Microsphere-mediated differentiation of embryonic stem cells

描述（由申请人提供）：用于各种退行性疾病和创伤性损伤的干细胞疗法可能能够恢复细胞损失并刺激组织再生。多能胚胎干细胞（ESC）能够分化成功能性神经元、心肌细胞和胰腺β细胞，因此代表了用于开发一系列不同再生细胞疗法的强大细胞来源。尽管多能干细胞具有明显的潜力，但一个关键的限制是不能以类似于胚胎发育的方式在空间和时间上控制形态发生因子向ESC的分子递送，所述胚胎发育有效地指导细胞分化为特定表型。由不同生物材料制成的微球可以被设计成局部控制生物活性分子的释放，从而同时提供生物分子递送至细胞和组织的空间、时间和剂量依赖性控制。将工程化微球整合到干细胞微环境中，用于在分化过程中将形态发生剂受控地局部递送到干细胞，是用于开发定向干细胞再生疗法的新颖且可转化的方法。该提议的目的是1）检查将不同类型的微球掺入经历分化的ESC的聚集体（称为胚状体（EB））内的效果，2）确定EB微环境内独立递送形态发生剂（例如Wnt 3a和BMP 2）的时空效果，和3）检查从EB内掺入的微球顺序递送不同形态发生剂（Wnt 3a和BMP 2）的效果。这些研究的完成将产生新的信息的能力，空间和时间控制的细胞外微环境的分子组成，通过生物材料为基础的交付方法调节ESC分化。此外，这种方法代表了一种通过局部分子因子的受控呈递来指导体外干细胞分化的全新途径，这可能是干细胞技术发展中广泛适用的原则。公共卫生相关性：寻求将活细胞恢复到患病或受伤组织的基于干细胞的疗法可以促进功能性组织再生，但目前受到不能有效控制干细胞分化的限制。该提议试图通过在空间上和时间上控制调节来自3D干细胞微环境内并入的微球的干细胞分化的形态发生因子的呈现来提高多能干细胞分化的效率和均一性。

DESCRIPTION (provided by applicant): Stem cell therapies for various degenerative diseases and traumatic injuries may be capable of restoring cell loss and stimulating tissue regeneration. Pluripotent embryonic stem cells (ESCs) are capable of differentiating into functional neurons, cardiomyocytes and pancreatic beta cells, thus representing a robust cell source for the development of an array of different regenerative cellular therapies. Despite the clear potential of pluripotent stem cells, a critical limitation is the inability to spatially and temporally control the molecular delivery of morphogenic factors to ESCs in a manner similar to embryonic development that efficiently directs the differentiation of the cells to specific phenotypes. Microspheres made of different biomaterials can be engineered to locally control the release of bioactive molecules, thereby simultaneously providing spatial, temporal and dose-dependent control of biomolecular delivery to cells and tissues. The integration of engineered microspheres into stem cell microenvironments for controlled, local delivery of morphogens to stem cells during the course of differentiation is a novel and translatable approach for the development of directed stem cell regenerative therapies. The objectives of this proposal are to 1) examine the effects of incorporating different types of microspheres within aggregates of ESCs undergoing differentiation referred to as embryoid bodies (EBs), 2) determine the spatiotemporal effects of independently delivering morphogens, such as Wnt 3a and BMP2, within EB microenvironments, and 3) examine the effects of sequential delivery of different morphogens (Wnt3a and BMP2) from microspheres incorporated within EBs. The completion of these studies will yield novel information about the ability to spatially and temporally control the molecular composition of the extracellular microenvironment regulating ESC differentiation via biomaterials-based delivery methods. In addition, this approach represents a fundamentally new route to directing the differentiation of stem cells in vitro through the controlled presentation of molecular factors locally, which may be a broadly applicable principle in the development of stem cell technologies. PUBLIC HEALTH RELEVANCE: Stem cell-based therapies seeking to restore viable cells to diseased or wounded tissues could promote functional tissue regeneration, but are currently limited by an inability to efficiently control the differentiation of the stem cells. This proposal seeks to improve the efficiency and homogeneity of pluripotent stem cell differentiation by spatially and temporally controlling the presentation of morphogenic factors regulating stem cell differentiation from microspheres incorporated within 3D stem cell microenvironments.