Preclinical Studies Of Therapy Severe Intrauterine Adhesions By Transplanting Menstrual Blood-derived Mesenchymal Stem Cells

Recent studies indicate that the development of intrauterine adhesions may be related to the reduction, deficient or dysfunction of endometrial stem cells (EnSCs), and therefore it is proposed that menstrual blood-derived mesenchymal stem cells (MenSCs) could be used for the treatment of intrauterine adhesions.A new study suggests that MenSCs is more suitable for being used as seed cells in cell therapy than mesenchymal stem cells (MSCs). In addition to the common merits of MSCs, MenSCs also have several other advantages, such as enormous proliferative capacity, differentiation to many types of tissues from three embryonic germ layers, high expression of matrix metalloproteinase and so on, due to its high expression of embryonic stem cell antigen Oct-4.This study focuses on the preclinical research of MenSCs transplantation in treatments of severe intrauterine adhesions, aimed to regain the reproductive capability of IUA patients. The main contents are divided into the following parts:1. Isolation, culture and identification of the EnSCs in menstrual blood to provide cells resource for stem cells transplantation to treat intrauterine adhesions.2.Induced differentiation of the MenSCs into endometrial cells in vitro, which provides a theoretical basis for the treatment of intrauterine adhesions using the MenSCs transplantation.3.While MenSCs are transplanted into the subrenal capsule or subcutaneous of NOD-SCID femal mice, differentiation from MenSCs to endometrium can be observed after estrogen therapy, which confirms that MenSCs can regenerate the endometrial tissues in vivo.4. Through a survey of comparing the difference of the EnSCs and MenSCs between severe IUA patients and healthy females which were diagnosed by hysteroscope, the reduction or loss of EnSCs and MenSCs in IUA patients is proved, which means that treating IUA using MenSCs should be necessary.Result:1) MenSCs cultured in low-glucose DMEM medium containing 10% fetal bovine serum proliferates in a clonal growth, exhibiting its stem-cell characterized growth. Flow cytometry was used to analyze the cultured MenSCs. The rates of OCT-4, CD45, STRO-1 and HLA-DR positive cells are 95.13%±0.81%,0.93%±0.42%,1.80%±0.92% and 1.00%±0.35% in respective, which means the vast majority of the cultured cells were OCT-4+ stem cells,and low immunogenicity, doubling population 24 times remain normal cell chromosome karyotype.2) The differentiation of the cultured MenSCs into endometrial cells could be induced under the joint action of conditioned medium and 17β-estradiol valerate. Immunocytochemical technique was employed to detect endometrium antigen CK and VIM, it turned out that the positive rate of CK and VIM after induction was relatively higher than before, furthermore the mRNA level and protein expression of CK and VIM was significantly increased (p<0.05). Therefore, it demonstrated that MenSCs could be induced to differentiate into endometrial cells in vitro.3) The cultured MenSCs were transplanted into subrenal capsule or arm pit of female NOD/SCID mice suffered bilateral oophorectomy or downregulation castration. CK, VIM, ER, PR and CD31 were detected after estrogen therapy. Results suggested that MenSCs were positive for CK, VIM, PR, while negative for CD31 and ER, demonstrating that MenSCs had the ability to reconstruct endometrial tissues. Negative expression of CD31 revealed blood supply to endometrial tissues during differentiation probably may come from hosts.4) The colony forming efficiency of cultured MenSCs from IUA patients was remarkably reduced compared with that from healthy females (0.74±0.11)×10-6 vs (6.8±.56)×10-6, p<0.001. The colony forming efficiency of cultured endometrial tissue cells in adhesion region was significantly reduced compared with that of healthy females 0 vs 1.21%±0.04%, p<0.001. The colony forming efficiency of cultured suspected normal tissue cells beside adhesion region was dramatically reduced compared with that of healthy females 0.14%±0.03% vs 1.21% ±0.04%. Immunohistochemical detection revealed that the percentage of OCT-4+ cells in IUA patients was significantly lower than that of healthy females (<0.1% vs 2%) and so was the percentage of CD 146 positive cells (<0.5% vs 1%). The percentage of CD31 positive cells in functional layer of normal females was 1% while 2% for the basal layer, compared with the percentage of <0.05% and<1% for IUA. The percentage of EpCAM in IUA patients and normal females turned out to be the same as <0.1%.Conclusion:This preclinical research shows that MenSCs can be cultured and be amplified to enough amounts sufficient for clinical therapy. In response to conditioned medium and moderate amounts of estrogen, MenSCs can differentiate into endometrial cells in vitro, and also can reconstruct the endometrial tissues in NOD/SCID mice. Compared with normal uterine cavity females, the number of MenSCs and EnSCs of intrauterine adhesions diagnosised by hysteroscope significantly decreased. In conclusion, using MenSCs for treatments of intrauterine adhesions is quite feasible and necessary.