Establishment of Marmoset iPS-derived Cranial Neural Crest Cells

描述（由申请人提供）：成人细胞可以重新编程为胚胎样诱导多能干细胞（iPS）的突破性发现为未来创新的患者特异性细胞和组织再生疗法带来了兴奋。iPS细胞可以在适当的条件下发育成任何所需的细胞类型。对于再生牙科目标，从iPS细胞中获得颅神经嵴（CNC）细胞是未来开发iPS衍生颅面结构再生疗法的重要一步。为了评估iPS衍生的细胞疗法的安全性和功效，非常需要开发非人灵长类动物模型系统。普通绒猴（Callithrix jacchus）是一种广泛用于生物医学研究的非人灵长类动物，最近从绒猴中产生了iPS细胞。拟开展的研究将探讨绒猴诱导多能干细胞（iPS）体外发育颅神经嵴细胞的潜力，并评估其体外下游分化潜力。CNC细胞是多能干细胞，其在脊椎动物发育期间产生颅许旺细胞、外周神经元、面骨、软骨和牙齿发生所需的牙齿间充质。iPS衍生的CNC细胞及其下游谱系特异性后代细胞成为牙齿再生目标的理想细胞，如骨、牙本质、牙髓、全牙和外周神经的再生。绒猴iPS衍生的CNC细胞的开发将通过非人灵长类动物细胞治疗建模加速人类iPS衍生的颅面结构再生疗法的开发。我们的总体假设是，绒猴iPS细胞将产生在神经和间充质细胞谱系中具有分化潜力的颅神经嵴干细胞。该假设将通过以下具体目的进行检验：1）建立绒猴iPS衍生的CNC细胞，2）确定绒猴iPS衍生的CNC细胞在体外的神经分化潜能，和3）确定绒猴iPS衍生的CNC细胞在体外的间充质分化潜能。这些研究将提供iPS重编程、干细胞维持和分化、定量基因表达、免疫细胞化学、流式细胞术、FACS分选、动物建模和神经元电生理学方面的培训。该提议是创新的，因为CNC分化尚未使用非人灵长类多能干细胞证明。这项研究意义重大，因为CNC细胞是决定早期颅面发育的关键群体。这些研究将通过分离CNC细胞和富集CNC衍生的细胞类型（如雪旺细胞前体、成骨细胞和成牙本质细胞），影响用于颅面结构再生的iPS衍生细胞疗法的发展。未来在绒猴中的自体细胞疗法将通过评估体内细胞疗法的长期安全性和有效性来影响iPS衍生疗法的发展。这项研究计划符合申请人的职业目标，成为一名研究再生牙科转化研究的牙科科学家。

DESCRIPTION (provided by applicant): The groundbreaking discovery that adult cells can be reprogrammed into embryonic-like induced pluripotent stem (iPS) cells has developed excitement for future innovative patient-specific cell and tissue regeneration therapies. iPS cells can develop into any desired cell type under appropriate conditions. For regenerative dentistry goals, deriving cranial neural crest (CNC) cells from iPS cells is an important step for future development of iPS-derived cranial-facial structure regeneration therapies. In order to assess the safety and efficacy of iPS-derived cell therapies, the development of a non-human primate model system is highly desirable. The common marmoset (Callithrix jacchus) is a non-human primate used widely in biomedical research and iPS cells were recently generated from the marmoset. The proposed studies will address the potential of marmoset induced pluripotent stem (iPS) cells for in vitro development of cranial neural crest cells and assess their downstream differentiation potential in vitro. CNC cells are multipotent stem cells that during vertebrate development give rise to cranial Schwann cells, peripheral neurons, facial bone, cartilage, and the dental mesenchyme required in tooth genesis. iPS-derived CNC cells and their downstream lineage-specific progeny cells make ideal cells for dental regenerative goals such as the regeneration of bone, dentin, dental pulp, whole teeth, and peripheral nerves. The development of marmoset iPS-derived CNC cells will accelerate the development of human iPS-derived therapies for cranial-facial structure regeneration through non-human primate cell therapy modeling. Our overall hypothesis that marmoset iPS cells will generate cranial neural crest stem cells with differentiation potential in both neural and mesenchymal cell lineages. This hypothesis will be tested by the following specific aims: 1) to establish marmoset iPS-derived CNC cells, 2) to determine the neural differentiation potential of marmoset iPS-derived CNC cells in vitro and 3) To determine the mesenchymal differentiation potential of marmoset iPS-derived CNC cells in vitro. These studies will provide training in iPS reprogramming, stem cell maintenance and differentiation, quantitative gene expression, immunocytochemistry, flow cytometry, FACS sorting, animal modeling, and neuron electrophysiology. This proposal is innovative because CNC differentiation has not been demonstrated using non-human primate pluripotent stem cells. The proposed research is significant because CNC cells are a key population that defines proper early cranial-facial development. These studies will impact the development of iPS-derived cell therapies for cranial-facial structure regeneration through the isolation of CNC cells and enrichment of CNC-derived cell types such as Schwann cell precursors, osteoblasts, and odontoblasts. Future autologous cell therapy in the marmoset will impact the development of iPS-derived therapies by assessing the long-term safety and efficacy of cell therapy in vivo. This research proposal fits the applicant's career goal to become a dentist-scientist studying translational research in regenerative dentistry.