Following nuclear transfer (NT) the epigenetic state of a donor nucleus must be reprogrammed to an embryonic one. To evaluate the efficiency of nuclear reprogramming, we monitored the levels of histone H3 di/tri-methylated on lysine 4 (H3K4m2/3), a marker for transcriptionally active/permissive euchromatin, and of histone H3 tri-methylated on lysine 27 (H3K27m3), a modification associated with facultative heterochromatin, in embryos cloned using rabbit mesenchymal stem cells (MSC) and adult fibroblasts (RAF) isolated from the same animals. In vivo fertilized, in vitro cultured embryos served as controls. H3K27m3 was undetectable in all stages of control embryos except for weak staining in a few blastocyst cells. A similar situation was found in all NT embryos irrespective of the type of donor cells used, although both MSC and RAF stained substantially for H3K27m3. H3K4m2/3 levels were very high in one- and two-cell control embryos, but then decreased to reach a minimum at the eight-cell stage, and finally increased again to initial levels at the morula and blastocyst stage. Reprogramming of H3K4m2/3 differed remarkably among the different types NT embryos as well as between NT embryos and control embryos, and was apparently dependent on the type of donor cells. Interestingly, abnormal reprogramming of H3K4m2/3 was observed in NT embryos derived from both MSC and RAF, donor cell types with markedly different proliferation capacity. Our study demonstrates that the repressive chromatin modification, H3K27m3, is faithfully reprogrammed in NT embryos derived from MSC or RAF, while reprogramming of the activating chromatin modification, H3K4m2/3, is quite variable and does not reflect the situation observed in control embryos derived by fertilization.
Multipotent skin-derived progenitors (SKP) can produce both neural and mesodermal progeny in vitro, sharing the characteristics of embryonic neural crest stem cells. However, the molecular basis for the property of multiple lineage potential and neural crest origin of SKPs is still elusive. Here we report the cooperative expression of pluripotency related genes (POU5F1, SOX2, NANOG, STAT3) and neural crest marker genes (p75NTR, TWIST1, PAX3, SNAI2, SOX9, SOX10) in GFP-transgenic porcine skin-derived progenitors (pSKP). The proportion of cells positive for POU5F1, nestin, fibronectin, and vimentin were 12.3%, 15.1%, 67.9% and 53.7%, showing the heterogeneity of pSKP spheres. Moreover, pSKP cells can generate both neural (neurons and glia) and mesodermal cell types (smooth muscle cells and adipocytes) in vitro, indicating the multiple lineage potency. Four transcription factors (POU5F1, SNAI2, SOX9, and PAX3) were identified that were sensitive to mitogen (FBS) and/or growth factors (EGF and bFGF). We infer that POU5F1, SNAI2, SOX9, and PAX3 may be the key players for maintaining the neural crest derived multipotency of SKP cells in vitro. This study has provided new insight into the molecular mechanism of stemness for somatic-derived stem cells at the level of transcriptional regulation.
The purposes of this study were to examine technical details in deriving and maintaining rabbit embryonic stem (rES) cell lines and to analyze their characteristics. When STO cells were used as feeder cells, no rES cell lines were established using either intact blastocysts or inner cell masses (ICMs). On the mouse embryonic fibroblasts (MEF) feeder, rES cell lines were efficiently (24%) derived. Addition of leukemia inhibitory factor (LIF) to the cells cultured on the MEF feeders further increased the derivation efficiency (57%) of rES cells. The fact that LIF induced serine-phosphorylation of STAT3 suggested LIF-dependent maintenance of rES cells. Most of the rES cell lines expressed AP, SSEA-4, Oct4, TRA-1-60, and TRA-1-81. Western blot or RT-PCR analysis also confirmed the expression of Oct4, Nanog, and Sox2. When induced to form EBs in vitro or injected to the severe combined immunodeficiency ( SCID) mice, the rES cells generated embryoid bodies (EBs) and teratomas with three germ layers expressing the marker genes including MAP2, Desmin, and GATA4, respectively. In conclusion, rabbit ES cell lines can be efficiently established using our current protocols with LIF supplement. These ES cells express pluripotent stem cell markers and retain their capability to differentiate into different tissue cells. Furthermore, rES cells depend on LIF for self-renewal, likely via the JAK-STAT pathway.
