Induce Female Bone Marrow Stem Cells To Differentiate Into Stra8~+ Cells And FSHR~+ Cells

Objective:The aim is to establish the culture systems of sertoli cells(SCs) and female bone marrow stem cells(FBMSCs) in vitro,investigate whether adult FBMSCs are able to differentiate into Follicle-stimulating hormone receptor (FSHR, a GCs (granulose cells)specific marker in the ovary cells) positive GCs like cells and Stra8 (stimulated by retinoic acid gene 8, a specific expression gene in mammalian germ cell's transition from mitosis to meiosis) expression oogonia in vitro by two different treatments: all-trans retinoic acid (ATRA) and indirect co-cultured with Sertoli cells (SCs).Methods:SCs were isolated from 12-16d SD rat testes and FBMSCs were separated from adult female SD rat bone marrow. FBMSCs and SCs were cultured respectively in DMEM supplemented with 10% fetal bovine serum . All experiments were performed using FBMSCs from the 2nd passage adherent cell fraction and SCs from the primary cultivation. MTT colorimetric assay was used to observe the survival and proliferation of FBMSCs(control group) and of FBMSCs in indirect co-culture with SCs(co-culture group) and FBMSCs cultured with 10-6M ATRA(ATRA group) in vitro respectively. RT-PCR was performed to detect the expression of Stra8 mRNA and FSHR mRNA, Western blot and immunocytochemistry staining was utilized to examine the expression of FSHR in FBMSCs cultured with ATRA, indirect co-cultured with SCs and with SCs and ATRA, respectively.Results:1. The FBMSCs in co-culture group proliferated faster than that in the control group from the 1st day to the 5th day(P<0.01). But in 6th and in 7th days, the FBMSCs amount of the co-culture group was decreased sharply than the control group (P<0.01). The FBMSCs cultured with ATRA proliferated faster than that in the control group from the 3rd day to the 4th day (P<0.05). But in 6th and in 7th days, their amount was decreased than the control group (P<0.01). 2. In all of the three treatments groups, some of the FBMSCs were large and round 7 days after induction culture. Some FBMSCs were FSHR positive by immunocytochemistry 3 days after induction differentiation, the FSHR+ cells were small or middle and round and were found in small clusters. Following longer in culture(from day 5 to day 7), the most of FSH+ cells gradually became larger. In control group, the BMSC still kept undifferentiated relatively elongated or spindle-shaped cells that they were FSHR negative.3. The FBMSCs of the three treatments groups also expressed Stra8 mRNA and FSHR mRNA by RT-PCR 7days after induction differentiation. The FBMSCs of the three treatments groups also expressed FSHR protein by Western Blotting. The FBMSCs of the control group did not express Stra8 and FSHR.Conclusions:1. The FBMSCs in co-culture group and the FBMSCs cultured with ATRA proliferated faster than that in the control group in the early period of the induction differentiation, but in later period SCs and ATRA suppressed their proliferation or promoted the apoptosis .2. ATRA and the soluble factors secreted by SCs could all induce FBMSCs to express Stra8 mRNA, the RA-regulated gene specifically expressed in premeiotic germ cells and a marker of the stem cells transdifferentiated into germ cells. It suggests that Stra8 is not only expressed in embryonic germ cells of ovary, but also can be expressed in adult FBMSCs by induction medium. Adult FBMSCs contain pluripotent stem cells that could differentiate into germ cells .3. ATRA and the soluble factors secreted by SCs could induce FBMSCs to differentiate into FSHR positive cells in vitro. It suggests that FSHR is not only expressed in granulose cells in the ovary, but also can be expressed in adult FBMSCs by induction medium. Adult FBMSCs contain pluripotent stem cells that could differentiate into gonadal somatic cells.4. These results suggest that ATRA and the soluble factors secreted by SCs could similarly induce FBMSC to differentiate in vitro. One of the soluble factors secreted by SCs might be ATRA.