R. Dutta

ISOLATION AND CHARACTERIZATION OF EMBRYONIC STEM CELL-LIKE CELLS FROM IN VITRO PRODUCED GOAT (Capra hircus) EMBRYOS

The aim of the present study was to isolate and characterize goat embryonic stem cell-like cells from in vitro produced goat embryos. Inner cell mass (ICM) cells were isolated either mechanically or by enzymatic digestion from 150 blastocysts and 35 hatched blastocysts whereas 100 morulae were used for blastomeres isolation mechanically. The ICM derived cells or blastomeres were cultured on a feeder layer. The primary colony formation was significantly higher (P < 0.01) for hatched blastocysts (77.14%) than early/expanded blastocysts (54%) or morula (14%). When ICMs were isolated mechanically the primary colony formation for hatched blastocysts (90%) as well as blastocysts (66%) were significantly more than when ICMs were isolated by enzymatic digestion (60% and 30%, respectively). The colonies were disaggregated either mechanically or by enzymatic digestion for further subculture. When mechanical method was followed, the colonies remained undifferentiated up to 15 passages and three ES cell-like cell lines were produced (gES-1, gES-2, and gES-3). However, enzymatic disaggregation resulted in differentiation. The undifferentiated cells showed stem cell like morphological features, normal karyotype, and expressed stem cell specific surface markers like alkaline phosphatase, TRA-1-61, TRA-1-81, and intracellular markers Oct4, Sox2, and Nanog. Following prolonged culture of the ES cell-like cells were differentiated into several types of cells including neuron like and epithelium-like cells. In conclusion, goat embryonic stem cell-like cells can be isolated from in vitro produced goat embryos and can be maintained for long periods in culture.

A comparative study on efficiency of adult fibroblast, putative embryonic stem cell and lymphocyte as donor cells for production of handmade cloned embryos in goat and characterization of putative ntES cells obtained from these embryos

The main purpose of the experiment was to compare the efficiency of three cell types, namely adult fibroblast, putative embryonic stem (ES) cell, and lymphocyte, as donor cells for somatic cell nuclear transfer by handmade cloning in goats. The outcome clearly shows that putative embryonic stem cells, with a cleavage and blastocyst production rate of 74.69% ± 3.92 and 39.75% ± 3.86, respectively, performs better in comparison to adult fibroblast cell and lymphocyte. Between adult fibroblast cell and lymphocyte no statistically significant difference exists at P < 0.05. An overall cleavage and blastocyst formation rate of 67.41% ± 3.92 and 26.96% ± 3.86 was obtained using adult fibroblast donor cells. The study establishes beyond doubt the reprogrammability of lymphocyte by handmade cloning (HMC) protocol with a cleavage and blastocyst production rate of 56.47% ± 3.92 and 24.70% ± 3.86, respectively. PCR analysis of highly polymorphic 286 bp fragment of MHC II DRB genes of cloned embryos and three donor cells were performed to verify the cloned embryos. The amplified PCR products were subjected to SSCP to confirm their genetic identity. The karyotyping of the cloned embryos showed normal chromosomal status as expected in goat. Significantly, in the second stage of the experiment, the produced cloned embryos were successfully used to derive ntES-like cells. The rate of primary colony formation rate was 62.50% ± 4.62 for fibroblast donor cell derived embryos. The same was 60.60% ± 4.62 for putative ES donor cell derived embryos and 66.66% ± 4.62 for lymphocyte donor cell derived embryos, respectively. The putative ntES colonies were positively characterized for alkaline phosphatase, Oct-4, TRA-1-60, TRA-1-81, Sox-2, and Nanog by Immunocytochemistry and Reverse Transcription PCR. To further validate the stem ness, the produced putative ntES colonies were differentiated to embryoid bodies. Immunocytochemistry revealed that embryoid bodies expressed NESTIN specific for ectodermal lineage; GATA-4 for endodermal lineage and smooth muscle actin-I, and troponin-I specific for mesodermal lineage. The study has established an efficient protocol for putative ntES cell derivation from HMC embryos. It could be of substantial significance as patient specific ntES cells have proven therapeutic significance.

