Differentiation Of Bone Mesenchymal Stem Cell Into Leydig Cell Under Co-cultivation

BackgroundTestes loss caused by trauma or surgical procedure for tumor treatment was not rare, and the morbidity of hypoandrogenism has increasing recently as a result of congenital disease, environmental pollution, anabatic working pressure and an increased aging population. However, until now there was no reliable and effective therapic method for this kind of diseases. With the development of tissue engineering technology, construction of tissueengineered androgen-secreting tissue seems promising therapic method in treating hypoandrogenism or testes loss. Using Leydig cell as seed cells, the tissue-engineered androgen-secreting tissue could secrete testosterone under the control of the pituitary-gonad axis, thus avoided the adverse effect commonly followed soly hormone therapy. Another advantage of androgen-secreting tissue was the plasticity of its shape, which could be molded like a testes shape that could fulfill the patient's strongly psychological and social need.The limited source of seed cells had greatly hampered the further study and clinical perspective of tissue-engineered androgen-secreting tissue study. Bone Mesenchymal Marrow Cells (BMCs) contain several pluripotent progenitor cells, which differentiate into multiple lineages. It can be isolated from bone marrow aspirated by preferential adherence to tissue culture plastic or by differential centrifugation through a percoll or ficoll density gradient to obtain a nucleated cell fraction. Pittenger et al. showed that MSCs represent only 0.001–0.01% of the total number of nucleated cells in the bone marrow; however, this population can be expanded ex vivo and enriched by standard culture conditions. So BMSCs may provide new source for seed cells in androgen secretiong tissue construction. 2006 Takashi Yazawa, etl shows that by adenovirus -mediated forced expression of SF-1, it could transform mouse primary long-term cultured BMCs into steroidogenic cells. 2007 year, YanHe Lue, Transplanted Bone Marrow Stem Cells into the Testis, and find adult bone marrow cells, in a favorable testicular environment, differentiate into somatic and germ cell lineages and author indicate that the resident neighboring cells in the recipient testis may control site-appropriate stem cell differentiation. So,it provide theoretical basis for co-culture of leydig cell and BMSCs in vitro。These study results above-mentioned shows that the feasibility of the availability of leydig cell indued by BMSCs, however, it maybe a long way to go when come to clininc applicaton. The technical of transfected BMSCs by adenovirus may obtain a new source of a secretory cell of androgen hormone, but the potential problem of biological safety followed adenovirus transferction is worldwide obstacle, and may not be solved in near future. Transplation of BMSCs into testis is way of experimental study; the high qualification of recipient was needed. For low proliferation of leydig cell in patients with hypoandrogenism, the opportunity of the practical application is slim. The initial factors and mechanism for the differentiation of stem or progenitor cells to leydig are not clear; however, by the way of imitation of microenvironment of leydig cell, it is feasible to induce BMSCs to differentiate into leydig cell theropially.Not only a promising way to gain the sufficient seed cells for tissue reconstruction for thus kinds of dieases, but also a new way have been provided to investigate the detail mechanisms under the differentiation of stem or progenitor leydig cell to mature leydig cell in the process of testis development by this study.ObjectiveTo provide a new source for seed cell for tissue-engineered androgen secretiong tissue, we investigate the possibility of indued the BMSCsto differentiate into leydig cell by co-culture system with transwell culture equipment. We contrived a simple and quick technique for LC isolation via differential adhesion. This method had increased LC number recovered from the testes, reduced cell damage during isolation. Bone marrow cells obtained from a nucleated cell fraction which by gradient solution centrifugation through a Ficoll density gradient. The mononuclear cell fraction obtained at the interface is explanted ex vivo by plating. And in 10%FBS DMEM medium, it proliferates quickly, and passage 3 cells were obtained to co-culture with leydig cells. After 2 and 4 weeks co-culture, BMSCs were accept