Discovery of ligands for directed-differentiation of stem cells

描述（由申请人提供）：除了发现用于hESC/iPSC维持和定向分化的可溶性小分子之外，组合化学还可以用作鉴定细胞外基质（ECM）模拟物的极好工具，所述细胞外基质（ECM）模拟物可以进一步促进hESC/iPSC的维持或特异性分化。Lam博士发明的一珠一化合物（OBOC）组合化学特别适合于发现ECM模拟物或其他针对干细胞独特细胞表面受体的功能配体。我们最近通过在OBOC文库的每个珠的外层上掺入已知的细胞粘附肽（例如整联蛋白结合配体）开发了一种单珠双化合物（OB 2C）文库方法。因此，每个结合的细胞将暴露于展示在每个珠表面上的文库化合物。我们已经产生了基因标记的Olig 2-GFP敲入hESC/iPSC报告细胞系，其可以方便地用作所提出的组合文库筛选中的读出，用于向各种谱系定向分化。该提案的具体目标是：1。设计和合成几个OBOC和OB 2C组合库，用于发现可以诱导hESCs或iPSC定向分化的细胞表面作用分子。2.使用活的Olig 2-GFP敲入报告基因hESC/iPSC系结合免疫细胞化学方法筛选各种OB 2C文库（来自目的1）用于定向分化为少突胶质祖细胞。3.使用一系列生物学和生物化学方法充分表征目标2中鉴定的配体和分子对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的化学定义的自组装PVA基水凝胶作为人工ECM，以支持干细胞生长和定向分化为少突胶质祖细胞[OPC]。影响拟议的研究将使我们能够发现与hESC/iPSC表面独特受体结合的合成分子。这些分子中的一些可以支持hESC/iPSC的生长，同时保持其“干性”。其他可能诱导特定的细胞信号传导和少突胶质细胞分化。 我们的长期目标是直接分化少突胶质细胞前体细胞进一步用于治疗神经系统疾病。 公共卫生相关性：除了发现用于hESC/iPSC维持和定向分化的可溶性小分子之外，组合化学还可以用作鉴定细胞外基质（ECM）模拟物的极好工具，所述细胞外基质模拟物可以进一步促进hESC/iPSC的维持或特异性分化。Lam博士发明的一珠一化合物（OBOC）和一珠两化合物（OB 2C）组合化学特别适合于发现针对干细胞的独特细胞表面受体的ECM模拟物或其他功能性配体。遗传标记的Olig 2-GFP敲入hESC/iPSC报告细胞系将用作所提出的组合文库筛选中的读出物，用于定向分化为少突胶质祖细胞。

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. PUBLIC HEALTH RELEVANCE: 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) and one-bead-two- compound (OB2C) 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. Genetically-labeled Olig2-GFP knock-in hESC/iPSC reporter lines will be used as readouts in the proposed combinatorial library screening for directed-differentiation into oligodendroglial progenitor cells.