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Bone marrow mesenchymal stem cells (BMSCs) have multilineage differentiation potential and highly proliferative potential in vitro. Cell therapy with BMSCs has great promise in regenerating the damaged tissues, restoring their function and treating ischemic related failures, but the chief limitation may be the poor viability of BMSCs transplanted into the pre-existing ischemic environment, where massive death of transplanted cells has been detected. The serum deprivation (SD) is a component of ischemia. Lidocaine can be used for relief of pain and has been one of the most widely used local anesthetics. Intravenously, it has been used successfully for emergency treatment of ventricular arrhythmias. It reportedly also showed anti- proliferative, anti-inflammatory and protective effects when administered before or after the ischemic insult. Which effects has it on BMSCs during the BMSCs transplanted in the patient with lidocaine treatment? The experimental objective is to investigate the effects of lidocaine on the proliferation of BMSCs cultured in high glucose plus serum deprivation DMEM.BMSCs were separated from adult male SD rat bone marrow. The BMSCs were cultured in the high glucose (4.5g/L) DMEM containing 10% fetal bovine serum (FBS). All experiments were performed using BMSCs from the 3rd passage adherent cell fraction. The BMSCs were divided into 10 groups according to the concentrations of lidocaine and FBS: 0μg/ml, 2μg/ml, 20μg/ml, 200μg/ml, 2000μg/ml lidocaine, containing 10% FBS; 0μg/ml, 2μg/ml, 20μg/ml, 200μg/ml, 2000μg/ml lidocaine, containing 0% FBS. Cell proliferation of the BMSCs was assessed by MTT assay at the time points of 24h, 48h and 72h respectively.As the MTT colorimetric assay revealed, lidocaine with 2μg/ml, 20μg/ml, 200μg/ml, 2000μg/ml all inhibited the proliferation of the BMSCs at under the condition of 10% FBS and under the condition of 0% FBS. The cells cultured in 0μg/ml lidocaine, 10% FBS displayed a significant proliferation compared to those cultured in 0μg/ml lidocaine,0% FBS (serum deprivation) (P<0.01, n=5) from 24h to 72h. The number of vital cells cultured in 2μg/ml, 20μg/ml, 200μg/ml lidocaine,10% FBS was more significantly compared to that cultured in 2μg/ml, 20μg/ml, 200μg/ml lidocaine, 0% FBS (P<0.01, n=5) at 24 h, 48h, 72 h respectively. But in 2000μg/ml lidocaine, the number of vital cells was similar and nearly zero both the BMSCs cultured in 10% FBS and serum deprivation groups. The number of vital BMSCs of all groups showed having a proliferative potential from 24h to 72h.BMSCs cultured under serum deprivation with 0μg/ml, 2μg/ml, 20μg/ml, 200μg/ml lidocaine gave rise to cell cluster formation, but all groups of BMSCs cultured in the presence of serum did not form the cell clusters. The phenomenon that the number and the size of the cell clusters were more could be observed in the BMSCs cultured under serum deprivation with 20μg/ml, 200μg/ml lidocaine.The proliferation capacity of the BMSCs cultured in the high glucose DMEM without serum was significantly decreased compared to those cultured in 10% FBS. Lidocaine inhibited the proliferation capacity both of the BMSCs cultured in the high glucose DMEM without serum and with 10% FBS. The results suggest that lidocaine does not exert protective effects on BMSCs in serum deprivation under the high glucose DMEM. Cytotoxicity to lidocaine was maximum at 2000μg/ml. BMSCs cultured under serum deprivation can induce and lidocaine can promote the formation of cell clusters. It is a fascinating point to investigate the relationship between the protective effects and toxicity of lidocaine on BMSCs in high and low glucose microenvironment. It is interested to explore the function and cell characteristics of the cell clusters in a further step of the research work.This research was supported by the grant from Jiangxi Provincial Department of Education, P. R. China (No. GJJ09103).No relevant conflicts of interest to declare.

