Generation and Characterization of Embryonic Stem‐Like Cell Lines Derived from In Vitro Fertilization Buffalo (Bubalus bubalis) Embryos

Abstract

In the present study, buffalo embryonic stem-like (ES-like) cell lines were successfully isolated, cultured and characterized. From a total of 92 normal buffalo embryos obtained by in vitro fertilization, 18 were morulae, 33 were blastocyst and 41 were hatched blastocyst, the inside of morulae or inner cell masses of blastocysts were isolated mechanically and cultured onto mitomocin-C-inactivated buffalo embryonic fibroblasts as feeder layers. Alkaline phosphatase (AP) of ES-like cells, as well as the specific stage embryonic antigen SSEA-1, SSEA-3, SSEA-4 and transcription factor OCT-4, was used to evaluate the characterization of the cells. The spontaneous differentiation of ES-like cells was induced by culturing on leukaemia inhibitory factor-free medium for more than 2 weeks without passage. To evaluate mark gene expression, total RNA was extracted from cells, and specific primers were used for reverse transcriptase-polymerase chain reaction (RT-PCR). After 8-10 days of culture, primary ES-like cell colonies were formed in 0% (0/18) of morulae, 24.24% (8/33) of blastocysts and 60.98% (25/41) of hatched blastocysts, respectively. The forming rate of primary ES-like cells colonies in hatched blastocyst group was significantly (p < 0.05) higher than the obtained for other groups. Two ES-like cell lines could survive to eight passages at least by using the method of mechanical dissociation, but just three passages by using the method of enzymatic dissociation. The cells formed large, multicellular colonies with distinct boundaries, exhibited many important features of ES/ES-like cells, including positive AP, SSEA-1, SSEA-3 and SSEA-4 activity. Undifferentiated buffalo ES-like cells expressed Oct-4, Nanog, Sox2 gene mRNA. In vitro differentiation experiments had demonstrated that those cells were pluripotent.

Developmental Competence of Buffalo (Bubalus bubalis) Pluripotent Embryonic Stem Cells Over Different Homologous Feeder Layers and the Comparative Evaluation with Various Extracellular Matrices

“…Interestingly, maintenance of pluripotency in human (13) and bovine (14,15) ES cell lines does not exhibit a clear dependency on LIF further suggested that human and bovine ESCs require different signals to maintain self-renewal and pluripotency. However, in buffaloes (1,12,16) supplementation of 20 ng/ml LIF in ES cells culture medium was found able to maintain their pluripotency regardless of the presence or absence of feeder cells. Several studies have reported that the culture of mice (17), rhesus monkey (9) and human (6, 18) ESCs on homologous feeders implying that homologous cells can serve as feeder cells for prolong undifferentiated growth of ESCs.…”

“…Thus, advantages to use synthetic matrices are no DNA contamination while doing RT-PCR studies for marker genes besides easy manipulation of the ESC colonies. Very limited literature is available for the establishment of ESC culture in buffaloes (11,12). Finally, use of any feeder layer increases the work load and subsequently limits derivation of ESCs in larger scale.…”

Expression of Transcriptional Factor Genes (Oct-4, Nanog, and Sox-2) and Embryonic Stem Cell-Like Characters in Placental Membrane of Buffalo (Bubalus bubalis)

“…Efforts are being made to study various types of pluripotent stem cells (Huang et al 2010;Sritanaudomchai et al 2007;Verma et al 2007) in buffalo (Bubalus bubalis), the mainstay of the dairy and meat industries in many countries. The present study is a preliminary effort to investigate whether the cells in bubaline placental membrane cells can be cultured and whether they exhibit stem cell-like attributes.…”

Culture conditions for bovine embryonic stem cell-like cells isolated from blastocysts after external fertilization

“…Cao et al (2009) used mechanical separation for passaging of bovine embryonic stem cells. Huang et al (2010) cultured embryonic stem cell-like cells from buffalo embryos after external fertilization, using a mechanical method and trypsin digestion for passaging; they found that the cells could be cultured to the eighth passage with the mechanical method, but differentiated when grown to at most the fourth passage with trypsin digestion. Hamano et al (1998) obtained bovine embryonic stem cell-like cells with trypsin digestion.…”