Discovery of ligands for directed-differentiation of stem cells

描述(申请人提供)：除了发现用于hESC/IPSC维持和定向分化的可溶性小分子外，组合化学还可以作为鉴定细胞外基质(ECM)模拟物的极佳工具，进一步促进hESC/IPSCs的维持或特异性分化。林博士发明的一珠一化合物(OBOC)组合化学特别适合于发现针对干细胞独特细胞表面受体的ECM模拟物或其他功能配体。我们最近开发了一种一珠两化合物(OB2C)文库方法，方法是在OBOC文库的每个珠子的外层加入已知的细胞粘附肽(例如整合素结合配体)。因此，每个结合的细胞都将暴露在每个珠子表面显示的库化合物中。我们已经产生了基因标记的寡核苷酸GFP敲入hESC/IPSC报告系，可以方便地用作建议的组合文库筛选中的读数，用于定向分化不同的谱系。本研究的具体目的是：1.设计和合成几个OBOC和OB2C组合文库，以发现可以诱导hESCs或ipSCs定向分化的细胞表面作用分子。2.用活的寡核苷酸-GFP敲入报告基因hESC/IPSC结合免疫细胞化学方法筛选OB2C文库，定向分化为少突胶质前体细胞。3.利用一系列生物学和生化方法，全面表征目标2中确定的配体和分子对hESC/IPSC生长和分化的影响。为了优化和使用这些配体在一种新型的化学定义的自组装PVA水凝胶中作为人工ECM来支持干细胞生长和定向分化为少突胶质前体细胞[OPC]。这项拟议的研究将使我们能够发现与hESC/IPSCs表面独特受体结合的合成分子。这些分子中的一些可能支持hESC/IPSCs的生长，同时保持其“干性”。其他的可能诱导特定的细胞信号和少突胶质细胞分化。我们的长期目标是进一步定向分化少突胶质前体细胞，用于治疗神经疾病。

DESCRIPTION (provided by applicant): In addition to discovering soluble small molecule for hESC/iPSC maintenance and directed differentiation, combinatorial chemistry can also serve as an excellent tool for the identification of extracellular matrix (ECM) mimics that can further facilitate the maintenance or specific- differentiation of hESC/iPSCs. The one-bead-one-compound (OBOC) combinatorial chemistry invented by Dr. Lam is particularly suited for the discovery of ECM mimics or other functional ligands against unique cell surface receptors of stem cells. We have recently developed a one- bead-two-compound (OB2C) library method by incorporating a known cell adhesion peptide (e.g. an integrin binding ligand) on the outer layer of every bead of the OBOC library. As a result, every bound cell will be exposed to the library compound displayed on the surface of each bead. We have generated genetically-labeled Olig2-GFP knock-in hESC/iPSC reporter lines that can be conveniently used as readouts in the proposed combinatorial library screening for directed-differentiation toward various lineages. Specific aims of this proposal are: 1. To design and synthesize several OBOC and OB2C combinatorial libraries for the discovery of cell surface acting molecules that can induce directed-differentiation of hESCs or iPSCs. 2. To use live Olig2-GFP knock-in reporter hESC/iPSC lines in conjunction with immunocytochemical methods to screen various OB2C libraries (from aim 1) for directed-differentiation into oligodendroglial progenitor cells. 3. To fully characterize effects of the ligands and molecules identified in aim 2 on hESC/iPSC growth and differentiation using a range of biological and biochemical approaches. To optimize and use these ligands in a novel chemically-defined self- assemble PVA-based hydrogel as artificial ECM to support stem cell growth and directed- differentiation into oligodendroglial progenitor cells [OPCs]. Impact The proposed research will enable us to discover synthetic molecules that bind to unique receptors on the surface of hESC/iPSCs. Some of these molecules may support the growth of hESC/iPSCs while maintaining its "stem-ness". Others may induce specific cell signaling and oligodendrocyte differentiation. Our long term goal is to direct-differentiate oligodendrocyte precursor cells further for the treatment of neurological diseases.