Hand-Made Cloned Buffalo ( Bubalus bubalis ) Embryos: Comparison of Different Media and Culture Systems

Yadav

2015

Cytotechnology

The developmental ability and gene expression pattern at 8-to 16-cell and blastocyst stages of buffalo (Bubalus bubalis) nuclear transfer (NT) embryos from fetal fibroblasts (FFs), amnion mesenchymal stem cells (AMSCs) and in vitro fertilized (IVF) embryos were compared in the present studies. The in vitro expanded buffalo FFs showed a typical ''S'' shape growth curve with a doubling time of 41.4 h and stained positive for vimentin. The in vitro cultured undifferentiated AMSCs showed a doubling time of 39.5 h and stained positive for alkaline phosphatase, and these cells also showed expression of pluripotency markers (OCT4, SOX2, NANOG), and mesenchymal stem cell markers (CD29, CD44) and were negative for haematopoietic marker (CD34) genes at different passages. Further, when AMSCs were exposed to corresponding induction conditions, these cells differentiated into adipogenic, chondrogenic and osteogenic lineages which were confirmed through oil red O, alcian blue and alizarin staining, respectively. Donor cells at 3-4 passage were employed for NT. The cleavage rate was significantly (P \ 0.05) higher in IVF than in FF-NT and AMSC-NT embryos (82.6 ± 8.2 vs. 64.6 ± 1.3 and 72.3 ± 2.2 %, respectively). However, blastocyst rates in IVF and AMSC-NT embryos (30.6 ± 2.7 and 28.9 ± 3.1 %) did not differ and were significantly (P \ 0.05) higher than FF-NT (19.5 ± 1.8 %). Total cell number did not show significant (P [ 0.05) differences between IVF and AMSC-NT embryos (186.7 ± 4.2, 171.2 ± 3.8, respectively) but were significantly (P \ 0.05) higher than that from FF-NT (151.3 ± 4.1). Alterations in the expression pattern of genes implicated in transcription and pluripotency (OCT4, STAT3, NANOG), DNA methylation (DNMT1, DNMT3A), histone deacetylation (HDAC2), growth factor signaling and imprinting (IGF2, IGF2R), apoptosis (BAX, BCL2), metabolism (GLUT1) and oxidative stress (MnSOD) regulation were observed in cloned embryos. The transcripts or expression patterns in AMSC-NT embryos more closely followed that of the in vitro derived embryos compared with FF-NT embryos. The results demonstrate that multipotent amnion MSCs have a greater potential as donor cells than FFs in achieving enhanced production of cloned buffalo embryos.

Section: Discussionmentioning

confidence: 99%

“…The recipient cytoplast preparations from in vitro matured oocytes (cumulus/zona removal and manual enucleation) and the procedures for HMC were performed using standard protocols as described earlier (Shah et al 2008). …”

Section: Preparation Of Recipient Cytoplast and Hand-made Cloning (Hmc)mentioning

confidence: 99%

Differential developmental competence and gene expression patterns in buffalo (Bubalus bubalis) nuclear transfer embryos reconstructed with fetal fibroblasts and amnion mesenchymal stem cells

Yadav

2015

Cytotechnology

“…The recipient cytoplasts preparations from in vitro matured oocytes (cumulus/zona removal and manual enucleation) and the procedures for HMC were performed using standard protocols as described earlier (Shah et al 2008). …”

Section: Preparation Of Recipient Cytoplasts and Hand-made Cloning (Hmc)mentioning

confidence: 99%

A comparative study on expression profile of developmentally important genes during pre-implantation stages in buffalo hand-made cloned embryos derived from adult fibroblasts and amniotic fluid derived stem cells

Kataria

et al. 2015

Cytotechnology

Abnormal gene expression in somatic cell nuclear transfer embryos due to aberrant epigenetic modifications of the donor nucleus may account for much of the observed diminished viability and developmental abnormalities. The present study compared the developmentally important gene expression pattern at 4-cell, 8-to 16-cell, morula, and blastocyst stages of buffalo nuclear transfer (NT) embryos from adult fibroblasts (AFs) and amniotic fluid stem cells (AFSCs). In vitro fertilized embryos were used as control embryos. Alterations in the expression pattern of genes implicated in transcription and pluripotency (OCT4, STAT3, NANOG), DNA methylation (DNMT1, DNMT3A), histone deacetylation (HDAC2), growth factor signaling, and imprinting (IGF2, IGF2R), apoptosis (BAX, BCL2), oxidative stress (MnSOD), metabolism (GLUT1) regulation were observed in cloned embryos. The expression of transcripts in AFSC-NT embryos more closely followed that of the in vitro fertilized embryos compared with AF-NT embryos. It is concluded that AFSCs with a relatively undifferentiated genome may serve as suitable donors which could be reprogrammed more efficiently to reactivate expression of early embryonic genes in buffalo NT.

“…The procedure followed for producing the SCNT buffalo embryos was an protocol developed in-house (Shah et al, 2008). Briefly, the IVM COCs were subjected to cumulus removal and Pronase (2.0 mg mL -1 in T10) treatment for zona digestion.…”

Section: Production Of Ivf Par and Scnt Buffalo Embryosmentioning

confidence: 99%

Development, Characterization, and Pluripotency Analysis of Buffalo (Bubalus bubalis) Embryonic Stem Cell Lines Derived fromIn Vitro–Fertilized, Hand-Guided Cloned, and Parthenogenetic Embryos

Saini

Ashraf

et al. 2015

Cellular Reprogramming

Self Cite

We present the derivation, characterization, and pluripotency analysis of three buffalo embryonic stem cell (buESC) lines, from in vitro-fertilized, somatic cell nuclear-transferred, and parthenogenetic blastocysts. These cell lines were developed for later differentiation into germ lineage cells and elucidation of the signaling pathways involved. The cell lines were established from inner cell masses (ICMs) that were isolated manually from the in vitro-produced blastocysts. Most of the ICMs (45-55%) resulted in formation of primary colonies that were subcultured after 8-10 days, leading subsequently to the formation of three buESC lines, one from each blastocyst type. All the cell lines expressed stem cell markers, such as Alkaline Phosphatase, OCT4, NANOG, SSEA1, SSEA4, TRA-1-60, TRA-1-81, SOX2, REX1, CD-90, STAT3, and TELOMERASE. They differentiated into all three germ layers as determined by ectodermal, mesodermal, and endodermal RNA and protein markers. All of the cell lines showed equal expression of pluripotency markers as well as equivalent differentiation potential into all the three germ layers. The static suspension culture-derived embryoid bodies (EBs) showed greater expression of all the three germ layer markers as compared to hanging drop culture-derived EBs. When analyzed for germ layer marker expression, EBs derived from 15% fetal bovine serum (FBS)-based spontaneous differentiation medium showed greater differentiation across all the three germ layers as compared to those derived from Knock-Out Serum Replacement (KoSR)-based differentiation medium.