HO-1Modification Enhances Activity And Immunomodulation Of Rat Bone Marrow Mesenchymal Stem Cells

Objective:(1) To illustrate the effects of hemeoxygenase-1(HO-1) on the activity of bone marrow mesenchymal stem cells (BM MSCs).(2) To explore the influence of HO-1overexpressed BM MSCs (HO-1/BM MSCs) on T lymphocytes and the associated mechanisms in vitro.Methods:(1) Specefic pathogen free,4-5-week age, male Wistar rats were used in this study. The rats were sacrificed and the long bones were isolated. The medullary cavity was rinsed by using DMEM/F12medium containing10%fetal bovine serum (FBS) and the flushing fluid was collected. Density gradient centrifugation and adherent culture was combined to isolate BM MSCs. The cellular morphology was observed under microscope. The third passage of BM MSCs were incubated with fluorescence-labelled antibodies and the surface markers were detected by flow cytometry analyses. Also, the differentiation ability of BM MSCs to lipocytes and osteoblasts was detected.(2) BM MSCs were infected by recombinant adenovirus bearing HO-1to form HO-1/BM MSCs, and the infection efficiency was observed under fluorescence microscope. Total RNA and protein were extracted from HO-1/BM MSCs, and the HO-1expression level was detected by real time PCR and Western blot assays, respectively.(3) HO-1/BM MSCs and the control cells were cultured in E-plates. After adherence, the culture medium was replaced with low-serum medium (2%FBS) and BM MSCs were treated with CoCl2for12h to form ischemic and anoxic circumstance. Cell proliferation was measured by growth curve analysis on a real time cell analyser. Annexin V/propidium iodide (PI) staining and flow cytometry were used to detect apoptosis of BM MSCs.(4) Mixed lymphocyte culture was used to evaluate the effect of BM MSCs on T lymphocytes. Briefly, the HO-1/BM MSCs and control cells were co-cultured with rat spleen-derived lymphocytes with presence of Concanavalin (ConA). After cultured for0h,24h and48h, BM MSCs viability was detected by MTT assay, and cell cycle distribution was measured by PI staining and flow cytometry analyses. Furthermore, enzyme linked immunosorbent assay (ELISA) was performed to measure the cytokine levels. Fluorescence-labelled antibody incubation and flow cytometry were used to evaluate the T cell subsets and surface markers such as CD25and CD71.Results:(1) Rat BM MSCs were successfully isolated and cultured in vitro. Cells exhibited anchorage dependent growth and were spindle or scroll, which were typical morphological character of BM MSCs. According to the cell surface markers analyses, CD29, CD90and RT1A were expressed at BM MSCs surface, with positive rates of99.49%,90.12%and98.99%, respectively. CD34, CD45and RT1B were not expressed and the negative rates were>95%. Under special culture medium, BM MSCs were able to differentiate into lipocytes (red lipid droplet within cytoplasm by Oil Red O staining) and osteoblasts (black calcium salt deposit by von Kossa staining).(2) BM MSCs were infected by recombinant adenovirus bearing HO-1. After48h, more than80%of cells exhibited green fluorescence. Real time PCR showed that HO-1expression in the infected BM MSCs was up-regulated to about5-fold of that in the control cells. HO-1/BM MSCs also showed an elevated HO-1protein level detected by Western blot assay.(3) Under normal culture medium, HO-1exhibited no significant influence on BM MSCs proliferation. However, increased proliferation was detected in HO-1overexpressed BM MSCs compared with the control group when cells were cultured under ischemic and anoxic circumstance. Similarly, HO-1did not affect BM MSCs apoptosis in normal culture medium but reduced apoptosis in ischemic and anoxic condition.(4) When T lymphocytes were co-cultured with HO-1/BM MSCs, the proliferation and cell cycle progression of T lymphocytes were restrained. HO-1also inhibited the secretion of IL-2, TNF-α and IFN-γ, and promoted the secretion of IL-10by T lymphocytes. The T lymphocyte activation markers CD25and CD71were also downregulated by the HO-l/BM MSCs.Conclusions:(1) As a kind of stem cells with multi-differential potentialities, BM MSCs could be isolated by density gradient centrifugation and adherent culture methods. This strategy could be served as a normal method to culture rat BM MSCs.(2) When infected by the recombinant adenovirus bearing HO-1, BM MSCs exhibited increased HO-1expression level.(3) HO-1promoted proliferation and suppressed apoptosis of BM MSCs in ischemic and anoxic condition.(4) HO-1modified BM MSCs showed improved immunomodulation effects on T lymphocytes.