Effects And Possible Mechanism Of Regulatory T Cells On Osteogenic And Adipogenic Differentiation Of Bone Marrow Derived Mesenchymal Stem Cells In Mice

Backgrounds Aplastic Anemia(AA) is a heterogeneous disease characterized by failure of bone marrow hematopoiesis resulting in varying degrees of pancytopenia with a markedly hypocellular bone marrow. Despite the exact causes of AA are unknown, various lines of laboratory evidence point towards an immune-mediated inhibition of hematopoiesis, just like abnormal activation of T lymphocytes and inhibition of Regulate T cells(Treg cells). Bone marrow mesenchymal stem cells (BMMSCs) are nonhematopoietic progenitor cells of the bone marrow stromal cells and the progenitor of most cell components in the hematopoietic microenvironment. But it is unclear that the effect of Treg cells, BMMSCs and its differentiated cells in hematopoietic microenvironment in AA.Hypocellular with prominent adipocytes is the most characteristic pathology of AA. There is evidence that MSCs are responsible for creating adipocytes and osteoblasts within the bone marrow microenvironment.Osteogenesis and adipogenesis of MSCs is the natural balance. Investigating the mechanisms that fine-tune this balance is of medical importance. Identification of potential target gene which can be used to study the relationship between them could be really helpful for this purpose. The contributing factors of adipogenic differentiation are always the inhibitor of osteogenic differentiation, and vice versa. Peroxisome proliferation-activated receptor-gamma(PPAR-Y) is the major stimulating factor of adipogenic differentiation, which can inhibit osteogenic differentiation of MSCs. Osteogenesis or adipogenesis differentiation of MSCs is up to the interaction of many signal pathways and cell factors,including Bone morphogenetic proteins (BMP)> WntN hedgehogs, delta/jagged protein、FGF、IGF、PPAR-γ and Runx2. Of all, BMPs signal pathway is the very important.BMPs are the most potential inductive factors on osteogenesis differentiation of MSCs, they belong to the transformation growth factorβ (TGF-β) super-family, Of which, BMP-2is the hot point of research work. Recently, more and more data shows that BMPs signal transduction mostly depend on MAPKs、PI3K pathway, which bypass the transcription factors Smad activation. BMPs-MARKs pathway play the major role in signal transduction.Leptin and Adponectin are two important adipokines in the modulation of the host response to infection. Leptin modulates the proliferation and activation of peripheral T lymphocytes in mice and in humans and can enhance the production of cytokines. Adponectin is a secretary protein synthesized exclusively by adipocytes.Our team’ previous research has confirmed the activated T lymphocytes can alter MSCs’capacity to differentiate into adipocyte. Now the question is whether the Treg cells can affect the differentiate capacity of MSCs, osteogenesis or adipogenesis, and possible mechanism. The study is to reveal whether these deficiencies exist, thus to offer a new standpoint in the theoretical basis for the pathogenesy of AA.Objective (1) To establish a method for isolation, cultivation and expansion of mice BMMSCs, and study their biological features.(2) To isolate, purify and identify Treg cells in vitro.(3)To investigate the effects and possible mechanism of Treg cells on osteogenic and adipogenic differentiation of bone marrow derived mesenchymal stem cells in mice.(4) To establish the model of immune-mediated bone marrow failure in vivo. Infusion the MSCs and/or Treg cells, to investigate hematopoietic effect on the model of immune-mediated bone marrow failure.Methods (1) BMMSCs of BALB/c mice were separated and amplified in vitro by wall sticking method. The cells was digested by0.25%trypsin and passaged when cells fused80-90%. The phenotypes of BMMSCs were examined with flow cytometry. Their differentiation potential were examined by specific induction conditions.(2) Magnetic activated cell sorting to isolate and purify Treg cells, The phenotypes were examined with flow cytometry.(3) Co-culture Treg cells and BMMSCs for72hours, to detect the genes and protein of BMMSCs osteogenesis or adipogenesis by Real-time PCR and Western Blot; Co-culture Treg cells and BMMSCs for7days, to detect the activity of AKP by PNPP method and AKP staining, detect the activity of Collagen type Ⅰ by ELISA method and immunohistochemical staining; Co-culture Treg cells and BMMSCs for14days, and detect the mineralized nodule by Von Kossa staining, Lipid droplet formation by Oil red O staining (4) BMP-2gene silence by SiRNA technology, p38MAPK inhibition SB203580, ERK1/2inhibition PD98059and PKA inhibition H-89application, detecting the changes of BMMSCs osteogenesis or adipogenesis factors by Real-time PCR, Western Blot, Elisa and immunohistochemistry technology.