| Myostatin (MSTN), also named GDF-8, is known as a regulator of skeletal muscle growth and development. It was reported that MSTN was involved in fat deposition and adipogenic differentiation modulation. However, the effects of MSTN on adipogenic differentiation were inconsistent and poorly understood. It might be due to the difference of cell models and research methods. Recently, increasing evidence supports the concept that adult stem cells are part of a natural system for tissue growth and repair. It is unlikely that one kind of stem cell can be ideal cell source for all practical applications. In this study, four experiments were conducted to investigate the effects of MSTN on adipogenic differentiation and nutritional regulation in mesenchymal stem cells (MSCs).In experiment 1,6-week-old C57BL/6J mice and fetus on gestation days 16-17 were used to compare the differences of cell morphology, immune phenotype, proliferation and multiple differentiation potential among adult mouse adipose (ADSCs), skeletal muscle (MDSCs) and fetal muscle derived mesenchymal stem cells (FMSCs) with flow cytometry, MTT, multiplelineage induction. The mesenchymal nature (CD29-, CD44-, CD73-and CD 105-positive expression) were confirmed and with respect to nonhematopoietic, nonendothelial origin, flow cytometric assays showed that more than 95% cells of passage 3 was CD45-, CD34-, and CD31-negative. CD44 expression in FMSCs at passage 1 (27.1%) was lower than ADSCs (61.7%) and MDSCs (78.8%) (P< 0.0001), but it increased to 99.5% at passage 3. Besides, stem cell antigen 1 (Sca-1) expression of MDSCs and FMSCs at passage 3 increased in comparison with cells at passage 1, and MDSC expressed higher Sca-1 than FMSCs and ADSCs. It was suggested that Sca-1 is a potential marker of myogenic capability. The mRNA levels of Myodl, myogenin, desmin, CKM and MyHC in MDSCs after 28-day myogenic induction were higher than FMSCs (P <0.01). ADSCs expressed those skeletal specific transcription factors at the lowest levels. MyHC and M-Cadherrin were not detected by real-time PCR and immunofluorescence technique respectively. ADSCs expressed higher levels of adipogenic genes than MDSCs after 14-day adipogenic differentiation induction, but no obvious difference was observed between them after 14-day adipogenic induction. It is unexpected that FMSCs exhibited lowest adipogenic potency. Besides, MDSCs and FMSCs expressed higher OCN than ADSCs (P< 0.01), and FMSCs expressed higher OPN than MDSCs (P<0.05) and ADSCs (P<0.01). Take together, the direct comparison of multilineage differentiation showed that MDSCs and FMSCs had higher myogenic and osteogenic differentiation capacity than ADSCs, whereas ADSCs and MDSCs exhibited higher adipogenic differentiation potential than FMSCs. It was suggested that the difference of cells from various tissues should not be ignored and further investigations should be helpful to understand their unique biology better.MSTN, instead of classical adipogenic inducer dexamethasone (DEX), was reported to induct adipogesis differentiation of MSCs. In experiment 2, pig bone marrow (pBMSCs), adipose (backfat) (pADSCs), and skeletal muscle (longissimus dorsi) derived mesenchymal stem cells (pMDSCs) were identified with classical cell biology techniques and the effects of MSTN and dexamethasone (DEX) on adipogenic differentiation in pig mesenchymal stem cells from various tissues were studied. The advisable application of MSTN and DEX in adipogenic differentiation induction was investingated. Porcine MSCs cells from different tissues were identified and it was showed that, the effects of MSTN and DEX on adipogenesis in the cells from various tissues were different. It was indicated that, MSTN promoted adipogenic genes expression in pBMSCs, including PPARy2,LPL and aP2. However, under adipogenic system without DEX, endocellular triglyceride content of pBMSCs was not increased by supplemental MSTN. Regardless of DEX supplementation, MSTN suppressed lipid accumulation and adipogenic genes expression in pADSCs and pMDSCs (P< 0.01) The significant interaction of cell source and treatment (P< 0.001) showed that the responses of MSCs to MSTN and DEX were different. In this study, it was suggested that both appropriate cell sources and methods are important to further investigations.Experiment 3 was conducted to study the regulation of arginine and CLB on multilineage differentiation of MSCs, and MSTN expression was determined. The results showed that, intra-cellular triglyceride concentration was increased by arginine (P< 0.01). Triglyceride level of cells supplemented with 50μg/mL arginine was the highest and triglyceride levels were numerically declined as the level of arginine increasing. In addition, supplemental arginine promoted both myogenic (myogenin, desmin and CKM) and adipogenic genes (PPARy2, LPL and aP2) expression, but suppressed the expression of MSTN under myogenic conditions (P< 0.01). The results suggested that, arginine supplementation might promote myogenic differentiation through surppressing endogenous MSTN expression. Besides, further investigation on the correlation between adipogenic differentiation modulation of arginine and MSTN expression is necessary.In experiment 4, in order to investingate the effects of MSTN as well as arginine modulation on adipogensis, pADSCs and pMDSCs were respectively used as cell models of subcutaneous fat or intramuscular fat in vitro. Cells were incubated in adipogenic differentiation system supplemented with arginine separately and combined with MSTN or antibody to MSTN antibody. The results showed that, the response of cells to 100 ng/mL MSTN or 4μg/mL MSTN antibody, cells was greatest. Exogenous myostatin addition decreased intracellular triglyceride level, and it was alleviated by arginine supplementation (P<0.01). Exogenous myostatin suppressed ADD1, C/EBPa, PPARy2 and LPL expression, and ADD1, aP2 expression was promoted by supplemental arginine in pADSCs (P<0.01). Besides, exogenous myostatin suppressed PPARy2 and aP2 expression and ADD1 expression was promoted by supplemental arginine in pMDSCs (P<0.01). Furthermore, under adipogenic induction conditions, the inhibition of myostatin on ADD1 expression was alleviated by supplemental arginine (P<0.01). Taken together, it was suggested that, myostatin surppressed adipogenic differentiation in porcine MSCs and arginine reversed it. Both supplemental myostatin and arginine regulated ADD1 expression, but no evidence showed that they modulated adipogenic differentiation in the same way.In summary, the differences of cell phenotype, proliferation and multipotency among MSCs from different sources were confirmed. MSCs derived from porcine bone marrow, backfat, and longissimus dorsi responsed differently to MSTN. It was suggested that MSTN supplementation promoted adipogenic differentiation in pBMSCs but suppressed it in pADSCs and pMDSCs. Arginine supplementation promoted both myogenic and adipogenic differentiation and endogenous MSTN expression was surppressed by supplemental arginine under myogenic conditions in pADSCs. Both myostatin and arginine supplementation regulated ADD1 expression in pADSCs and pMDSCs, but it seems that they modulated adipogenic differentiation in different ways. |
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