Human embryonic stem cell (hESC) growth is dependent on various factors released by feeder cells. Some of them have already been elucidated, although much research is still needed to understand the biology of stem cell maintenance in culture. Traditionally, primary mouse embryonic fibroblasts (PMEFs) have been used as feeder layers, and both murine and human fibroblast cell lines have been shown to support pluripotency and self-renewal of hESC. Here we report the derivation of three new mouse embryonic fibroblast cell lines, MEFLU-M, MEFLU-T, and MEFLU-TB, with different properties regarding growth and support for undifferentiated hESCs. MEFLU-TB is able to support continuous growth of the newly derived Man-1, as well as H1, HUES-1, HUES-7, HUES-8, and HUES-9 human embryonic stem cell lines. After more than 50 passages and doublings, MEFLU-TB feeders compare to early passage primary mouse embryonic fibroblasts in their ability to support undifferentiated hESC growth. Our results contradict a previous paradigm that PMEFs tend to lose their capacity to support proliferation of hESCs with increasing passages, and show that the MEFLU-TB mouse embryonic fibroblast cell line and its conditioned medium have the potential to support the maintenance of hESC lines. Also, our results clearly show that spontaneous immortalization of primary fibroblasts can be achieved in culture without any chemical addition or genetic modification.
Animal cloning has been associated with developmental abnormalities, with the level of heteroplasmy caused by the procedure being one of its potential limiting factors. The aim of this study was to determine the effect of the fusion of hemicytoplasts or aggregation of hemiembryos, varying the final cytoplasmic volume, on development and cell density of embryos produced by hand-made cloning (HMC), parthenogenesis or by in vitro fertilization (IVF). One or two enucleated hemicytoplasts were paired and fused with one skin somatic cell. Activated clone and zona-free parthenote embryos and hemiembryos were in vitro cultured in the well-of-the-well (WOW) system, being allocated to one of six experimental groups, on a per WOW basis: single clone or parthenote hemiembryos (1 x 50%); aggregation of two (2 x 50%), three (3 x 50%), or four (4 x 50%) clone or parthenote hemiembryos; single clone or parthenote embryos (1 x 100%); or aggregation of two clone or parthenote embryos (2 x 100%). Control zona-intact parthenote or IVF embryos were in vitro cultured in four-well dishes. Results indicated that the increase in the number of aggregated structures within each WOW was followed by a linear increase in cleavage, blastocyst rate, and cell density. The increase in cytoplasmic volume, either by fusion or by aggregation, had a positive effect on embryo development, supporting the establishment of pregnancies and the birth of a viable clone calf after transfer to recipients. However, embryo aggregation did not improve development on a hemicytoplast basis, except for the aggregation of two clone embryos.
Here, we have now developed a new inducing system to promote the differentiation of human stem cells (hESCs) toward hematopoietic lineages by the treatment with cells extract of human fetal liver tissue (hFLT). The embryoid bodies (EBs) obtained from human H1 embryonic stem cells were exposed to buffer, hFLT cells extract, heated hFLT cell extract, and cell extract of human liver cells lines-LO2. Then, the feature of EBs in different groups was characterized by real-time RT-PCR and colony-forming assays. The results showed the treatment by hFLT cells extract could activate the hematopoietic genes expression and improve the capacity for hematopoietic progenitor development of hEBs. After that, we cocultured hFLT extract treated hEBs on the hFLSCs ( human fetal liver stromal cells) feeder to differentiate them into hematopoietic cells. As a control, untreated hEBs were cocultured on hFLSCs feeder with cytokines. The feature of induced cells from hEBs was characterized by flow cytometry, Wright-Giemsa staining, and colony-forming assays. The results demonstrated that hFLT cells extract was capable of inducing hEBs into hematopoietic cells and combing it with hFLSCs feeder could largely promote hematopoietic differentiation of hESCs. This method may supply a new way to substitute the cytokines required in hematopoietic induction of hESCs.
