Epigenetic control of functional maintenance and differentiation capacity of USSC

最近的证据表明，Dlk 1的表达区分两种类型的脐带血来源的干细胞，即Dlk 1 + USSC具有分化成骨谱系的潜力和Dlk 1- CB-MSC，其主要具有成脂分化潜力。Dlk 1是印迹dlk 1-dio 3基因座的一部分，最近与小鼠诱导多能干细胞（iPS）中不完全的表观遗传重编程和分化潜能受损有关。在该项目的第一部分中，我们的目标是剖析Dlk 1的扩展dlk 1-dio 3基因座的背景下，调节Dlk 1的表观遗传基础。高密度平铺阵列覆盖的正义和反义方向的完整基因座将用于比较分析的DNA甲基化和组蛋白修饰的脐带血衍生的体干细胞和iPS细胞。对Dlk 1-Dio 3基因座的其他组分如miRNA、snoRNA和大型非编码RNA的分析将有助于全面了解干细胞中DLK 1的调控。在项目的第二部分，我们将探索使用表观遗传修饰剂对USSC进行表观遗传重编程的潜力。在这种情况下，我们已经表明，Dlk 1 + USSC可以可重复地转化为Dlk 1-细胞，采用球形表型（spheUSSC），并可以分化为脂肪细胞的高效率。重要的是，spheUSSC可以繁殖到大的细胞数量，而无需进一步用表观遗传修饰剂处理。我们将进一步表征spheUSSC的分化潜力，其通过外源因子诱导多能性的适用性及其“营养活性”的潜力。最后，我们将进一步阐明使用新型表观遗传药物组合对USSC进行定向表观遗传重编程的潜力。

Recent evidence suggests that expression of Dlk1 distinguishes between two types of cord bloodderived stem cells, namely Dlk1+ USSC that have the potential to differentiate to the osteogenic lineage and Dlk1- CB-MSC, which have mainly adipogenic differentiation potential. Dlk1 is part of the imprinted dlk1-dio3 locus, which was recently associated with incomplete epigenetic reprogramming and impaired differentiation potential in murine induced pluripotent stem (iPS) cells. In the first part of the project, we aim to dissect the epigenetic basis for the regulation of Dlk1 in the context of the extended dlk1-dio3 locus. High-density tiling arrays covering the complete locus in sense and antisense direction will be used for comparative analysis of DNA methylation and histone modifications in cord blood-derived somatic stem and iPS cells. Analysis of other components of the Dlk1-Dio3 locus such as miRNAs, snoRNAs, and large non-coding RNAs will add to a comprehensive picture of the regulation of DLK1 in stem cells. In the second part of the project we will explore the potential for epigenetic reprogramming of USSC using epigenetic modifiers. In this context, we have already shown that Dlk1+ USSC can be reproducibly converted to Dlk1- cells that adopt a sphere-like phenotype (spheUSSC) and can be differentiated to adipocytes with high efficiency. Importantly, spheUSSC can be propagated to large cell numbers without further treatment with epigenetic modifiers. We will further characterize the differentiation potential of spheUSSC, their suitability for induction of pluripotency by exogenous factors and their potential for “trophic activity”. Finally, we will further elucidate the potential for directed epigenetic reprogramming of USSC using novel combinations of epigenetic drugs.