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为成熟细胞类型的能力是 干细胞。组织源干细胞(TDSCs)具有多向分化潜能，是目前世界上 使其成为治疗疾病或损伤的宝贵细胞来源的关键特性。 大量的初级干细胞可以直接从组织中分离出来；因此，初级干细胞的体外扩增 为了获得大量用于治疗的细胞，细胞是必需的。 TDSCs会导致细胞失去分化能力，从而产生大量的 这极大地阻碍了TDSCs的治疗应用。我们的长- 学期目标是：(A)阐明导致TDSCs分化丧失的分子机制 扩张。(B)制定方法，以保持其分化能力，使大量高- 体外扩增可获得高效的TDSCs，用于细胞治疗。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-2(Crispld2)的表达显著增加 当细胞在体外扩增后失去成骨能力时，主要的TDSCs减少。更多 重要的是，该基因在高潜能成骨tdscs中的敲除会导致细胞失去其成骨能力。 辨别能力。据报道，Crispld2基因敲除对胚胎是致命的，这表明它在 发展。我们的中心假设是TDSCs的体外扩增导致了Crispld2的失调， 这反过来又会导致它们分化能力的丧失。这一假设将在两个特定的目标中进行检验， 人骨髓干细胞(HBMSCs)：目的1.鉴定和鉴定转录因子 人骨髓间充质干细胞中Crispld2的表达。目的2.确定强制或诱导表达Crispld2是否会影响 人骨髓间充质干细胞的分化。我们将结合新的和现代的技术，CRISPR/CAS9和生物信息学， 与传统的细胞和分子生物学方法，如细胞培养，生物检测，基因表达分析， 并通过细胞分化试验来实现这些目标。体外细胞实验和体内动物研究将是 在我们的方法中使用。这一提议意义重大，因为它将克服产生 通过了解维持分化能力的分子调控大量培养TDSCs 干细胞的数量。我们希望找出调节Crispld2表达的转录因子(激活因子和抑制因子)，并 确定是否需要高水平的Crispld2表达以保持细胞的分化能力 TDSCs。该项目的完成将开始阐明Crispld2的调控网络。因为 Crispld2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、细胞信号转导等方面，本项目的研究结果明确了 Crsipld2将产生广泛而重大的影响和影响。此外，这项研究将探索潜在的 用雌孕激素维持TDSCs的分化能力。

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.