Nuclear Transfer in Rabbits with Different Types of Donor Cells

Production of cloned transgenic rabbits from somatic nuclei was hampered since the nuclear transfer technique had extremely low rate of success. The objectives of the present study were to evaluate the developmental potential of cloned embryos from different types of donor cells and to produce cloned rabbits from cultured somatic cells. In vivo matured MII oocytes were treated with demecolcine to induce small cytoplasmic extrusion containing all maternal chromatin. Enucleation was carried out by the removal of the cytoplasmic extrusion. Various types of cells were used as nuclear donors. After electric pulses, embryonic blastomeres were introduced into enucleated oocyte cytoplasm having escaped from meiotic arrest by electric pulses followed by treatment with 6-DMAP/CHX. Nuclei from in vitro cultured cumulus cells (RCCs) and fetal fibroblast cells (RFFs) either grown to confluency or deprived of serum from the culture medium (starved-RFFs) or treated with sodium butyrate (NaBu-RFFs) were introduced into enucleated oocytes by electric pulses, and the reconstructed oocytes were activated by electric pulses followed by 6-DMAP/CB. The developmental potential of nuclear transfer embryos was assessed by the rates of in vitro blastocyst formation and in vivo development to term. The results showed that the chemically assisted enucleation protocol was successfully used with MII oocytes since high extrusion and enucleation rates were obtained. High cleavage rates were obtained in any type of nuclear donors (87% for embryonic blastomere, 78% for RCC, 93% for confluent RFF, 87% for starved-RFF, 95% for NaBu-RFF). However, higher blastocyst rates were obtained from embryonic blastomere and NaBu-RFF derived embryos (59% and 49%, respectively) than that from other type of somatic donors (RCC: 32.3%, confluent RFF: 33.1%, starved-RFF: 32.5%). When cloned embryos were transferred into pseudo-pregnant mothers, live offspring derived from embryonic blastomeres and day 27-fetuses derived from cultured RCCs were produced. No full-term development was achieved when RFFs were used as nuclear donors. Additionally, When 4-10-cell stage cloned embryos were aggregated with one or two blastomeres of 6-12-cell stage in vivo fertilized embryos, although a greater proportion of aggregated embryos developed to blastocysts as compared with cloned, non-aggregated embryos (78% vs. 61%, P0.05) and two of them developed to term, although the development to blastocyst was similar to that of cloned embryos (58% vs. 61%, P>0.05), and even lower than that of cloned-in vivo fertilized embryos (58% vs. 78%, P<0.05). One of the two newborns, an overgrowth pup, died of respiratory failure within one hour while another one lived about two weeks until an accidental death. Genotypic analyses confirmed that both pups are clones derived from fetal fibroblasts. Overall, results from our study show that embryos cloned from embryonic cells had higher developmental potential than those cloned from somatic cells. Embryos cloned from somatic cells did not differ in their developmental capacity. After successive optimisation of all steps of nuclear transfer, we produced the first cloned rabbits from cultured somatic cells including cumulus cell and fetal fibroblast cells. These achievements will open a new way to more wide use of rabbit model for basic and applied research

Evidence for conserved DNA and histone H3 methylation reprogramming in mouse, bovine and rabbit zygotes

<p>Abstract</p> <p>Background</p> <p>In mammals the parental genomes are epigenetically reprogrammed after fertilization. This reprogramming includes a rapid demethylation of the paternal (sperm-derived) chromosomes prior to DNA replication in zygotes. Such active DNA demethylation in the zygote has been documented for several mammalian species, including mouse, rat, pig, human and cow, but questioned to occur in rabbit.</p> <p>Results</p> <p>When comparing immunohistochemical patterns of antibodies against 5-methyl-cytosine, H3K4me3 and H3K9me2 modifications we observe similar pronuclear distribution and dynamics in mouse, bovine and rabbit zygotes. In rabbit DNA demethylation of the paternal chromosomes occurs at slightly advanced pronuclear stages. We also show that the rabbit oocyte rapidly demethylates DNA of donor fibroblast after nuclear transfer.</p> <p>Conclusion</p> <p>Our data reveal that major events of epigenetic reprogramming during pronuclear maturation, including mechanisms of active DNA demethylation, are apparently conserved among mammalian species.</p

Comparative Analysis of Tenogenic Gene Expression in Tenocyte-Derived Induced Pluripotent Stem Cells and Bone Marrow-Derived Mesenchymal Stem Cells in Response to Biochemical and Biomechanical Stimuli

