In vitro differentiation of bovine bone marrow‐derived mesenchymal stem cells into male germ cells by exposure to exogenous bioactive factors

Abstract

Mesenchymal stem cells (MSC) are multipotent progenitor cells defined by their ability to self-renew and give rise to differentiated progeny. Previous studies have reported that MSC may be induced in vitro to develop into different types of specialized cells including male gametes. In vitro gamete derivation technology has potential applications as an alternative method for dissemination of elite animal genetics, production of transgenic animals and conservation of endangered species. This study aimed at investigating the in vitro effect of BMP4, TGFβ1 and RA on the potential for germ cell (GC) differentiation of bovine foetal MSC (bfMSC) derived from bone marrow (BM). The effect of BMP4, TGFβ1 and RA was analysed on the expression of pluripotent, GC and male GC markers on bfMSC during a 21-day culture period. bfMSC cultured under in vitro conditions expressed OCT4, NANOG and DAZL, but lacked expression of mRNA of VASA, STELLA, FRAGILIS, STRA8 and PIWIL2. Treatment with exogenous BMP4 and TGFβ1 induced a transient increase (p < .05) in DAZL and NANOG mRNA levels, respectively. However, exposure to RA was more effective in increasing (p < .05) expression of DAZL and regulating expression of OCT4 and mRNA levels of NANOG. These data suggest that bfMSC may possess potential for early GC differentiation, where OCT4, NANOG and specially DAZL may play significant roles in controlling progression along the GC lineage.

Overexpression of DAZL, STRA8, and BOULE Genes and Treatment With BMP4 or Retinoic Acid Modulate the Expression of MSC Overexpressing Germ Cell Genes

“…Moreover, RA treatment repressed the expression of OCT4 and NANOG in these cells. Our previous analyses in bovine fetal BM-MSC showed that the NANOG levels were not affected by RA; however, this treatment increased the expression of DAZL and reduced the expression of OCT4 (7). RA is a multipurpose factor that controls the meiotic entrance of PGCs in the genital cords by controlling the Cyp26b1 enzyme expression (15).…”

“…Pooling tissue samples was performed in an effort to reduce individual biological variation. Furthermore, transfected and control AT-MSCs were treated with 100 ng/mL of BMP4 or 0.1 μM of RA for 14 days ( 7 , 8 ). Cells samples at Day 14 were analyzed for the expression of pluripotent genes NANOG and OCT4 and GC genes DAZL, STRA8, BOULE, PIWIL2, c-KIT, and FRAGILIS using Q-PCR.…”

“…Plasticity of bfMSCs is not limited to mesodermal derivatives since these cells were also able to differentiate into neuroectodermal and endodermal cell types ( 6 ). Recently, it has been reported that bfMSCs exposed to bioactive factors, including retinoic acid (RA) and bone morphogenetic protein (BMP) 4 or cocultured with Sertoli cells, are able to upregulate the expression of specific markers associated to the early stages of GC differentiation ( 7 , 8 ). In addition, bfMSCs can be easily isolated and expanded under in vitro conditions to obtain a sufficient number for transplantation.…”

Gametes from stem cells: Status and applications in animal reproduction

“…In mouse, transplantation of PGCLCs differentiated in vitro from MSCs into germline‐depleted testes resulted in morphologically correct gametes, although their functionality was unproven (Nayernia et al, ). Expression of germ cell markers upon in vitro differentiation of MSCs isolated from different tissues of livestock species, including sheep (Ghasemzadeh‐Hasankolaei, Sedighi‐Gilani, & Eslaminejad, ), goat (Zhang et al, ) and cattle (Cortez et al, ), has been reported. In sheep, the use of RA and TGFβ1 as differentiation factors in MSC cultures from bone marrow has led to the generation of germ‐like cells that were unable to differentiate after transplantation into testes (Ghasemzadeh‐Hasankolaei, Eslaminejad, & Sedighi‐Gilani, ).…”

“…In sheep, the use of RA and TGFβ1 as differentiation factors in MSC cultures from bone marrow has led to the generation of germ‐like cells that were unable to differentiate after transplantation into testes (Ghasemzadeh‐Hasankolaei, Eslaminejad, & Sedighi‐Gilani, ). In cattle, MSCs isolated from foetal bone marrow have shown potential for generation of early germ cells under culture with BMP4, TGFβ1 and RA (Cortez et al, ). However, results were only partially positive since no expression was detected for VASA, STELLA, FRAGILIS, STRA8 or PIWIL2.…”