Abstract 4582

One of the most devastating adverse effects of cancer treatments including bone marrow transplantation is damage to the reproductive system, which in young girls and women less than 40 years old is frequently associated with premature menopause and infertility, although adult bone marrow stem cells (BMSCs) are able to generate cells of all three germ layers under appropriate experimental conditions. These outcomes seem to be a result of cytotoxic effects on oocytes housed within the ovaries. Recent studies showed that female BMSCs (FBMSC) could differentiate into granulosa cells (GCs) and oocyte and form primordial follicles in ovary after FBMSCs transplantation and might have repaired chemotherapy-induced damage to the niche or the ovarian stroma. The formation of new primordial follicles in adult mammal ovaries is a controversial issue because some new studies have concluded that all offspring were derived from the recipient germ line after FBMSCs transplantation. To explore the effects of FBMSCs on female reproductive function, the experiment is to investigate whether adult female BMSCs are able to differentiate into Follicle-stimulating hormone receptor (FSHR, a GCs specific marker in the ovary cells) positive GCs 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 vitroinduced by all-trans retinoic acid (RA).

One of the most devastating adverse effects of cancer treatments including bone marrow transplantation is damage to the reproductive system, which in young girls and women less than 40 years old is frequently associated with premature menopause and infertility, although adult bone marrow stem cells (BMSCs) are able to generate cells of all three germ layers under appropriate experimental conditions. These outcomes seem to be a result of cytotoxic effects on oocytes housed within the ovaries. Recent studies showed that female BMSCs (FBMSC) could differentiate into granulosa cells (GCs) and oocyte and form primordial follicles in ovary after FBMSCs transplantation and might have repaired chemotherapy-induced damage to the niche or the ovarian stroma. The formation of new primordial follicles in adult mammal ovaries is a controversial issue because some new studies have concluded that all offspring were derived from the recipient germ line after FBMSCs transplantation. To explore the effects of FBMSCs on female reproductive function, the experiment is to investigate whether adult female BMSCs are able to differentiate into Follicle-stimulating hormone receptor (FSHR, a GCs specific marker in the ovary cells) positive GCs 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 induced by all-trans retinoic acid (RA). Methods: FBMSCs were separated from adult female SD rat bone marrow. FBMSCs were cultured in DMEM supplemented with 10% fetal bovine serum. All experiments were performed using FBMSCs from the 2nd passage adherent cell fraction. FBMSCs were cultured above medium with 10−6 M RA or without RA(control group). 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 RA. Results: Some of the FBMSCs were large and round and had a oocyte-like cell appearance 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 FSHR+ cells gradually became larger. The FBMSCs also expressed FSHR mRNA by RT-PCR and FSHR protein by Western Blotting 7days after induction differentiation. In control group, the FBMSC still kept undifferentiated relatively elongated or spindle-shaped cells that they were FSHR negative. The FBMSCs expressed Stra8 mRNA by RT-PCR 7days after induction differentiation, the RA-regulated gene specifically expressed in premeiotic germ cells and a marker of the stem cells transdifferentiated into germ cells. In control group, the FBMSCs were stra8 negative. Recent reports indicated that Stra8 was expressed in embryonic germ cells of ovary in mice and was not expressed after birth in ovary, it implied that there was not oocyte production in postnatal ovary. Our results is not support the view and suggest that adult FBMSCs could be able to generate oocytes. Conclusions: These results suggest that RA could induce FBMSC to differentiate into GC-like cells and oogonia-like cells in vitro. Stra8 is not only expressed in embryonic germ cells of ovary, but also can be expressed in adult FBMSC induced by RA. Adult FBMSCs contain pluripotent stem cells that could differentiate into germ cells and gonadal somatic cells. It is a point to investigate the function of GC-like cells derived from FBMSCs and the differentiation capable of oogonia-like cells derived from FBMSCs in vitro in a further step of the research work.