells in KSOM-H with nocodazole and fused by 2 pulses of 1.3 kV cm–1 DC for 30 μs in 0.3 M mannitol medium. Following 10- to 15-min incubation in KSOM-H, the fusion was assessed and repeated if the constructs were nonfused. Cloned embryos were activated in 1 mM SrCl2 in Ca2+-free KSOM medium for 2 to 2.5 or 5 to 6 h and cultured in 20-μL KSOM droplets using the well-of-the-well (WOW) method (Vajta et al. 2000) under mineral oil at 37°C and 5% CO2. Day 4 compacted morulae and blastocysts were surgically transferred into the uterus of Day-2.5 pseudopregnant recipients that were sacrificed on Day 19.5 to examine fetal development. The donor mice age was important for oocyte survival: ~16% of oocytes of 7- to 10-week-old mice lysed before or during fusion in 33% of experiments (n experiments = 15), whereas oocytes of older mice were not sensitive to enzymatic treatment and electric impulses even after 3 fusion rounds (n = 19). The time of pronase treatment did not affect oocyte survival, whereas extending the time between hyaluronidase treatment and enucleation revealed self-activation in ~25% of oocytes. The fusion efficiency of ES cells was significantly lower compared with serum-starved fibroblasts (61%, n = 623 v. 100%, n = 80). The duration of SrCl2 treatment did not affect embryo development (cleavage: 82% v. 84%; Day 4 blastocysts: 49% v. 52%). ES cell culture with 2i increased Day 4 blastocyst development (60.7% v. 50.4%; P = 0.07), and their ability to implant (52.6% v. 38.2%; P = 0.06). Moreover, only NT embryos derived from 2i-ES cells developed to term (8.2%, n = 5; P = 0.08), and produced live fetuses (4.9%, n = 3). In light of these results, the fusion of ES cells remains the critical step in the mouse zona-free protocol.Partially supported by grant Superpig from Regione Lombardia.

Effective contribution of transplanted vascular progenitor cells derived from embryonic stem cells to adult neovascularization in proper differentiation stage

Blood ◽  
2003 ◽  
Vol 101 (7) ◽  
pp. 2675-2678 ◽  
Author(s):  
Takami Yurugi-Kobayashi ◽  
Hiroshi Itoh ◽  
Jun Yamashita ◽  
Kenichi Yamahara ◽  
Hideyo Hirai ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Mural Cells ◽  
Vascular Walls ◽  
Vascular Regeneration ◽  
Vascular Progenitor Cells ◽  
Differentiation Stage ◽  
Endothelial Growth Factor

We demonstrated that Flk-1+ cells derived from mouse embryonic stem (ES) cells can differentiate into both endothelial cells (ECs) and mural cells (MCs) to suffice as vascular progenitor cells (VPCs). In the present study, we investigated the importance of the stage of ES cell differentiation on effective participation in adult neovascularization. We obtained Flk-1+LacZ-expressing undifferentiated VPCs. Additional culture of these VPCs with vascular endothelial growth factor (VEGF) resulted in a mixture of ECs and MCs (differentiated VPCs). We injected VPCs subcutaneously into tumor-bearing mice. Five days after the injection, whereas undifferentiated VPCs were often detected as nonvascular cells, differentiated VPCs were more specifically incorporated into developing vasculature mainly as ECs. VPC-derived MCs were also detected in vascular walls. Furthermore, transplantation of differentiated VPCs augmented tumor blood flow in nude mice. These results indicate that a specific vascular contribution in adult neovascularization can be achieved by selective transplantation of ES cell–derived VPCs in appropriate differentiation stages, which should be the basis for vascular regeneration schemes.

294 PRODUCTION AND CHARACTERIZATION OF PUTATIVE NUCLEAR TRANSFER EMBRYONIC STEM CELLS DERIVED FROM HANDMADE CLONED EMBRYOS USING EMBRYONIC STEM CELLS, LYMPHOCYTES, AND ADULT FIBROBLAST CELLS AS DONOR CELLS IN GOAT

2011 ◽  
Vol 23 (1) ◽  
pp. 244
Author(s):  
R. Dutta ◽  
D. Malakar ◽  
K. Khate ◽  
J. Akshay
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Nuclear Transfer ◽  
Es Cells ◽  
Embryonic Stem ◽  
Donor Cell ◽  
Patient Specific ◽  
Fibroblast Cells ◽  
Donor Cells ◽  
Adult Fibroblasts

