Molecular basis for the loss of differentiation capability in human bone marrow stem cells during expansion

项目摘要 自我更新的能力和分化成成熟细胞类型的能力是细胞的两个关键性质。 干细胞组织源性干细胞（Tissue-derived stem cells，TDSCs）具有多向分化潜能，是目前研究最多的干细胞。 这是一种重要的特性，使它们成为治疗疾病或损伤的有价值的细胞来源。通常，只有有限的 可以直接从组织中分离大量的原代干细胞;因此， 需要细胞来获得用于治疗应用的大量细胞。然而，在体外扩增的 TDSC导致细胞失去其分化能力，这使得产生大量的 高潜能干细胞的研究很困难。这极大地阻碍了TDSC的治疗应用。我们长久以来- 长期目标是：（a）阐明在TDSC过程中引起TDSC分化丧失的分子机制， 扩张. (b)为了开发保持其分化能力的方法，使得大量的高- 有效的TDSC可以从体外扩增获得，用于基于细胞的治疗。在初步研究中，我们 发现富含半胱氨酸的分泌蛋白LCCL结构域包含-2（Crispld 2）的表达显著增加， 当细胞在体外扩增后失去其成骨能力时，主要TDSC中的成骨能力降低。更 重要的是，在高潜能成骨TDSC中敲低该基因会导致细胞失去其成骨能力， 分化能力。据报道，Crispld 2敲除是胚胎致死的，这表明其在胚胎发育中的关键作用。 发展我们的中心假设是TDSC的体外扩增导致Crispld 2的失调， 这又导致其分化能力的丧失。这一假设将在两个具体目标中得到检验， 人骨髓干细胞（hBMSCs）：目的1.识别和表征转录因子（TF）调节 hBMSCs中Crispld 2的表达。目标2.确定Crispld 2的强制或诱导表达是否影响 hBMSCs的分化。我们将结合联合收割机、CRISPR/Cas9和生物信息学等现代新技术， 利用传统的细胞和分子生物学方法，例如细胞培养，生物测定，基因表达分析， 和细胞分化测定来实现这些目的。将进行体外细胞实验和体内动物研究。 在我们的方法中使用。这一建议意义重大，因为它将导致克服困难， 通过了解维持分化能力的分子调控， 的干细胞。我们期望鉴定调节Crispld 2表达的TF（激活剂和抑制剂）， 确定高水平的Crispld 2表达是否是保持细胞分化能力所必需的。 TDSC。该项目的完成将开始阐明Crispld 2的调控网络。因为 crispld 2在许多组织中广泛表达，具有多种功能，包括发育，细胞防御， 干细胞分化、细胞信号转导等，本项目的结果，以确定监管因素， 第二次检讨将产生广泛而重大的影响和影响。此外，该研究还将探索 在使用雌激素和孕激素来保持TDSCs的分化能力方面。

Project Summary The ability of self-renewal and the capability to differentiate into mature cell types are two critical properties of stem cells. Tissue-derived stem cells (TDSCs) possess multipotent differentiation capability, which is the most crucial property to make them a valuable cell source for treatments of diseases or injuries. Usually, only limited quantities of primary stem cells can be directly isolated from tissues; therefore, in vitro expansion of primary stem cells is needed to obtain large quantities of cells for therapeutic applications. However, in vitro expansion of TDSCs results in the cells to lose their differentiation capability, which makes a generation of large quantities of high-potential stem cells difficult. This dramatically hampers the therapeutic applications of the TDSCs. Our long- term goals are: (a) To elucidate the molecular mechanisms causing the loss of differentiation in TDSCs during expansion. (b) To develop methods to preserve their differentiation capability, such that large quantities of high- potency TDSCs can be obtained from in vitro expansion for cell-based therapies. In preliminary studies, we found that expression of cysteine-rich secretory protein LCCL domain-containing-2 (Crispld2) is dramatically decreased in major TDSCs when the cells lose their osteogenic capability after in vitro expansion. More importantly, knockdown of this gene in high potential osteogenic TDSCs causes the cells to lose their osteogenic differentiation ability. Crispld2 knockout was reported to be embryonic lethal, suggesting its crucial role in development. Our central hypothesis is that in vitro expansion of TDSCs results in dysregulation of Crispld2, which in turn leads to loss of their differentiation ability. The hypothesis will be tested in two specific aims with human bone marrow stem cells (hBMSCs): Aim 1. Identify and characterize transcription factors (TFs) regulating Crispld2 expression in hBMSCs. Aim 2. Determine if enforced or induced expression of Crispld2 affects differentiation of hBMSCs. We will combine novel and modern technologies, CRISPR/Cas9, and bioinformatics, with traditional cell and molecular biology methods, such as cell culture, bioassays, gene expression analysis, and cell differentiation assays to achieve these Aims. In vitro cell experiments and in vivo animal studies will be used in our approaches. This proposal is significant because it will lead to overcoming the difficulty in generating large quantities of TDSCs by understanding the molecular regulation for maintaining the differentiation capability of the stem cells. We expect to identify TFs (activators and repressors) that regulate Crispld2 expression and determine whether high-level Crispld2 expression is necessary to preserve the differentiation capability of TDSCs. Accomplishment of this project will begin to elucidate the regulatory network of Crispld2. Because Crispld2 is widely expressed in many tissues and has multiple functions, including development, cellular defense, stem cell differentiation, cell signal transduction, etc., the results of this project to define the regulatory factors of crsipld2 will have a broad and significant impact and implication. Furthermore, the study will quest the potential in using estrogen and progesterone to preserve the differentiation ability of TDSCs.