immunohistochemistry and RT-PCR analysis, several specific marker of leydig cell were contained.Methods1.Identification,isolation and culture of Bone mesenchymal cells: BMSCs were harvested from femurs of adult wistar rats aged 3weeks by flushing the shaft with Dulbecco's Modified Eagle's Medium (DMEM) using a syringe with a 26-gauge needle. The aspirate was washed with phosphate buffered saline (PBS) with 2% fetal bovine serum, and then separated by Ficoll density gradient centrifugation to obtain mononuclear cells. After 4 day, nonadherent cells were removed, and adherent cells were thoroughly washed twice with PBS. Medium was replaced every 3–4 days. After 6 days culture, the colony-forming unit fibroblastic,CFU-Fs were formed. 2 weeks after cultured by the sepecial indued culture medium, BMSCs differentiate into adipocytes, and osteogenic cells.2.Isolation of leydig cell by differential adhesion: 3w old rats obtained by differential adhesion as discribed above, 3β-HSD and LHR expression in cultured cells were tested by immunohistochemistry 1 day culture. Testosterone productivity in isolated Leydig cells with or without HCG stimulation were also tested. Testes of 3w old Wister rats were harvested, after collagenase digestion, the seperated cells were filtrated by 33μm stainlesss-teel mesh, centrifuged and then cultured for 2 hours. After 2 hours'selective adhesion, the superanant was discarded, the adherent cells were than washed with PBS, and cultured under standard condition.3. In vitro induced BMSCs into leydig like cell by co-culture of leydig cell and BMSCs: leydig cell and BMSCs from 3w old rats were obtained by differential adhesion and Ficoll density gradient centrifugation, the cells were co-cultured in DMEM/F12 media with 3% FBS, Medium was replaced every 1–2 days .2 weeks after co-culture, 3β-HSD and LHR expression of BMSCS were tested by immunohistochemistry and the mRNA transcription of several key steroid genetic synthetases were examined by RT-PCR. The results show that BMSCs express the LHR, and the mRNA of stAR,3β-HSD and LHR, were transcripted after 2 weeks co-cultivation.ResultsPartⅠIdentification,isolation and culture of Bone mesenchymal cells: BMSCs from 3w old rats were obtained by Ficoll density gradient centrifugation and adhesion. After 6 days culture, the colony-forming unit fibroblastic (CFU-Fs) were formed, 2 weeks after cultured by the special inducing culture medium, BMSCs differentiate into adipocytes, and osteogenic cells. The ability of the forming of CFU-Fs and differentiation of adipocytes, and osteogenic cells, shows that the cells gained from this method can self-renew and be capacable of differentiate into at least one high differentiated cell.PartⅡIsolation of leydig cell by differential adhesion: Leydig cell of 3w old rats can be obtained by differential adhesion together with administration contain: collagenase digestion, filtration with 33μm mesh, centrifugalization and After 2 hours'selective adhesion. After culture 1 day, the cells were test by 3β-HSD and LHR immunohistochemistry. It shows that most of all cells cultured were positive for 3β-HSD and LHR. So it is confirm that it is effective way to gain leydig cells with high purity, and can be used as cell in co-cultivation system.PartⅢIn vitro induced BMSCs into leydig like cell by co-culture of leydig cell and BMSCs: Both Leydig cells and proliferated BMSCs showed good compatibility with in transwell co-cultured system with medium of DMEM/F12 with 3%FBS.2 weeks later, BMSCs were show positive for LHR and 3β-HSD, and is mRNA also tested by RT-PCR。So the co-culture system is a new way to induce BMSCs to differentiate into leydig-like cell.ConclusionThis study primitively proved that BMSCs can be induced to differentiate into leydig-like cells, for 3β-HSD and LHR both two specific marker for leydig cell.were ditected in BMSCs after co-cultured for 2 and 4 weeks; secondly, we testify Leydig cell lineage, could be successfully obtained by differential adhesion. In the further studys, We will add several growth factors or neutrition materials which were needed during leydig cell differentiation, and investigate the mechanism of differentiation of leydig stem cell to adult leydig cell。We will find safe and effective way to obtain the proper seed cells for constructed tissue-engineered androgen secretiong tissue or hormone substitution in deficiency of androgen secretiong tissue study, and may provide a promising method for the diseases of shortage of testerone.