Induction of Ram Bone Marrow Mesenchymal Stem Cells into Germ Cell Lineage using Transforming Growth Factor‐β Superfamily Growth Factors

• Published in: Reproduction in domestic animals 2014-08, Vol.49 (4), p.588-598
• Main Authors: Ghasemzadeh‐Hasankolaei, M, Sedighi‐Gilani, MA, Eslaminejad, MB
• Format: Article
• Language: English
• Subjects: alkaline phosphatase; Alkaline Phosphatase - metabolism; Animals; bone marrow; Bone Marrow Cells - cytology; Bone Morphogenetic Protein 4 - pharmacology; Bone Morphogenetic Proteins - pharmacology; Cell Differentiation - drug effects; Cells, Cultured; germ cells; growth factors; immunocytochemistry; Immunohistochemistry - veterinary; Male; male fertility; Mesenchymal Stromal Cells - cytology; Osteogenesis; rams; retinoic acid; reverse transcriptase polymerase chain reaction; Reverse Transcriptase Polymerase Chain Reaction - veterinary; Sheep; Spermatogenesis; Spermatogonia - cytology; stem cells; Transforming Growth Factor beta1 - pharmacology
• ISSN: 0936-6768; 1439-0531
• DOI: 10.1111/rda.12327

Several studies have proposed that in vitro generation of germ cells (GCs) from stem cells can be considered a future option for infertility treatment. Mesenchymal stem cells (MSCs) have the capability to differentiate into male GCs with the use of inducers such as retinoic acid. Transforming growth factor‐beta 1 (TGFb1) has been shown to play important roles in male fertility and spermatogenesis. Bone morphogenic protein 4 (BMP4) and BMP8b are also involved in the derivation of primordial GCs (PGCs) from epiblast cells. Therefore, this study aims to determine whether TGFb1, BMP4 and BMP8b can initiate transdifferentiation of MSCs into GCs in vitro and to determine the type of changes that occur in the expression of GC‐specific markers. In this study, we have divided passage‐3 ram bone marrow (BM)‐MSCs into three main groups (BMP4, BMP8b and TGFb1) which were separately treated with 10 ng/ml TGFb1, 100 ng/ml BMP4 and 100 ng/ml BMP8b for a period of 21 days. We have evaluated the ability of these groups to differentiate into GCs by assessing expressions of GC‐specific markers with reverse transcription PCR (RT‐PCR), quantitative RT‐PCR (qRT‐PCR), immunocytochemistry, morphological changes and alkaline phosphatase (ALP) activity. Our results showed that BMP4 and BMP8b induced PGCs properties in some BM‐MSCs and TGFb1 formed spermatogonial stem cells (SSCs) and spermatogonia‐like cells in BM‐MSCs culture. The important results of this study provide the basis for additional studies to determine the exact mechanism of GCs differentiation and possibly solve the problem of infertility.