(5) Male B6(C57BL/6) mice and female BALB/c mice were used at the ages of6-16 weeks, then hybrid the cByB6F1mice. The lymphnode and spleen cells of normal BALB/c mice were injected to hybrid CByB6F1recipients with a sublethal dose of y irradiation (4.5Gy) to establish the model of immune-mediated bone marrow failure. Compare the hematopoietic effect on the five groups:Treg cells infusion, BMMSCs infusion,Treg cells and BMMSCs infusion, bone marrow failure models and blank. The number of white blood cells, red blood cells, platelets and haematoglobin bere recorded. HE stain of bone was used to understand bone marrow adipogenesis.Results (1) Both primary and passage BMMSCs appearance presented cambiform or fibroblasts-like, vortex-like and triangular-shaped. The third or the fourth generation in the cultured cells was showed by FACS:expression of CD44, CD73, CD90and CD105was strong positive, and expression of CD14, CD34, CD45and HLA-DR was negative. Under suitable conditions, BMMSCs have the similar ability of differentiation into adipocyte, osteoblast and chondrocyte.(2) Treg cells treated by Magnetic activated cell sorting system expressed CD4, CD25and Foxp3.(3) When BMMSCs co-cultured with Treg cells in the ratio of1:5for72hours, the BMP-2, osteocalcin(OCN),osterix (OSX) gene of BMMSCs were highly expressed while the PPAR-Y, Leptin and adiponectin gene were slightly decreased, compared to BMMSCs co-cultured with normal MNCs by Real-time PCR analysis. Western Blot analysis showed the same tendency. The change of BMP-2was the most obvious. Expression of Alkaline phosphatase(AKP) and type I collagen were increased. In osteogenic environment, formation of calcified nodule was enhanced in Treg group, while in adipogenic environment, the formation of lipid drops was restrained.(4) BMP-2gene silence by SiRNA technology, OCN,OSX,osteogenic gene of BMMSCs, were reduced, while the PPAR-γ and adiponectin gene were highly expressed. Western Blot analysis showed the same tendency. In Treg cells group, levels of phosphorylation of p38MARK, ERK1/2were increased, the change of JNK was not obvious. With p38MAPK inhibition SB203580and/or ERK1/2inhibition PD98059application, the expression of type I collagen and AKP were obviously decreased. In osteogenic environment, the expression of OCN, osteopontin (OPN) and formation of calcific nodules were decreased. The levels of cAMP and protein kinase A (PKA) were higher in Treg group, with application of PKA inhibition H-89, the expression of type I collagen was reduced. ELISA and immunohistochemical method showed the similar results.(5) Infusion of lymphnode and spleen cells of a parent animal into sublethally irradiated recipients produced severe BM failure. Marrow cavities were essentially empty in these affected mice in comparison to untreated control mice. Residual BM contained a very large proportion of adipocytes in the immune-related bone marrow failure mice while few adipocytes existed in the controls bone marrow. Compare the improvement of adipogenesis effect on the five groups:infusion of Treg cells and BMMSCs and BMMSCs infusion can improve the severe BM failure.Conclusions(1) BMMSCs could be isolated and purified by adhesiveness delection. Derived cells could be cultured stably and expanded quickly. Expanded cells we harvested had the characteristic markers of BMMSCs, and had the potential to differentiate to adipocyte, osteoblast and chondrocyte.(2) Magnetic activated cell sorting system could get enough cell.(3) The Treg cells could increase the expression of BMP-2、OCN、OSX genes and proteins on BMMSCs, decrease the expression of adiponectin,PPAR-γ,Leptin.(4) The Treg cells could promote the osteogenic differentiation of BMMSCs, inhibit adipogenic differentiation of BMMSCs by BMP-MARKs pathway and cAMP-PKA pathway.(5) Infusion of lymph node and spleen cells of a parent animal into sub lethally irradiated recipients produced immune-related BM failure. In immune-related BM failure mice, residual BM contained a very large proportion of adipocytes. The improvement of adipogenesis effect, co-infusion of Treg cells and BMMSCs were obviously remarkable.This study had suggested that the Treg cells could facilitate BMMSCs osteogenic differentiation. Treg cells and BMMSCs co-infusion could improve the adipogenesis of immune-related BM failure model. It was maybe the way to heal the AA.