Somatic cell nuclear transfer ( SCNT) efficiency is still low in rabbit. Previous studies indicated that trichostatin A (TSA) treatment could improve cloning efficiency and term development in the mouse, and cotransfer of parthenogenetic (PA) embryos benefited the pregnancy of cloned embryos in porcine and the mouse. In this study we investigated the effect of TSA treatment on the term development of the SCNT rabbit embryos, and the possibility of the pregnancy maintenance of clones by cotransfer of PA embryos. The SCNT embryos were produced by fusing cumulus cells with enucleated cytoplasts before activation by electrical stimulation, and Dimethylaminopurine (6-DMAP) and Cyclohexamide (CHX) treatments. They were cultured in EBSS-complete medium regardless of their treatment with or without TSA. In vitro developmental data showed no differences in the cleavage and the blastocyst rates, and the blastocyst cell number between the TSA-treated and the untreated SCNT embryos. Two of the six recipients became pregnant after the embryo transfer ( ET) in the TSA-treated group, and one pregnant female delivered seven live and three stillborn pups. The death of all live pups occurred within an hour to 19 days. Four of the seven recipients became pregnant in the TSA-untreated group. Three of them gave birth to six live and eight stillborn pups. Four pups of the TSA-untreated group have grown into adulthood, and three of them produced progeny. Cotransfer of three to four PA embryos with 26-32 SCNT embryos to the same recipient resulted in pregnancy and birth rates statistically no different compared to the control SCNT ET group. In conclusion, our results indicate that TSA treatment has a limited effect on the in vitro development of the SCNT embryos; furthermore, both the TSA-treated and the untreated clones can develop to term in rabbits, but none of the offspring from TSA-treated embryos survived to adulthood in our experiment.
Multipotent mesenchymal stromal cells ( MSC) display a high potential for the development of novel treatment strategies for cartilage repair. However, the pathways involved in their differentiation to functional non hypertrophic chondrocytes remain largely unknown, despite the work on embryologic development and the identification of key growth factors including TGF beta, Hh, Wnt and FGF. In this study, we asked if we could identify specific biological networks common to the growth factors used (TGF beta 3 or BMP-2). To address this question, we used DNA microarrays and performed large-scale expression profiling of MSC at different time points during their chondrogenic differentiation. By comparing these data with those obtained during the differentiation of MSC into osteoblasts and adipocytes, we identified 318 genes specific for chondrogenesis and developed a new algorithm to classify the genes according to their kinetic profile. We distributed the selected genes in five classes according to their kinetic of expression. We could reconstruct three phases characterized by functional pathways. The first phase corresponds to cell attachment and apoptosis induction; the second phase is characterized by a proliferation/differentiation step, and the third phase is characterized by a differentiation/hypertrophy pathway. Indeed, these data propose new pathways to understand the complexity of MSC differentiation to chondrocytes.
Recent findings identifying the transcription factors involved in the regulation of pluripotency and self-renewal in embryonic stem cells (ESC) may provide keys that enable the derivation of ESC in domestic species. In this study we monitored the expression of pluripotency-related genes in bovine inner cell mass (ICM) explants during the critical first steps in establishment of primary cultures. The expression of NANOG and POU5F1 transcripts and proteins in intact, in vitro produced (IVP) blastocysts was confirmed by quantitative RT-PCR and fluorescent immunocytochemistry. NANOG was localized to the nucleoplasm as well as the nucleoli in the ICM, whereas it appeared to be restricted to the nucleoli in trophectoderm cells. POU5F1 was localized in the nuclei of ICM and trophectoderm cells. ICM explants were analyzed by quantitative PCR and semiquantitative RT-PCR. The three major pluripotency-related transcription factors, NANOG, POU5F1, and SOX2, were expressed initially in the ICM explants, but were downregulated with subsequent culture. Markers of differentiation (BMP4, HNF4, NCAM, CDX2) and genes involved in LIF, BMP, and WNT signaling pathways were also expressed. ICM explants were cultured in the presence of various concentrations of cytokines belonging to the TGF-beta superfamily. Noggin, a cytokine inhibiting the BMP4 pathway, successfully upregulated the relative expression of NANOG mRNA in the ICM explants with respect to controls.
