New Tools for Identifying, Tracking, and Isolating Human Progenitor Cells

描述（由研究者提供）： 人胚胎干（hES）细胞提供了潜在的无限替代细胞来源，用于治疗人退行性疾病，例如心血管疾病、黄斑变性、糖尿病、帕金森病、阿尔茨海默病以及伤口愈合和脊髓损伤，仅举几例。此外，在重编程体细胞（诱导多能干（iPS）细胞）方面的进展是患者特异性多能细胞的潜在未来来源。近期，这些最新进展无疑将显著增加研究级ES样细胞系的数量，从而增加用于鉴定、跟踪和分离具有治疗意义的分化细胞类型的祖细胞的研究工具的市场。多能干细胞向所需分化细胞类型的定向分化通常是低效的，通常仅产生几个百分比的起始细胞。此外，hES细胞难以大规模生长。因此，需要鉴定有用细胞类型的祖细胞的试剂，所述祖细胞可以在分化前分离和扩增，以允许成本有效的规模扩大并增加分化产物的产率。此外，这种试剂将是基础干细胞研究和发育生物学的有价值的研究试剂。我们建议开发一种称为延时噬菌体展示的新技术平台，用于鉴定祖细胞结合肽，以用作靶向、跟踪和分离治疗有用细胞的祖细胞的工具。在第1阶段，我们将通过分离靶向内皮谱系的CD31（PECAM 1）阳性细胞祖细胞的肽来测试该技术的可行性，所述内皮谱系的CD31（PECAM 1）阳性细胞我们将从hES细胞（NIH注册系WA 09）衍生。将对所得肽的特异性进行表征，并用于追踪靶向祖细胞的谱系。在第二阶段，我们将使用该技术来鉴定主要是心血管以及其他谱系的额外祖细胞的肽。将优化用于细胞追踪和分离的肽的使用。近期收入将通过肽作为试剂的商业化产生，例如用于祖细胞鉴定、细胞谱系追踪和细胞分离的缀合荧光探针和试剂盒。选定的肽将在内部用于开发ACT感兴趣的再生疗法，如缺血性和心血管疾病，其他肽将通过向企业合作伙伴授权进行商业化。我们预期本文提出的技术将普遍适用于鉴定靶向源自hES细胞以及其他类型的多能或多能干细胞（包括诱导的iPS和成体干细胞）的祖细胞的肽。公共卫生相关性：人类胚胎干细胞（hES）提供了用于治疗人类损伤和疾病的多能细胞的潜在无限来源，因为它们具有持续自我更新以及分化成几乎任何成熟细胞类型的能力。来自重编程成体细胞的ES样细胞的其他来源可能很快就会被证明比它们的胚胎对应物更具成本效益和更少的限制。然而，无论多能细胞的来源如何，关键问题仍然是如何获得商业数量的用于移植的临床级细胞。此外，在大多数情况下，用于移植的最佳细胞尚未确定。我们建议通过开发工具来识别，跟踪和分离来自多能hES细胞的祖细胞来解决这些问题。我们将首先使用一种称为延时噬菌体展示的新型肽选择方法来鉴定靶向人类血管细胞祖细胞的肽，并表征这些肽的细胞特异性靶向、细胞分离及其在祖细胞分化时跟踪祖细胞的能力。长期目标是将所提出的技术应用于鉴定心血管和其他细胞谱系的祖细胞以用于治疗开发，并将所得的细胞靶向试剂、细胞追踪和细胞分离试剂盒商业化。

DESCRIPTION (provided by investigator): Human embryonic stem (hES) cells offer a potentially unlimited source of replacement cells for treating human degenerative diseases such as cardiovascular disease, macular degeneration, diabetes, Parkinson's disease, Alzheimers, as well as wound healing and spinal cord injury to name a few. In addition, advances in reprogramming somatic cells (induced pluripotent stem (iPS) cells) are a potential future source of patient specific pluripotent cells. Near term, these recent advances will no doubt increase the number of research grade ES-like cell lines dramatically and thus increase the market for research tools to identify, track and isolate progenitors of differentiated cell types of therapeutic interest. The directed differentiation of pluripotent stem cells to a desired differentiated cell type is generally inefficient often yielding only a few percent of the starting cells. Moreover, hES cells are difficult to grow on a large scale. Thus there is a need for reagents that identify progenitors of useful cell types that can be isolated and expanded before differentiation to allow cost effective scale up and increase the yield of differentiated product. Moreover, such reagents would be valuable research reagents for basic stem cell research and developmental biology. We propose here to develop a novel technology platform called time-lapse phage display for identifying progenitor cell binding peptides for use as tools to target, track, and isolate progenitors of therapeutically useful cells. In phase 1, we will test the feasibility of the technology by isolating peptides that target progenitors of CD31 (PECAM1) positive cells of endothelial lineage that we will derive from hES cells (NIH registered line WA09). The resulting peptides will be characterized for specificity and used to trace the lineage of the targeted progenitor cells. In phase II, we will use the technology to identify peptides for additional progenitors primarily of cardiovascular as well as other lineages. The use of the peptides for cell tracking and isolation will be optimized. Near term revenue will be generated through the commercialization of the peptides as reagents such as conjugated fluorescent probes and kits for progenitor identification, cell lineage tracking, and cell isolation. Selected peptides will be used internally to develop regenerative therapies of interest to ACT such as ischemic and cardiovascular disease and other peptides will be commercialized by licensing to corporate partners. We anticipate the technology proposed here will be generally applicable for identifying peptides that target progenitors derived from hES cells as well as other types of pluripotent or multipotent stem cells including induced iPS and adult stem cells. PUBLIC HEALTH RELEVANCE: Human embryonic stem (hES) cells provide a potentially unlimited source of pluripotent cells for treating human injury and disease because of their ability to continually self renew as well as differentiate into virtually any mature cell type. Additional sources of ES-like cells from reprogrammed adult cells may soon prove to be more cost-effective and less restrictive than their embryonic counterparts. However, regardless of the source of pluripotent cells, a key problem remains of how to obtain commercial quantities of clinical grade cells for transplantation. Moreover the optimal cells for transplantation in most cases have yet to be determined. We propose to address these issues by developing tools to identify, track, and isolate progenitor cells derived from pluripotent hES cells. We will initially use a novel peptide selection method called time-lapse phage display to identify peptides that target progenitors of human vascular cells and characterize the peptides for cell-specific targeting, cell isolation, and their ability to track the progenitors as they differentiate. The long term goals are to apply the proposed technology to identifying progenitors of cardiovascular and other cell lineages for therapeutic development and to commercialize the resulting cell targeting reagents, cell tracking and cell isolation kits.