Toward Xeno-free Stem Cell Culture: Nanofiber-directed Differentiation of mESC to Neurons

 描述：在干细胞扩增和分化过程中，使用异种、昂贵和复杂的培养液和底物极大地限制了它们的广泛使用和翻译相关性。为了解决这些障碍，我们开发了一种合成纳米纤维基质，可以在1天内诱导小鼠胚胎干细胞分化为神经前体细胞。我们提出的纳米纤维组合使用了地形、排列和最多三个单独的生物活性多肽的连接，其中呈现和浓度可以精确控制，以影响细胞膜信号相互作用。因此，本建议旨在开发这种多肽系留培养底物，用于神经元和神经胶质细胞的定向分化和成熟。我们在开发能够为小鼠胚胎干细胞定向分化提供多个功能化位点的合成培养底物方面具有得天独厚的优势；到目前为止，还没有其他组织提供超过1个栓系多肽功能化位点。在这份R15提案中，我们将通过(I)表征表面束缚的生物活性分子对神经谱系承诺(神经胶质或神经元)分化和成熟的影响，以及(Ii)通过受控的表面相互作用而不是可溶性因素引导mESCs向神经谱系的分化来指导干细胞分化。这两个目标的结果将为成熟神经细胞的分化和长期培养提供一种新的、合成的培养基质。这些结果具有广泛的意义，因为这些培养底物可以进一步修改，以控制其他谱系的分化和成熟。PIs在研究培训方面有着良好的记录，大多数在实验室接受培训的本科生都进入了以研究为基础的、与健康相关的研究生课程。如果得到资助，拟议的项目将大大改善阿克伦大学两所学院的研究环境。两个PI都积极地将本科生纳入他们的研究实验室，在过去的5年里产生了18篇合著论文(包括5篇第一作者论文)。

 DESCRIPTION: The use of xenogenic, costly, and complicated culture media and substrates during stem cell expansion and differentiation procedures has greatly limited their widespread use and translational relevance. To address these barriers, we have developed a synthetic nanofiber substrate that induces differentiation of mouse embryonic stem cells to neural progenitors within 1 day. Our proposed nanofibers use combinations of topography, alignment, and the tethering of up to three individually bioactive peptides where the presentation and concentration can be controlled precisely to influence cell membrane signaling interactions. Therefore, this proposal seeks to develop this multi-peptide tethered culture substrate for the directed differentiation and maturation of neurons and glia. We are uniquely positioned to develop a synthetic culture substrate capable of providing multiple functionalization sites for directed mouse embryonic stem cell differentiation; no other group to date offers more than 1 tethered peptide functionalization site. In this R15 proposal, we will direct stem cell differentiation by (i) characterizing the impact of surface-tethered bioactive molecules on neural lineage commitment (glial or neuronal) differentiation and maturity, and (ii) directing differentiation of mESCs into neural lineages via controlled surface interaction rather than soluble factors. The outcomes of these two aims will provide a novel, synthetic culture substrate for differentiation and long-term culture of mature neural cells. These outcomes have broad implications, as these culture substrates could be further modified to control the differentiation and maturity of other lineages. The PIs have excellent track records with research training, with the majority of the undergraduate students trained in their laboratories advancing into research-based, health-related graduate programs. If funded, the proposed project would significantly enhance the research environment in two colleges at The University of Akron. Both PIs have actively integrated undergraduates into their research laboratories, resulting in 18 co-authored papers (including 5 first-authored papers) over the past 5 years.