We previously reported a feeder-free culture method for pure production of subculturable vascular endothelial cells (VECs) from cynomolgus monkey embryonic stem cells (cmESCs) without as using cell-sorting technique. By this method, canonical vascular endothelial (VE)-cadherin/platelet-endothelial cell adhesion molecule 1 (PECAM1)-positive VECs (c-VECs) and atypical VE-cadherin/PECAM1-negative VECs (a-VECs) were generated without a contamination by pericytes, lymphatic endothelial cells, or immature ES cells. More recently, we established a unique culture technique to maintain human ESCs (hESCs) under a feeder-free and recombinant cytokine-free condition. Combining these two systems, we have successfully generated pure VECs from two lines of hESCs, khES-1 and khES-3, under a completely feeder-free condition. Our method is very simple: spheres generated from hESCs by floating culture using differentiation media supplemented with vascular endothelial growth factor, bone morphogenetic protein 4, stem cell factor, FMS-related tyrosine kinase-3 ligand, and interleukin 3 (IL3) and IL6 were cultured on gelatin-coated plates. Cell passage was performed by an ordinary enzymatic treatment. The hESC-derived differentiated cells demosntrated cord-forming activities and acetylated low-density lipoprotein-uptaking capacities. Moreover, they exclusively expressed von Willebrand factor and endothelial nitric oxide synthase. Flow cytometric analyses indicate that khES-3 generated both c-VECs and a-VECs as in the case of cmESCs. By contrast, khES-1 produced only a-VECs, which nonetheless demonstrated effective recruitment into neovascularity in vivo. Interestingly, a-VECs turned to express PECAM1 after transplantation into immunodeficient mice. The hESC-derived VECs were subculturable at least up to 10 passages without functional depression. Our method does not require a presorting processes to enrich progenitor fractions such as CD34-positive or kinase insert domain receptor (KDR)-positive cells, providing the most efficient and easiest technique for VEC production from hESCs.
Somatic cell nuclear transfer (SCNT) was used to generate patient-specific embryonic stem cells (ESCs) from blastocysts cloned by nuclear transfer (ntESCs). In this study, a total of 135 oocytes were obtained from 12 healthy donors (30-35 years). Human oocytes, obtained within 2 h following transvaginal aspiration, were enucleated using a Spindle Imaging System to position the spindle and chromosomes that lay on the metaphase plate, and a Zona Infrared Laser Optical System was used to open a single hole in the zona pellucida at the ~ 2 o'clock position. Human fibroblasts and lymphocytes were used to construct SCNT embryos. Nearly half (26 of 58) of the oocytes were fused after electrofusion and embryo development rates were 96.2% (two-cell, 25 of 26), 92.3% (four-cell, 24 of 26), 61.5% (eight-cell, 16 of 26), 34.6% (16-cell, 9 of 26), 26.9% (morula, 7 of 26), and 19.2% (blastocyst, 5 of 26), respectively, following incubation in improved G-series sequential medium. One cloned blastocyst was used for STR-DNA identification and genetic polymorphism analysis of mtDNA, and STR-DNA analysis of all cloned blastocysts indicated they were derived from SCNT. Quantitative analysis showed that mtDNA of cloned embryos reflected the change tendency of those observed in human IVF embryos. Our research provides an alternative enucleation approach for producing human SCNT-derived blastocysts, and may aid in providing a new method for human therapeutic cloning.
This study was designed to examine the effect of the age of rabbit oocytes on the developmental potential of cloned embryos. The metaphase II oocytes used for nuclear transfer (NT) were collected at 10, 12, 14, and 16 h post-hCG injection (hpi). The total number of oocytes collected per donor (21.4-23.7) at 12 to 16 hpi was similar, but significantly higher than that collected at 10 hpi (16.2). Additionally, a significant improvement in blastocyst development was achieved with embryos generated by electrically mediated cell fusion (56.0%), compared to those from nuclear injection (13.1 %) (Experiment 1). Markedly higher blastocyst development (45.8-54.5%) was also achieved with oocytes collected at 10-12 hpi than from those collected 14-16 hpi (8.3-14.3%) (Experiment 2). In Experiment 3, the blastocyst rates of NT embryos derived from oocytes harvested 12 hpi (39.2-42.8 %) were significantly higher than from those collected at 16 hpi (6.8-8.4 %) (p < 0.05), regardless of the donor cell age. Kinase activity assays showed variable changes of activity in rabbit oocytes over the period of 10-16 hpi; however, there was no correlation with preimplantational development (blastocyst rate vs. MPF, R = 0.326; blastocyst rate vs. MAPK, R = -0.131). Embryo transfer of NT embryos utilizing 12 hpi oocytes resulted in one full-term but stillborn, and one live cloned rabbit; thus, an efficiency of 1.7 % (n = 117) (Experiment 4). These results demonstrated that NT utilizing relatively young rabbit oocytes, harvested at 10-12 h after hCG injection, was beneficial for the development of NT embryos.