Hand-Made Cloned Goat (Capra hircus) Embryos—A Comparison of Different Donor Cells and Culture Systems

Nuclear transfer is a very effective method for propagation of valuable, extinct, and endangered animals. Hand-made cloning (HMC) is an efficient alternative to the conventional micromanipulator-based technique in some domestic species. The present study was carried out for the selection of suitable somatic cells as a nuclear donor and development of an optimum culture system for in vitro culture of zona-free goat cloned embryos. Cleavage and blastocyst rates were observed 72.06 ± 2.94% and 0% for fresh cumulus cells, 81.95 ± 3.40% and 12.74 ± 2.12% for cultured cumulus cells, and 92.94 ± 0.91% and 23.78 ± 3.33% for fetal fibroblast cells, respectively. There was a significant (p < 0.05) increase in blastocyst production in goats when cultured on a flat surface (FS) (23.78 ± 3.33 %) than well of wells (WOW) (15.84 ± 2.12 %) and microdrops (MD) (0.7 ± 0.7%). Furthermore, cleavage and blastocyst production rates were significantly (p < 0.05) more in the WOW (15.84 ± 2.12%) than the MD (0.7 ± 0.7%) system. The quality of HMC blastocysts was studied by differential staining. Genetic similarity was confirmed by polymerase chain reaction (PCR)-based amplification of the second exon of the MHC class II DRB gene, which gave similar bands in electrophoresis (286 bp) both in cloned embryos and donor cells. In conclusion, the present study describes that the fetal fibroblast cell is a suitable candidate as nuclear donor, and the flat surface culture system is suitable for zona-free blastocyst development by the hand-made cloning technique in the goat.

Cardiomyocytes rhythmically beating generated from goat embryonic stem cell

The aim of present investigation was isolation, characterization and differentiation into cardiomyocytes of putative goat embryonic stem cells produced from in vitro fertilized goat embryos. Goat blastocysts were produced in vitro by standard methods of in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) techniques. The ICMs isolated from IVF blastocysts were cultured on 10 μl/ml mitomycin-C inactivated fetal fibroblast feeder layer with LIF. The putative ES colonies were characterized for extracellular markers like alkaline phosphatase, TRA-1-60, TRA-1-81, SSEA-1, SSEA-4 by immunocytochemistry and intracellular markers like Oct4, Sox2 and Nanog with reverse-transcription-PCR. The ES cells were successfully subcultured up to 22nd passage with feeder layer and LIF and up to 12th passage without feeder layer with LIF only. They exhibited normal karyotyping (20th passage) and maintained the expression of specific surface markers like alkaline phosphatase, SSEA-4, TRA-1-61, TRA-1-81 and intracellular markers Oct4, Sox2 and Nanog. The embryoid bodies (EBs) were generated from goat ES cells of 20th passage and were analyzed with markers like Gata4, BMP4 and Nestin. Differentiation was induced by medium containing 100 ng/ml Activin-A, 10 ng/ml FGF-2 and 100 ng/ml BMP-4. The embryoid bodies were analyzed with markers like Gata4, BMP4 and Nestin. The rhythmic beating of cardiomyocytes was observed after 30 d and the beating was still continuing even after 160 d of culturing. Similarly, 2nd and 3rd batches of EBs were also beating and the beating continues after 75 d and on. The beating cells were observed positive for cardiac specific markers like α Actinin, C-Troponin and α-Myosin heavy chain. Histological studies also revealed morphology similar to cardiomyocytes. Prominent contractions typical of cardiac tissue have been maintained in the differentiated cells up to 160 d and still continuing beating at the rate of 30 beats/min. It could be concluded that ES cells generated from goat embryos were maintained undifferentiated up to 22nd passage on feeder layer and to 12th passage without feed layer using LIF and that the differentiation protocol induced rhythmic beating cells.

Effect of roscovitine treated donor cells and different activation methods on development of handmade cloned goat (Capra hircus) embryos.

The aim of the present investigation was to find out the effects of roscovitine treatment of donor cells and different activation methods on development of HMC goat embryos. Goat fetal fibroblast cells were cultured and divided into three treatment groups-contact inhibition group, roscovitine treatment group and serum starvation group. There was a significant decrease in blastocyst yield in serum starvation group (6.82%) compared to roscovitine treatment group (19.31%) and contact inhibition group (18.52%), however, no significant difference was found between roscovitine treatment group and contact inhibition group. To see the effect of different methods of activation, the reconstructed embryos were randomly divided into two groups and activated by two methods-one half by 2 μM Ca ionophore and another half by 2.31 kV/cm for 15 μSec electrical pulse. Subsequently, cloned embryos were cultured in TCM-199 based embryo development medium supplemented with 10 mg/mL bovine serum albumin in WOW culture system. There was a significant increase in the rate of cleavage and blastocyst production in electric pulse activation of 78.57% and 21.43% than Ca ionophore activation of 62.63% and 10.61% respectively. In conclusion, treatment of donor cells with roscovitine yields a significantly increased blastocyst than serum starved donor cells but equivalent blastocyst to contact inhibition group and electrical pulse activation (EPA) improves the production of HMC goat embryos.

