Molecular regulation underlying differentiation of dental pulp stem cells

 描述（由申请人提供）：利用干细胞修复患病或受损的组织/器官代表了再生医学补救的新方法。虽然已经研究了胚胎干细胞（ESC）和诱导多能干细胞（iPS），但是使用ESC和iPS存在主要缺点和问题;例如，在移植后可能发生肿瘤发生和畸胎瘤形成。目前尚不清楚， 与ESC和iPS有关的障碍可以被克服，以便将它们用于治疗;至少还有很长的路要走。相比之下，成体干细胞（AdSC）用于治疗 人类疾病。一个例子是移植骨髓干细胞来治愈或改善某些疾病，如白血病。这种基于细胞的疗法通常需要大量的干细胞.由于AdSC在体外培养时失去了它们的干细胞特性（干性），包括分化能力，因此原代分离的AdSC只能用于有限的传代，并且难以从原代分离中繁殖大量高潜能干细胞。因此，干细胞分离需要大量的组织，并且多次治疗需要连续分离AdSC。然而，组织，例如，可用于AdSC分离的牙组织通常是有限的。从拔牙和脱落牙齿的牙髓中分离的干细胞已被证明是再生医学干细胞的良好来源。然而，这些牙髓干细胞（DPSCs）在体外培养时也失去了其分化潜能，这阻碍了它们的应用。本研究的长期目标是阐明DPSCs中调控分化能力的基因和分子。初步研究表明，在培养的DPSCs中，当细胞丧失分化能力时，热休克蛋白B8（HspB 8）表达下调，在早期传代的DPSCs中敲低HspB 8表达可导致细胞丧失分化能力。进一步的研究表明microRNAs（miRNAs）下调了DPSC中HspB 8的表达。核心假设是，在晚期传代DPSC中某些miRNA的上调表达下调HspB 8和其他调控基因，进而导致DPSC在体外培养期间丧失分化能力。该项目的目标是：（1）鉴定其表达在晚期传代DPSC中显著增加的miRNA，称为晚期传代上调的miRNA（LPU-miRNA）;（2）鉴定下调HspB 8表达的LPU-miRNA;即，（3）研究LPU-miRNA和HspB 8-靶向miRNA对DPSC分化和调节其他潜在基因的作用。这些目标的实现对于理解miRNAs和HspB 8在调节长期培养的DPSC中观察到的分化丧失中的作用具有重要意义，并且也将为其他AdSC提供启示。这将有助于开发用于在培养DPSC和AdSC中维持干细胞性质的尖端方法，使得可以从原代分离的干细胞的扩增中获得大量具有高分化潜能的干细胞。

 DESCRIPTION (provided by applicant): Utilization of stem cells to repair diseased or damaged tissues/organs represents a novel approach for remedies in regenerative medicine. Although embryonic stem cells (ESC) and induced pluripotent stem cells (iPS) have been studied, there are major drawbacks and concerns in using ESC and iPS; for example, tumorigenesis and teratomas formation can occur after their transplantation. It is not clear if the obstacles pertaining to ESC and iPS can be overcome in order to use them for therapeutics; at least there is still long way to go. In contrast, adult stem cells (AdSC) are used for treatment of human diseases. One example is the transplantation of bone marrow stem cells to cure or improve certain disorders, such as leukemia. Such cell- based therapies usually require large numbers of stem cells. Because AdSC lose their stem cell properties (stemness) including differentiation capability when cultured in vitro, primary isolated AdSC can only be used for limited passages, and propagation of a large quantity of high-potential stem cells from primary isolation is difficult. Thus, large amounts of tissues are needed for stem cell isolation, and continuous isolation of AdSC is required for multiple treatments. However, tissues, e.g., dental tissues that can be used for AdSC isolation are often limited. Stem cells isolated from dental pulp of extracted and exfoliated teeth have been shown to be a good source of stem cells for regenerative medicine. However, like other AdSC, these dental pulp stem cells (DPSCs) also lose their differentiation potential when cultured in vitro, which hampers their applications. The long-term goal of this research is to elucidate the genes and molecules regulating differentiation capability in the DPSCs. Preliminary studies showed that heat shock protein B8 (HspB8) was downregulated in cultured DPSCs when the cells lost differentiation capability, and knockdown of HspB8 expression in the early passage DPSCs caused the cells to lose differentiation capability. Further studies suggested that microRNAs (miRNAs) are responsible for downregulation of HspB8 expression in DPSCs. The central hypothesis is that upregulated expression of certain miRNAs in late passage DPSCs downregulates HspB8 and other regulatory genes, and in turn causes DPSCs to lose differentiation capability during in vitro culture. The project Aims: (1) to identify the miRNAs whose expression is substantially increased in late passage DPSCs, designated as late-passage upregulated miRNAs (LPU-miRNAs); (2) to identify the LPU-miRNAs that downregulate HspB8 expression; i.e., HspB8-targeting miRNAs, and (3) to study the effects of LPU-miRNAs and HspB8-targeting miRNAs on DPSC differentiation and on regulating other potential genes. Accomplishment of the Aims would be significant in understanding the roles of miRNAs and HspB8 in regulating the loss of differentiation seen in the long-term cultured DPSCs and will shed light on other AdSC as well. This would facilitate the development of cutting- edge methods for maintaining the stem cell properties in culturing DPSCs and AdSC, such that a large quantity of stem cells with high-differentiation potential can be obtained from expansion of primary isolated stem cells.