A comparative study on efficiency of adult fibroblasts and amniotic fluid-derived stem cells as donor cells for production of hand-made cloned buffalo (Bubalus bubalis) embryos

Abstract

The efficiency of two cell types, namely adult fibroblasts, and amniotic fluid stem (AFS) cells as nuclear donor cells for somatic cell nuclear transfer by hand-made cloning in buffalo (Bubalus bubalis) was compared. The in vitro expanded buffalo adult fibroblast cells showed a typical "S" shape growth curve with a doubling time of 40.8 h and stained positive for vimentin. The in vitro cultured undifferentiated AFS cells showed a doubling time of 33.2 h and stained positive for alkaline phosphatase, these cells were also found positive for undifferentiated embryonic stem cell markers like OCT-4, NANOG and SOX-2, which accentuate their pluripotent property. Further, when AFS cells were exposed to corresponding induction conditions, these cells differentiated into osteogenic, adipogenic and chondrogenic lineages which was confirmed through alizaran, oil red O and alcian blue staining, respectively. Cultured adult fibroblasts and AFS cells of passages 10-15 and 8-12, respectively, were used as nuclear donors. A total of 94 embryos were reconstructed using adult fibroblast as donor cells with cleavage and blastocyst production rate of 62.8 ± 1.8 and 19.1 ± 1.5, respectively. An overall cleavage and blastocyst formation rate of 71.1 ± 1.2 and 29.9 ± 2.2 was obtained when 97 embryos were reconstructed using AFS cells as donor cells. There were no significant differences (P > 0.05) in reconstructed efficiency between the cloned embryos derived from two donor cells, whereas the results showed that there were significant differences (P < 0.05) in cleavage and blastocyst rates between the cloned embryos derived from two donor cell groups. Average total cell numbers for blastocyst generated using AFS cells (172.4 ± 5.8) was significantly (P < 0.05) higher than from adult fibroblasts (148.2 ± 6.1). This study suggests that the in vitro developmental potential of the cloned embryos derived from AFS cells were higher than that of the cloned embryos derived from adult fibroblasts in buffalo hand-made cloning.

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

“…Primary ear fibroblast culture from an adult Murrah buffalo was established and prepared for cloning as reported earlier by Sadeesh et al (2014b). Briefly, ear skin biopsies from adult Murrah buffalo were obtained aseptically in sterile phosphate buffered saline (PBS) with 1 % antibiotic-antimycotic solution and transferred to laboratory within 10 min.…”

“…For characterization and differentiation of AFSCs the same culture protocols were used as described earlier (Sadeesh et al 2014b). …”

“…Donor cell preprations for HMC were done as described earlier (Sadeesh et al 2014b). Briefly, after a minimum of 1 week of cryopreservation, one vial of cryopreserved cells was thawed at 37°C in a water bath for approximately 15 s, then transferred into a centrifuge tube and the cells were suspended in the same growth medium as described for isolation and primary expansion.…”

“…In vitro culture of NT embryos was performed according to the methods described previously (Sadeesh et al 2014b). Briefly, the activated embryos were cultured in 400 ll of Research Vitro Cleave medium (K-RVCL-50, Cook, Brisbane, QLD, Australia) supplemented with 1 % fatty acid-free BSA in a four well dish (15-20 embryos/well) covered with mineral oil and kept undisturbed in a humidified CO 2 incubator at 38.5°C.…”

“…The adult fibroblast derived from ear pinna and NT-ES cell-like cells derived from cloned blastocysts generated using this adult fibroblast as donor cells, when used for NT, gave almost similar cleavage and blastocyst rates (George et al 2011). It has been recently demonstrated an enhanced in vitro developmental potential of the cloned embryos derived from amniotic fluid stem cells (AFSCs) than that of the cloned embryos derived from adult fibroblasts (AFs) in buffalo hand-made cloning (Sadeesh et al 2014b).…”