The tendon is highly prone to injury, overuse, or age-related degeneration in both humans and horses. Natural healing of injured tendon is poor, and cell-based therapeutic treatment is still a significant clinical challenge. In this study, we extensively investigated the expression of tenogenic genes in equine bone marrow mesenchymal stem cells (BMSCs) and tenocyte-derived induced pluripotent stem cells (teno-iPSCs) stimulated by growth factors (TGF-β3 and BMP12) combined with ectopic expression of tenogenic transcription factor MKX or cyclic uniaxial mechanical stretch. Western blotting revealed that TGF-β3 and BMP12 increased the expression of transcription factors SCX and MKX in both cells, but the tenocyte marker tenomodulin (TNMD) was detected only in BMSCs and upregulated by either inducer. On the other hand, quantitative real-time PCR showed that TGF-β3 increased the expression of EGR1, COL1A2, FMOD, and TNC in BMSCs and SCX, COL1A2, DCN, FMOD, and TNC in teno-iPSCs. BMP12 treatment elevated SCX, MKX, DCN, FMOD, and TNC in teno-iPSCs. Overexpression of MKX increased SCX, DCN, FMOD, and TNC in BMSCs and EGR1, COL1A2, DCN, FMOD, and TNC in teno-iPSCs; TGF-β3 further enhanced TNC in BMSCs. Moreover, mechanical stretch increased SCX, EGR1, DCN, ELN, and TNC in BMSCs and SCX, MKX, EGR1, COL1A2, DCN, FMOD, and TNC in teno-iPSCs; TGF-β3 tended to further elevate SCX, ELN, and TNC in BMSCs and SCX, MKX, COL1A2, DCN, and TNC in teno-iPSCs, while BMP12 further uptrended the expression of SCX and DCN in BMSCs and DCN in teno-iPSCs. Additionally, the aforementioned tenogenic inducers also affected the expression of signaling regulators SMAD7, ETV4, and SIRT1 in BMSCs and teno-iPSCs. Taken together, our data demonstrate that, in respect to the tenocyte-lineage-specific gene expression, BMSCs and teno-iPSCs respond differently to the tenogenic stimuli, which may affect the outcome of their application in tendon repair or regeneration

Motor neurons and endothelial cells additively promote development and fusion of human iPSC-derived skeletal myocytes

Abstract Background Neurovascular cells have wide-ranging implications on skeletal muscle biology regulating myogenesis, maturation, and regeneration. Although several in vitro studies have investigated how motor neurons and endothelial cells interact with skeletal myocytes independently, there is limited knowledge about the combined effect of neural and vascular cells on muscle maturation and development. Methods Here, we report a triculture system comprising human-induced pluripotent stem cell (iPSC)-derived skeletal myocytes, human iPSC-derived motor neurons, and primary human endothelial cells maintained under controlled media conditions. Briefly, iPSCs were differentiated to generate skeletal muscle progenitor cells (SMPCs). These SMPCs were seeded at a density of 5 × 104 cells/well in 12-well plates and allowed to differentiate for 7 days before adding iPSC-derived motor neurons at a concentration of 0.5 × 104 cells/well. The neuromuscular coculture was maintained for another 7 days in coculture media before addition of primary human umbilical vein endothelial cells (HUVEC) also at 0.5 × 104 cells/well. The triculture was maintained for another 7 days in triculture media comprising equal portions of muscle differentiation media, coculture media, and vascular media. Extensive morphological, genetic, and molecular characterization was performed to understand the combined and individual effects of neural and vascular cells on skeletal muscle maturation. Results We observed that motor neurons independently promoted myofiber fusion, upregulated neuromuscular junction genes, and maintained a molecular niche supportive of muscle maturation. Endothelial cells independently did not support myofiber fusion and downregulated expression of LRP4 but did promote expression of type II specific myosin isoforms. However, neurovascular cells in combination exhibited additive increases in myofiber fusion and length, enhanced production of Agrin, along with upregulation of several key genes like MUSK, RAPSYN, DOK-7, and SLC2A4. Interestingly, more divergent effects were observed in expression of genes like MYH8, MYH1, MYH2, MYH4, and LRP4 and secretion of key molecular factors like amphiregulin and IGFBP-4. Conclusions Neurovascular cells when cultured in combination with skeletal myocytes promoted myocyte fusion with concomitant increase in expression of various neuromuscular genes. This triculture system may be used to gain a deeper understanding of the effects of the neurovascular niche on skeletal muscle biology and pathophysiology