Bones Morphogenic Protein‐4 and retinoic acid combined treatment comparative analysis for in vitro differentiation potential of murine mesenchymal stem cells derived from bone marrow and adipose tissue into germ cells

Abstract

Nowadays, infertility is no longer considered as an unsolvable disorder due to progresses in germ cells derived from stem lineage with diverse origins. Technical and ethical challenges push researchers to investigate various tissue sources to approach more efficient gametes. The purpose of the current study is to investigate the efficacy of a combined medium, retinoic acid (RA) together with Bone Morphogenic Protein-4 (BMP4), on differentiation of Bone Marrow Mesenchymal Stem Cells (BMMSCs) and adipose-derived mesenchymal stem cells (ADMSCs) into germ cells. Murine MSCs were obtained from both Bone Marrow (BM) and Adipose Tissue (AT) samples and were analyzed for surface markers to get further verification of their nature. BMMSCs and ADMSCs were induced into osteogenic and adipogenic lineage cells respectively, to examine their multipotency. They were finally differentiated into germ cells using media enriched with BMP4 for 4 days followed by addition of RA for 7 days (11 days in total). Analyzing of differentiation potential of BMMSCs- and ADMSCs were performed via Immunofluorescence, Flowcytometry and Real time-PCR techniques for germ cell-specific markers (Mvh, Dazl, Stra8 and Scp3). Mesenchymal surface markers (CD90 and CD44) were expressed on both BMMSCs and ADMSCs, while endothelial and hematopoietic cell markers (CD31 and CD45) had no expression. Finally, all germ-specific markers were expressed in both BM and AT. Although germ cells differentiated from ADMSCs showed faster growth and proliferation as well as easy collection, they significantly expressed germ-specific markers lower than BMMSCs. This suggests stronger differentiation potential of murine BMMSCs than ADMSCs.

Comparison of the efficacy of bone morphogenetic protein-4 on in vitro differentiation of murine adipose and bone marrow mesenchymal stem cells into primordial germ cells

“…The multi-potency of BMD-MSC and AD-MSC has made them a proper alternative to treat and control mesoderm defects and also the lineages of other tissues ( 16 17 ). Also, previous studies showed that BMD-MSC and AD-MSC could be differentiated under appropriate conditions to germ cells ( 15 18 19 20 21 22 ).…”

“…Based on our previous research, the combined addition of retinoic acid and BMP4 has led to the development of late germ cells in vitro ( 18 ). Consequently, it seems that to continue the development, more time or the use of other stimuli combined with BMP4 is needed.…”

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“…In another study, RA and BMP4 were combined and applied to both BM-MSCs and adipose tissue-derived MSCs (AD-MSC) to compare their differentiation potency to germ cells by determining the expression levels of Mvh, Dazl, Stra8 and Scp3 germ cell specific genes. Although there was an increase in the expression of these genes in both BM-MSCs and AD-MSCs, the expression rate in BM-MSCs was higher than in AD-MSCs, indicating that the potential for BM-MSCs to differentiate into germ cells is stronger [33]. The properties of AD-MSCs such as antioxidants and immune modulators are known.…”

“…Recent advances in stem cell technology have also brought a new perspective to treatment of infertility. Nowadays, infertility is no longer a desperate disease due to the studies on obtaining germ cells from cells of different origins [33]. It is thought that infertility will be treated by transplanting germ cells obtained from differentiated stem cells in vitro to gonads in patients of non-obstructive azoospermia [34].…”

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“…Mesenchymal stromal cells (MSCs) can be isolated from a variety of adult and neonatal tissues and can transdifferentiate into multiple cell lineages including PGCLCs. MSCs have been shown to express multiple germ cell markers following treatment with retinoic acid and/ or various growth factors (Dissanayake et al, 2018;Ghasemzadeh-Hasankolaei et al, 2014;Hua et al, 2009;Luo et al, 2019;Shirzeyli et al, 2017;Wei et al, 2016;Yan et al, 2015). Haploid spermatid-like cells have been observed from MSC-derived PGCLC cultures and transplants (Ghasemzadeh-Hasankolaei et al, 2016a,b;Shlush et al, 2017;Zhang et al, 2014).…”