The handmade cloning technique has been a relatively recent addition in the field of nuclear transfer. In the present study, attempts were made to efficiently derive stem cells from handmade cloned (HMC) embryos in goat using adult fibroblast cells, embryonic stem (ES) cells, and lymphocytes as donor cells, and to characterise the derived putative nuclear transfer ES (ntES) cells for their stemness. Efficiency of the donor cells for nuclear transfer was also compared, and an overall cleavage and morula formation rates of 62.44 ± 3.9% and 35.30 ± 3.86%, 75.45 ± 3.92% and 45.84 ± 3.86%, and 56.38 ± 3.92% and 29.09 ± 3.86% were obtained from adult fibroblasts, ES cells, and lymphocytes, respectively. A significant difference was found between ES cells and the other 2 donor cells in terms of cleavage and morula formation. However, no such difference existed between fibroblasts and lymphocyte donor cells. Stem cell colonies were successfully derived from HMC embryos obtained from all 3 different donor cells. The rate of primary colony formation was 61.66 ± 4.62% for fibroblast-donor-cell-derived embryos. This rate was 59.91 ± 4.62% for ES-donor-cell-derived embryos and 62.49 ± 4.62% for lymphocyte-donor-cell-derived embryos. The putative ntES colonies were positively characterised for TRA-1-60, TRA-1-81, SSEA-1, SSEA-4, OCT-4, SOX-2, and Nanog by immunocytochemistry and RT-PCR. Results indicated that ES cells had better efficiency as donor cells in cloned embryo production than did adult fibroblasts and lymphocytes. The finding also suggested that terminally differentiated cell-like lymphocytes can also be reprogrammed. Moreover, there was no difference between the different donor-cell-derived HMC embryos in terms of ntES cell derivation. The study has established an efficient protocol for putative ntES cell derivation from HMC embryos. This could be of substantial significance because patient-specific ntES cells have proven therapeutic significance. The authors acknowledge N.D.R.I for the financial and infrastructural assistance.

scholarly journals Comparison of Teratoma Formation between Embryonic Stem Cells and Parthenogenetic Embryonic Stem Cells by Molecular Imaging

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Hongyan Tao ◽  
Xiaoniao Chen ◽  
Anbang Wei ◽  
Xianghe Song ◽  
Weiqiang Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Growth Factor Receptor ◽  
Renilla Luciferase ◽  
Alternative Source ◽  
Teratoma Formation

With their properties of self-renewal and differentiation, embryonic stem (ES) cells hold great promises for regenerative therapy. However, teratoma formation and ethical concerns of ES cells may restrict their potential clinical applications. Currently, parthenogenetic embryonic stem (pES) cells have attracted the interest of researchers for its self-renewing and pluripotent differentiation while eliciting less ethic concerns. In this study, we established a model with ES and pES cells both stably transfected with a double-fusion reporter gene containing renilla luciferase (Rluc) and red fluorescent protein (RFP) to analyze the mechanisms of teratoma formation. Transgenic Vegfr2-luc mouse, which expresses firefly luciferase (Fluc) under the promoter of vascular endothelial growth factor receptor 2 (Vegfr2-luc), was used to trace the growth of new blood vessel recruited by transplanted cells. Bioluminescence imaging (BLI) of Rluc/Fluc provides an effective tool in estimating the growth and angiogenesis of teratoma in vivo. We found that the tumorigenesis and angiogenesis capacity of ES cells were higher than those of pES cells, in which VEGF/VEGFR2 signal pathway plays an important role. In conclusion, pES cells have the decreased potential of teratoma formation but meanwhile have similar differentiating capacity compared with ES cells. These data demonstrate that pES cells provide an alternative source for ES cells with the risk reduction of teratoma formation and without ethical controversy.

231 HISTOCOMPATIBLE PARTHENOGENETIC EMBRYONIC STEM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 232
Author(s):  
A. Yabuuchi ◽  
K. Kitai ◽  
A. Takeuchi ◽  
P. Lerou ◽  
K. Ng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Full Complement ◽  
Teratoma Formation ◽  
High Level ◽  
Selection Of

Organ or tissue transplantation is the preferred treatment for numerous diseases but is hindered by immunologic barriers. Genetically matched pluripotent embryonic stem cells generated via nuclear transfer (ntES cells) or parthenogenesis (pES cells) are possible sources of histocompatible cells and tissues. We have developed two ways of isolating pES cells that carry the full complement of major histocompatibility complex (MHC) antigens of the oocyte donors. One method entails activation of oocytes after blockade of karyokinesis in meiosis II, followed by selection of predominantly homozygous pES cells that have undergone recombination in their MHC antigen region to restore the heterozygous maternal MHC genotype (parthenote recombinant, or prES cells). The second method involves activation of immature oocytes after blockade of karyokinesis of meiosis I, followed by selection of predominantly heterozygous pES lines that retain the MHC genotype of the oocyte donor (parthenote clone recombinant, or pcrES cells). The cells are pluripotent by several criteria: teratoma formation, in vitro differentiation into hematopoietic elements, and high-level skin chimerism in blastocyst chimeras. Breeding of 8 founder females and examination of over 700 progeny failed to demonstrate germ line transmission of the pES cells. Injection of over 50 tetraploid embryos with these lines and embryo transfer have failed to support full gestational development. However, differentiated tissues from these pluripotent ES cells engraft when transplanted into genetically matched immunocompetent recipients, demonstrating that selected pES cells can serve as a source of histocompatible tissues for transplantation.

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