Study of the efficiency of chemically assisted enucleation method for handmade cloning in goat (Capra hircus).

The present investigation was carried out to find an efficient chemically assisted procedure for enucleation of goat oocytes related to handmade cloning (HMC) technique. After 22-h in vitro maturation, oocytes were incubated with 0.5 μg/ml demecolcine for 2 h. Cumulus cells were removed by pipetting and vortexing in 0.5 mg/ml hyaluronidase, and zona pellucida were digested with pronase. Oocytes with extrusion cones were subjected to oriented bisection. One-third of the cytoplasm with the extrusion cone was removed with a micro blade. The remaining cytoplasts were used as recipients in HMC. Goat foetal fibroblasts were used as nuclear donors. The overall efficiency measured as the number of cytoplasts obtained per total number of oocytes used was significantly (p < 0.05) higher in chemically assisted handmade enucleation (CAHE) than oriented handmade enucleation without demecolcine (OHE) (80.02 ± 1.292% vs. 72.9 ± 1.00%, respectively, mean ± SEM). The reconstructed and activated embryos were cultured in embryo development medium (EDM) for 7 days. Fusion, cleavage and blastocyst development rate were 71.63 ± 1.95%, 92.94 ± 0.91% and 23.78 ± 3.33% (mean ± SEM), respectively which did not differ significantly from those achieved with random handmade enucleation and OHE. In conclusion, chemically assisted enucleation is a highly efficient and reliable enucleation method for goat HMC which eliminates the need of expensive equipment (inverted fluorescence microscope) and potentially harmful chromatin staining and ultraviolet (UV) irradiation for cytoplast selection.

Production and Characterization of Putative ntES Cells from Handmade Cloned Goat Embryos Derived from Adult Fibroblast Donor Cells

In the field of nuclear transfer handmade cloning (HMC) technique has been a relatively recent addition. In the present investigation attempts were made to efficiently derive stem cell from hand made cloned (HMC) embryos in goat using adult fibroblast donor cell and to characterize the derived putative ntES cell for their stemness. Oocytes were collected from slaughterhouse ovaries and matured in maturation medium containing TCM199 (HEPES modified), 1 μg/ml FSH, 10 μg/ml LH, 1 μg/ml Estradiol-17β, 3% BSA and 5% FBS. The efficiency of the donor fibroblast cell for nuclear transfer was also assessed and an overall cleavage and morula formation rate of 62.44%±3.9 & 35.30%±3.86 was obtained respectively. Genetic relationship between the cloned embryos and donor cells was established by PCR amplification of highly polymorphic goat major histocompatibility complex (MHC) class II DRB gene. The PCR products were further subjected to SSCP for confirming the genetic relationship conclusively. Stem cell colonies wer...

Effect of leukaemia inhibitory factor and different types of feeder layers on growth and pluripotent nature of embryonic stem cells from in vitro produced goat (Capra hircus) blastocysts

Feeder layer and leukaemia inhibitory factor (LIF) play an important role in maintaining the embryonic stem cells in undifferentiated state and help in their unlimited proliferation. The aim of the present investigation was to see the effect of LIF and different types of feeder layers on growth and pluripotent nature of goat embryonic stem cells. Goat blastocysts were produced in vitro by standard methods of in vitro maturation (IVM), in vitro fertilisation (IVF) and in vitro culture (IVC) techniques. Inner cell mass (ICM) cells were isolated mechanically and cultured in embryonic stem (ES) cell medium on inactivated feeder layer supplemented with or without LIF. When ICM cells were cultured on goat fibroblast feeder layer in ES cell culture medium supplemented with LIF they maintained undifferentiated state up to 15 passages. The undifferentiated ES cells showed stem cell specific morphological features, normal karyotype and expressed stem cell specific surface markers like alkaline phosphatase,TRI-1-61, TRI-1-81 and intracellular markers Oct4, Sox2 and Nanog. It could be concluded that goat embryonic stem cells were successfully cultured on feeder layers which were not of goat origin but to maintain the undifferentiated state for long time was found the best on goat fetal fibroblast with LIF.