Background: Germ cells undergo towards male or female pathways to produce spermatozoa or oocyte respectively which is essential for sexual reproduction. Mesenchymal stem cells (MSCs) have the potential of trans-differentiation to the multiple cell lineages. Methods: Herein, rat MSCs were isolated from bone marrow and characterized by their morphological features, expression of MSC surface markers, and in vitro differentiation capability. Results: Thereafter, we induced these cells only by retinoic acid supplementation in MSC medium and, could able to show that bone marrow derived MSCs are capable to trans-differentiate into male germ cell-like cells in vitro. We characterized these cells by morphological changes, the expressions of germ cell specific markers by immunophenotyping and molecular biology tools. Further, we quantified these differentiated cells. Conclusions: This study suggests that only Retinoic acid in culture medium could induce bone marrow MSCs to differentiate germ cell-like cells in vitro . This basic method of germ cell generation might be helpful in the prospective applications of this technology.

Some tissues retain extensive regeneration potential through out adult life and remain as active sites of cell production. Various cell types present in tissues are being produced through proliferation and progressive specialization from a pool of stem cells. In this regard, adult stem cells (ASCs) are multipotent progenitor cells with an ability to proliferate in vitro and undergo extensive self-renewal and differentiation into a wide range of cell types, including adipocytes, chondrocytes, osteocytes, myocytes, cardiomyocytes and neurons. In addition, recent studies showing the abilities of ASCs in generating oocytes-like cells (OLCs) present new perspectives to understand the specification and interaction during the germ cell formation and oogenesis. In the present study, ASCs were established from skin, adipose and ovarian tissues of minipigs. Isolated cells exhibited a fibroblast-like morphology with higher proliferation potential and stronger alkaline phosphatase (AP) activity. ASCs from all tissues expressed pluripotent transcriptional factors, such as Oct-3/4, Nanog and Sox-2 and phenotypic markers, including CD29, CD44, CD90 and vimentin. Further, ASCs were successfully dIfferentiated into osteocytes, adipocytes and neuron-like cells. Upon induction in oogenesis specific media, all ASCs were capable of differentiation into OLCs by exhibiting distinct morphological features. Generated OLCs expressed a range of germ cell specific markers, such as Vasa, deleted in Azoospermia-like (DAZL) factor, stella, c-kit, c-Mos, synaptonemal complex protein 3 (SCP-3), growth differentiation factor 9b (GDF- 9b), zona pellucida C (ZPC) and follicle stimulating hormone receptor (FSHR) at different time points of induction. Differentiated OLCs were also positive for the expression of Vasa and DAZL protein markers. Our findings showing that OLCs can be generated from ASCs of different tissue origin may offer pig as a suitable model for designing transgenic application strategies for reproductive tissue therapy. However, further studies are needed to understand the cellular and molecular mechanisms involved in germ cell differentiation from tissue specific stem cells.

The conserved THO/TREX complex is participated in pre-mRNA processing and mRNA nuclear export. In Metazoa, it has been reported that THO/TREX is loaded on nascent RNA transcribed by RNA polymerase II in a splicing-dependent manner; however, how THO/TREX functions is poorly understood. To understand the role of THO/TREX in eukaryotic gene expression, we investigated the role of THO/TREX in Drosophila germline, and found that lack of THOC5, a component of THO/TREX, showed defects in the biogenesis of piRNA, a distinct class of small non-coding RNAs that control expression of transposable elements (TE) in the Drosophila germline. Genome wide RNA-seq showed that THOC5 and other TREX components are essential for the biogenesis of piRNA. THO/TREX components are enriched on piRNA precursors transcribed from dual-strand piRNA clusters and co-localize in distinct nuclear foci that overlap with sites of piRNA transcription. The localization of TREX in nuclear foci and its loading on piRNA precursor transcripts depends on Cutoff, a protein associated with chromatin of piRNA clusters. We also show that TREX is required for accumulation of nascent piRNA precursors, suggesting that TREX is required for their efficient transcription. In addition to the biogenesis of piRNA, both THO and piRNA mutants showed over-proliferation of germline stem cell-like cells outside of stem cell niche. Taken together our study reveals a novel splicing-independent mechanism for THO/TREX loading on nascent RNA and its importance in piRNA biogenesis as well as a role of piRNA in the differentiation of germline stem cell.

난자 및 정자형성과정은 성에 따라 차이가 일부 있지만 생식줄기세포 (germ-line stem cells)의 증식을 통해 그 수를 유지하고, 그 중 일부 또는 전부가 감수분열에 진입하여 생식세포인 정자와 난자를 생산한다. 따라서 생식세포의 형성과정은 세포의 증식과 성장, 분화 기작을 연구할 수 있는 매우 훌륭한 시스템으로 여겨져 과거로부터 많은 연구가 진행되어왔다. 더욱이 인간에서 보조생식술과 동물 생명공학의 발전을 통해 그 중요성은 더욱 커지고 있으나, 그 체외배양 모델시스템이 없어 최근까지의 연구에는 많은 어려움이 있었다. 과거에 많은 연구자들에 의해 생식세포의 배양, 정소와 난소의 기관 배양 등이 시도된 바 있으나, 대부분 단기간 내 성공일 뿐 장기간 배양에는 어려움이 있었다. 하지만 교토대학의 Shinohara 교수팀에 의해 체외에서 정원줄기세포의 장기간 증식 배양법이 확립되었고, 또 배양된 정원줄기세포가 정소 세정관 내에 이식을 통해 정자형성과정을 재현할 수 있음이 확인되어 이 분야의 연구와 응용연구는 활성화되기 시작했다. 또한, 최근 들어 배아줄기세포, 성체줄기세포와 같은 여러 종류의 전분화능 줄기세포에서 생식줄기세포 또는 생식세포로의 분화가 일부 성공됨에 따라 생식세포의 증식과 분화의 조절기작이 일부 밝혀지고 있다. 따라서 이러한 연구성과 는 현재까지 치료가 불가능한 비폐쇄성 무정자증환자의 불임치료 방법으로의 발전 가능성을 제시할 뿐 아니라 동물생명공학분야로의 응용가능성을 더욱 높여주고 있다. 본 연제에서는 실험동물과 인간의 줄기세포로부터 생식세포의 분화에 관한 최근 연구를 정리하고, 이를 이용한 기초연구, 산업, 임상치료에의 적용가능성을 제시하고자 한다.