Isolation and characterization of EG‐like cells from Chinese swamp buffalo (Bubalus bubalis)

Abstract

There have been few studies done on the isolation and characterization of Chinese swamp buffalo embryonic germ cells (EG cells). Here, we first report on EG-like cells isolated from Chinese swamp buffalo fetuses. The results showed the cells grew in large, multilayered colonies, which were densely packed with an obvious border resembling mouse embryonic stem cells (ES cells) and EG cells. The buffalo EG-like cells expressed AP, SSEA-1, SSEA-3, SSEA-4 and OCT-4. By RT-PCR, we found that undifferentiated swamp buffalo EG-like cells expressed the OCT-4, NANOG, SOX2, FOXD3, GP130, STAT3, and HEB gene mRNA, but not Fgf4. When these cells were cultured for more than 2weeks without passage, they could differentiate into several types of cells including fibroblast-like, neuron-like, smooth muscle-like, and epithelial-like cells. Some cells formed simple embryoid bodies (EBs) and cystic EBs by suspension culture. By RT-PCR, we found cystic EBs expressed FOXD3, GP130, STAT3 and HEB gene mRNA, but not OCT-4, NANOG, and SOX2 gene mRNA, which could be detected in undifferentiated buffalo EG-like cells. At the same time, the expression of KERATIN-14 (Endoderm), GATA4, ACTA2 (Mesoderm) and TUBB3 (Ectoderm) gene mRNA were also detected in cystic EBs. The results suggested that these cells were capable of forming three germ layers in in vitro differentiation. The expression of OCT-4, NANOG and SOX2 might be essential for Chinese swamp buffalo EG-like cells in a pluripotent state. During the isolation and culture of Chinese swamp buffalo EG-like cells, we found the fetuses that were at 30-80days post-coitus were more efficient than others; and the mechanical method was better than trypsin digestion. The maximal passage of the mechanical method was eight, but the trypsin digestion was just three passages. So it seemed like that the buffalo EG-like cells were sensitive to trypsin. In summary, we were the first to isolate and characterize Chinese swamp buffalo EG-like cells that had morphology and characterization similar to those of established EG/EG-like cells in mouse and human.

Induced Multilineage Differentiation of Chicken Embryonic Germ Cells via Embryoid Body Formation

“…(2) subcutaneous injection of these cells into syngenetic animal induces teratomas that may include cells of endodermal, ectodermal, or mesodermal origin; (3) In vitro differentiation via embryoid body (EB) formation, which results in collections of precursors and differentiated cells from a wide variety of lineages. During the last decade, EG cell lines that share most of these characteristics have been mostly reported for the mouse [9,10] and human [5,11].…”

“…In vivo, PGCs can give rise to embryonic carcinoma (EC) cells, the stem cells of testicular tumors [1]. In vitro, when cultured PGCs are exposed to a specifi c cocktail of growth factors, they continue to proliferate and form large colonies that can be expanded indefinitely [2,3]. Those cells, which termed as embryonic germ (EG) cells, are an important alternative source of pluripotent stem cells that can differentiate into a variety of cell types in vitro including musculoskeletal and neuronal cells [4,5].…”

Pluripotent cells in farm animals: state of the art and future perspectives

“…The cells can be maintained in vitro indefinitely in the undifferentiated state when the medium is supplemented by specific growth factors such as stem-cell factor (SCF), LIF and bFGF (Matsui et al 1992). Attempts towards the successful isolation and characterisation of EGCs were performed in fetuses from human (Shamblott et al 1998), rabbits (Kakegawa et al 2008), goats (Kühholzer et al 2000), sheep (Ledda et al 2010), pigs (Shim et al 1997), cattle (Choi and Anderson 1998), buffalo (Huang et al 2007), horse (Curran et al 1997), laboratory rat (Leitch et al 2010) and chicken (Park and Han 2000). The following paragraphs summarise the current status of the derivation of germline stem cells in farm animals.…”

Reproductive biotechniques in buffaloes (Bubalus bubalis): status, prospects and challenges

“…These stem cells, termed parthenogenetic ES cells (PGES), were capable of differentiating in vitro and in vivo into a variety of cell types and were thus suitable for use as an in vitro model in which to investigate the effects of imprinting on cell differentiation, as well as in which to conduct extensive molecular studies on imprinted genes (Sritanaudomchai et al 2007). Huang et al (2007) reported for the first time on embryonic germ cells isolated from very early stage buffalo fetuses. The cells were found to grow in large multilayered colonies, with features resembling mouse ES cells.…”