Isolation and characterization of embryonic stem cell‐like cells from in vitro‐produced buffalo (Bubalus bubalis) embryos

Abstract

This study was carried out to isolate and characterize buffalo embryonic stem (ES) cell-like cells from in vitro-produced embryos. Inner cell mass (ICM) cells were isolated either mechanically or by enzymatic digestion from 120 blastocysts whereas 28 morulae were used for the isolation of blastomeres mechanically. The ICM cells/ blastomeres were cultured on mitomycin-C-treated feeder layer. Primary cell colony formation was higher (P < 0.05) for hatched blastocysts (73.1%, 30/41) than that for early/expanded blastocysts (25.3%, 20/79). However, no primary cell colonies were formed when blastomeres obtained from morulae were cultured. Primary colonies were formed in 14.1% (12/85) of intact blastocyst culture, which was significantly lower (P < 0.05) than that of 41.6% for ICM culture. These colonies were separated by enzymatic or mechanical disaggregation. Using mechanical disaggregation method, the cells remained undifferentiated and two buffalo ES cell-like cell lines (bES1, bES2) continued to grow in culture up to eight passages. However, disassociation through enzymatic method resulted in differentiation. Undifferentiated cells exhibited stem cell morphological features, normal chromosomal morphology, and expressed specific markers such as alkaline phosphatase (AP) and Oct-4. Cells formed embryoid bodies (EBs) in suspension culture; extended culture of EBs resulted in formation of cystic EBs. Following prolonged in vitro culture, these cells differentiated into several types of cells including neuron-like and epithelium-like cells. Furthermore, the vitrified-thawed ES cell-like cells also exhibited typical stem cell characteristics. In conclusion, buffalo ES cell-like cells could be isolated from in vitro-produced blastocysts and maintained in vitro for prolonged periods of time.

Derivation of buffalo embryonic stem-like cells from in vitro-produced blastocysts on homologous and heterologous feeder cells

“…In a present study, the morphology of BuES-like cells on BEF was observed to be similar to our previous study [16], and closely resembled porcine [39] and cat [30] ES cells. However, the colony morphology was found to vary on heterologous feeder layers where they appeared compact, thinner and flatter but with clear margins on GEF feeder layers and appeared to carry loose cells with increased intercellular spacing and showed lack of clear margins on SEF feeder layers.…”

“…As stated earlier in various reports, homologous feeder cell layers supported self renewal and maintained pluripotent characteristics of human and monkey ES cells, with characteristics similar to those of ES cells cultured on mouse fetal fibroblast (MEF) [9,15]. Recently, buffalo embryonic stem cell-like cells have been generated on buffalo fetal fibroblasts [16,17]; although the buffalo ES (BuES) cell-like cells grew on homologous feeder cell layers but cell colonies did not maintain an undifferentiated state over a long period of time. Whether the absence of specific factors or other unknown attributes of buffalo embryonic fibroblast (BEF) affected the self-renewal of BuES-like cells is not known.…”

“…Corresponding results were reported by [30], in which cat ES-like cells expressed the "stemness" transcription factors and the levels of expression of OCT-4, NANOG and C-MYC were reduced in cat ES-like colonies as compared to that seen in blastocysts. In other study, expression of transcription factors like Oct 4 and Nanog were used as molecular markers for ES cells in various species [16,17,[41][42][43]. Moreover, when undifferentiated primary colonies were analyzed for the expression of pluripotent transcription factors, Gómez et al [30] found that OCT-4 was down regulated regardless of the expression of NANOG.…”

“…The present study show that homologous feeder layers allow efficient derivation of BuES-like cells from hatched and expanded blastocysts than heterologous feeder layers. Successful use of homologous embryonic fibroblast feeder layers to produce ES cells has been previously done in mink [19], marsupials [20], human [21][22][23], pig [24,25] and buffalo [16,17,26]. In contrast, in an early study, Piedrahita et al [27] determined that porcine embryonic fibroblasts (PEF) did not promote attachment of the porcine ICM to the feeder layer.…”

“…leading to an increased inclination for BuES-like cell derivation from hatched blastocysts compared to expanded blastocysts. In earlier studies too, the isolation of ICMs either from morula, early stage of blastocyst or late stage of blastocyst gave an unsatisfactory result as compared to hatched blastocyst in various species [16,17,35,36]. In our earlier study from our laboratory [17], the comparison of ICM isolation method on buffalo ES cell generation was evaluated.…”