Cell cycle synchronization by serum starvation (SS) induces apoptosis in somatic cells. This side effect of SS is hypothesized to negatively affect the outcome of somatic cell nuclear transfer (SCNT). We determined whether apoptotic fibroblasts affect SCNT yields. Serum-starved, adult, bovine fibroblasts were stained with annexin V-FITC/propidium iodide to allow apoptosis detection by flow cytometry. Positive and negative cells sorted by fluorescence activated cell sorting (FACS) and an unsorted control group were used as nuclear donors for SCNT. Reconstructed embryos were cultured in vitro and transferred to synchronized recipients. Apoptosis had no effect on fusion and cleavage rates; however, it resulted in reductions in blastocyst production and quality measured by apoptotic index. However, reconstructed embryos with apoptotic cells resulted in pregnancy rates similar to that of the control on day 30, and generated one live female calf. In conclusion, we showed that apoptotic cells present in serum-starved cultures negatively affect embryo production after SCNT without compromising full-term development. Further studies will evaluate the ability of the oocyte to reprogram cells in specific phases of apoptosis.
Oct4 and Nanog are crucial for maintaining pluripotency in embryonic stem (ES) cells and early-stage embryos. In the present study, the status of DNA methylation and of histone modifications in the regulatory regions of Oct4 and Nanog in rhesus nuclear transfer-derived ES (ntES) cells was compared with in vitro fertilized embryo-derived ES (IVFES) cell counterparts. Dynamic changes in DNA methylation during differentiation into neural lineage were also monitored and correlated with mRNA abundance and protein levels of both genes. In ntES cells Oct4 exhibited mono-allelic methylation along with relatively lower mRNA levels, and its transcription was seen predominantly from the unmethylated allele. In contrast, in IVFES cells Oct4 was hypomethylated on both alleles and had relatively higher transcript levels, suggesting incomplete reprogramming of DNA methylation on the Oct4 gene following somatic cell nuclear transfer. During neuronal differentiation, Oct4 underwent biallelic methylation and reduced amounts of Oct4 mRNA were detected in both types of ES cells. Analysis of Nanog regulatory regions revealed that both alleles were hypomethylated and similar levels of Nanog transcripts were expressed in ntES cells and IVFES cells. During neuronal differentiation both alleles were methylated and reduced amounts of Nanog mRNA were detected. Other epigenetic modifications including histone 3 lysine 4, 9, and 27 trimethylation (H3K4me3, H3K9me3, and H3K27me3) showed similar patterns around the regulatory regions of Oct4 and Nanog in both kinds of ES cells. During neural differentiation, dramatic enrichment of H3K27me3 and H3K9me3 (repressive marks) was observed on Oct4 and Nanog regulatory regions. Differentiation of ntES and IVFES cells correlated with the silencing of Oct4 and Nanog, reactivation of the neural marker genes Pax6, N-Oct3, and Olig2, and dynamic changes in histone modifications in the upstream regions of Pax6 and N-Oct3. In short, although ES cells derived from somatic cell nuclear transfer showed a different epigenetic status in the Oct4 regulatory region than the IVF-derived counterparts, based on the parameters tested, the neural differentiation potential of ntES and IVFES cells is equivalent.
Interspecies somatic cell nuclear transfer (iSCNT) has the potential to become a useful tool to address basic questions about the nucleus-cytoplasm interactions between species. It has also been proposed as an alternative for the preservation of endangered species and to derive autologous embryonic stem cells. Using chimpanzee/bovine iSCNT as our experimental model we studied the early epigenetic events that take place soon after cell fusion until embryonic genome activation (EGA). Our analysis suggested partial EGA in iSCNT embryos at the eight-cell stage, as indicated by Br-UTP incorporation and expression of chimpanzee embryonic genes. Oct4, Stella, Crabp1, CCNE2, CXCL6, PTGER4, H2AFZ, c-MYC, KLF4, and GAPDH transcripts were expressed, while Nanog, Glut1, DSC2, USF2, Adrbk1, and Lin28 failed to be activated. Although development of iSCNT embryos did not progress beyond the 8- to 16-cell stage, chromatin remodeling events, monitored by H3K27 methylation, H4K5 acetylation, and global DNA methylation, were similar in both intra- and interspecies SCNT embryos. However, bisulfite sequencing indicated incomplete demethylation of Oct4 and Nanog promoters in eight-cell iSCNT embryos. ATP production levels were significantly higher in bovine SCNT embryos than in iSCNT embryos, TUNEL assays did not reveal any difference in the apoptotic status of the nuclei from both types of embryos. Collectively, our results suggest that bovine ooplasm can partially remodel chimpanzee somatic nuclei, and provides insight into some of the current barriers iSCNT must overcome if further embryonic development is to be expected.
