Microarray Gene Expression for Undifferentiated Mesenchymal Stem Cells, Adipogenically Differentiated and Dedifferentiation cells

Bone marrow mesenchymal stem cells (MSC) were adipogenically differentiated followed by dedifferentiation. We are interested to know the new fat markers, adipogenic signaling pathways and dedifferentiation signaling pathways.Furthermore we are also intrested to know that how differentiated cells convert into dedifferentiated progenitor cells. To address these questions, MSC were adipogenically differentiated, followed by dedifferentiation. Finally these dedifferentiated cells were used for adipogenesis, osteogenesis and chondrogenesis. Histology, FACS, qPCR and GeneChip analyses of undifferentiated, adipogenically differentiated and dedifferentiated cells were performed. Regarding the conversion of adipogenically differentiated cells into dedifferentiated cells, gene profiling and bioinformatics demonstrated that upregulation (DHCR24, G0S2, MAP2K6, SESN3) and downregulation (DST, KAT2, MLL5, RB1, SMAD3, ZAK) of distinct genes play a curcial role in cell cycle to drive the adipogenically differentiated cells towards an arrested state to narrow down the lineage potency. However, the upregulation (CCND1, CHEK, HGF, HMGA2, SMAD3) and downregulation (CCPG1, RASSF4, RGS2) of these cell cycle genes motivates dedifferentiation of adipogenically differentiated cells to reverse the arrested state. We also found new fat markers along with signaling pathways for adipogenically differentiated and dedifferentiated cells, and also observed the influencing role of proliferation associated genes in cell cycle arrest and progression. We have differentiated bone marrow derived mesenchymal stem cells(MSC) into adipogenically differentiated cells followed by dedifferentiation. We are intrested to know new fat markers, signaling pathways for adipogenically differentiated and dedifferentiated cells, along to observe the genes associated with cell cycle arrest and progression. Results are also provide molecular insight into the process of adipogenesis and dedifferentiation. To study the adipogenic differentiation and dedifferentiation process along with "how adipogenically differentiated cells convert into dedifferentiated cells" on the molecular level, gene expression profiling with genome-wide Affymetrix HG-U133 Plus 2.0 olgonucleotide microarrays (Affymetrix, Santa Clara, CA, USA) was applied. In total, 12 GeneChips were performed for n=3 donors and 4 times (3x4=12): 3x P4 MSC (undifferentiated state), 3x adipogenically differentiated cells at day 15 (differentiated state), 3x dedifferentiated cells at day 7 (dedifferentiated state) and 3x dedifferentiated cells at day 35 (dedifferentiated state)骨髓间充质干细胞（bone marrow mesenchymal stem cells, MSC）先经成脂分化后进行去分化。本研究旨在探索新型脂肪标志物、成脂信号通路及去分化信号通路，同时明确分化细胞向去分化祖细胞转化的机制。为解答上述科学问题，我们将MSC先诱导为成脂分化细胞，再进行去分化；最终利用这些去分化细胞开展成脂、成骨及成软骨分化实验。我们对未分化状态、成脂分化状态及去分化状态的细胞分别进行了组织学检测、流式细胞术（FACS）、实时定量PCR（qPCR）及基因芯片（GeneChip）分析。关于成脂分化细胞向去分化细胞的转化过程，基因表达谱分析与生物信息学研究显示，特定基因的上调（DHCR24、G0S2、MAP2K6、SESN3）与下调（DST、KAT2、MLL5、RB1、SMAD3、ZAK）在细胞周期中发挥关键作用，可驱使成脂分化细胞进入周期停滞状态，从而限制其谱系潜能；而另一组细胞周期基因的上调（CCND1、CHEK、HGF、HMGA2、SMAD3）与下调（CCPG1、RASSF4、RGS2）则可促进成脂分化细胞发生去分化，逆转其周期停滞状态。本研究还鉴定了成脂分化与去分化细胞的新型脂肪标志物及相关信号通路，并观察到增殖相关基因在细胞周期停滞与进程中的调控作用。我们通过诱导骨髓来源MSC发生成脂分化并进一步去分化，旨在探索新型脂肪标志物、成脂与去分化相关信号通路，同时观察细胞周期停滞与进程相关基因的表达变化。本研究结果可为成脂分化与去分化过程提供分子层面的机制见解。为在分子水平研究成脂分化与去分化过程，以及成脂分化细胞向去分化细胞的转化机制，我们采用全基因组Affymetrix HG-U133 Plus 2.0寡核苷酸微阵列（Affymetrix，美国加利福尼亚州圣克拉拉市）开展基因表达谱分析。本次实验共计使用12张基因芯片，对应3名供体的4组样本（3×4=12）：3份第4代未分化MSC（未分化状态）、3份诱导15天的成脂分化细胞（分化状态）、3份诱导7天的去分化细胞（去分化状态）以及3份诱导35天的去分化细胞（去分化状态）。