Embryonic stem cells (ESC) hold tremendous potential for therapeutic applications, including regenerative medicine, as well as for understanding basic mechanisms in stem cell biology. Since numerous experiments cannot be conducted in human ESC because of ethical or practical limitations, nonhuman primate ESC serve as invaluable clinically relevant models. The novel marmoset (Callithrix jacchus) ESC line cjes001 was characterized using different stem cell markers. The cells were stained positively with Oct4, SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, and Sox-2 underscoring their status as undifferentiated ESC. ESC are typically grown on mouse embryonic fibroblasts (MEF) as feeder cells whose proliferation is arrested either by treatment with Mitomycin C or by gamma-irradiation. To assess the impact of these treatments on the ability of MEF to support the growth of undifferentiated ESC, we used an MTT assay to evaluate the cellular metabolic activity of growth arrested feeder cells. There was a significant (p < 0.02) difference in gamma-irradiated cells displaying a higher metabolic activity compared to Mitomycin C inactivation. Also we quantified 69 soluble factors in the supernatant of both Mitomycin-treated and gamma-irradiated MEF by bead-based multiplex analysis, and thus established a profile of MEF-secreted factors. The time course of secretion was analyzed by monitoring the supernatant at 0, 6, 12, and 24 h after changing the medium. Comparing gamma-irradiated and Mitomycin-treated MEF suggested higher amounts of some cytokines including FGF or SCF by the former. We also assessed whether the method of inactivation had an effect on growth kinetics and differentiation of primate ESC. There appeared to be a trend to a lower number of differentiated ESC colonies on the gamma-irradiated feeder cells, suggesting that this may be the preferable method of growth arrest.
Among many factors that potentially affect somatic cell nuclear transfer (SCNT) embryo development is the donor cell itself. Cloning potentials of somatic donor cells vary greatly, possibly because the cells have different capacities to be reprogrammed by ooplasma. It is therefore intriguing to identify factors that regulate the reprogrammability of somatic donor cells. Gene expression analysis is a widely used tool to investigate underlying mechanisms of various phenotypes. In this study, we conducted a retrospective analysis investigating whether donor cell lines with distinct cloning efficiencies express different levels of genes involved in epigenetic reprogramming including histone deacetylase-1 (HDAC1), -2 (HDAC2); DNA methyltransferase-1 (DNMT1), -3a (DNMT3a),-3b (DNMT3b), and the bovine homolog of yeast sucrose nonfermenting-2 (SNF2L), a SWI/SNF family of ATPases. Cell samples from 12 bovine donor cell lines were collected at the time of nuclear transfer experiments and expression levels of the genes were measured using quantitative polymerase chain reaction (PCR). Our results show that there are no significant differences in expression levels of these genes between donor cell lines of high and low cloning efficiency defined as live calving rates, although inverse correlations are observed between in vitro embryo developmental rates and expression levels of HDAC2 and SNF2L. We also show that selection of stable reference genes is important for relative quantification, and different batches of cells can have different gene expression patterns. In summary, we demonstrate that expression levels of these epigenome regulatory genes in bovine donor cells are not correlated with cloning potential. The experimental design and data analysis method reported here can be applied to study any genes expressed in donor cells.
To improve intersubspecies cloning efficiency, this paper provides six kinds of SCNT embryos with different nuclear-cytoplasmic relatedness to compare the relatedness on the cloning efficiency. Three kinds of SCNT embryos with different relatedness are produced by using Boer goat fibroblast cells as nuclear donors and oocytes of Sannen goat, crossbred F1 (Sannen goat x Boer goat) and Boer goat as cytoplast recipients. Four kinds of SCNT embryos with different relatedness are produced by using Sannen goat oocytes as recipients and fibroblast cells of Boer goat, crossbred F2 (crossbred F1 x Boer goat), crossbred F1, and Sannen goat as nuclear donors. Results show that no obvious differences were observed for preimplantation development of these SCNT embryos. However, different nuclear-cytoplasmic relatedness resulted in obvious differences for postimplantation development of these SCNT embryos. The relatedness is complementary: improving either cytoplasmic compatibility relatedness to nucleus or nuclear relatedness to cytoplast could reduce the gestation loss rate, and increase the birth rate of cloned intersubspecies embryos significantly. But a further amelioration of the relatedness did not improve the postimplantation development in direct proportion. These results suggested that close nuclear-cytoplasmic relatedness can improve the postimplantation development rate of cloned intersubspecies embryos.
There is renewed interest in using animal oocytes to reprogram human somatic cells. Here we compare the reprogramming of human somatic nuclei using oocytes obtained from animal and human sources. Comparative analysis of gene expression in morula-stage embryos was carried out using single-embryo transcriptome amplification and global gene expression analyses. Genomic DNA fingerprinting and PCR analysis confirmed that the nuclear genome of the cloned embryos originated from the donor somatic cell. Although the human-human, human-bovine, and human-rabbit clones appeared morphologically similar and continued development to the morula stage at approximately the same rate (39, 36, and 36%, respectively), the pattern of reprogramming of the donor genome was dramatically different. In contrast to the interspecies clones, gene expression profiles of the human-human embryos showed that there was extensive reprogramming of the donor nuclei through extensive upregulation, and that the expression pattern was similar in key upregulation in normal control embryos. To account for maternal gene expression, enucleated oocyte transcriptome profiles were subtracted from the corresponding morula-stage embryo profiles. t-Test comparisons (median-normalized data @ fc > 4; p < 0.005) between human in vitro fertilization (IVF) embryos and human-bovine or human-rabbit interspecies somatic cell transfer (iSCNT) embryos found between 2400 and 2950 genes that were differentially expressed, the majority (60-70%) of which were downregulated, whereas the same comparison between the bovine and rabbit oocyte profiles found no differences at all. In contrast to the iSCNT embryos, expression profiles of human-human clones compared to the age-matched IVF embryos showed that nearly all of the differentially expressed genes were upregulated in the clones. Importantly, the human oocytes significantly upregulated Oct-4, Sox-2, and nanog (22-fold, 6-fold, and 12-fold, respectively), whereas the bovine and rabbit oocytes either showed no difference or a downregulation of these critical pluripotency-associated genes, effectively silencing them. Without appropriate reprogramming, these data call into question the potential use of these discordant animal oocyte sources to generate patient-specific stem cells.
Multipotent mesenchymal stromal cells ( MSC) are progenitor cells able to differentiate into several lineages including chondrocytes, and thus represent a suitable source of cells for cartilage engineering. However, the control of MSC differentiation to hypertrophy is a crucial step for the clinical application of MSC in cartilage repair where a stable chondrogenic phenotype without transition to terminal differentiation is the goal to achieve. This study aims at identifying new factors that may regulate this process. Using microarrays, we compared the transcriptional profiles of human MSC and MSC-derived chondrocytes obtained after culture in micropellets. After chondrogenesis induction, 676 genes were upregulated, among which five transcription factors not yet associated with chondrocyte differentiation of adult stem cells. These factors, in particular Foxo3A, are strongly expressed at day 21 and in mature chondrocytes. We investigated the role of Foxo3A using RNA interference. Our results revealed an important role of Foxo3A in the differentiation process of MSC toward chondrogenic fate, both in early and late stages. Indeed, stable Foxo3A knockdown tends to increase cell survival and decrease apoptosis, mainly in early stages of chondrogenesis. Importantly, we show that the loss of Foxo3A in MSC results in an increased expression level of markers specific for mature ( aggrecan, collagen II) and hypertrophic ( collagen X) chondrocytes. Therefore, our findings suggest that upregulation of Foxo3A over the course of chondrogenic differentiation plays a dual role, mainly inhibiting the differentiation process toward hypertrophy and promoting cell apoptosis.
