Establishment of Marmoset iPS-derived Cranial Neural Crest Cells

描述(申请人提供)：成体细胞可以被重新编程为胚胎样诱导多能干细胞(IPS)这一突破性发现，为未来创新的针对患者的细胞和组织再生疗法带来了兴奋。在适当的条件下，iPS细胞可以发育成任何所需的细胞类型。为了达到再生牙科的目的，从iPS细胞中分离出颅神经脊细胞是iPS来源的颅面结构再生疗法未来发展的重要一步。为了评估iPS来源的细胞疗法的安全性和有效性，开发一种非人类灵长类动物模型系统是非常必要的。常见的绒猴(Callithrix Jacchus)是一种广泛应用于生物医学研究的非人灵长类动物，最近从绒猴身上产生了iPS细胞。这些研究将探讨绒猴诱导的多能干细胞(IPS)在体外发育为颅神经脊细胞的潜力，并评估其体外向下分化的潜力。Cnc细胞是一种多能干细胞，在脊椎动物发育过程中可分化出颅骨雪旺细胞、外周神经元、面骨、软骨和牙齿发生所需的牙齿间充质。IPS来源的CNC细胞及其下游谱系特定的子代细胞是牙齿再生目标的理想细胞，例如骨、牙本质、牙髓、整个牙齿和周围神经的再生。绒猴iPS来源的CNC细胞的发展将通过非人类灵长类细胞治疗模型加速人类iPS来源的头面部结构再生治疗的发展。我们的总体假设是，绒猴iPS细胞将在神经和间充质细胞系中产生具有分化潜能的颅神经脊干细胞。这一假说将通过以下具体目标来验证：1)建立绒猴iPS来源的NC细胞，2)体外测定绒猴iPS来源的NC细胞的神经分化潜能，3)体外测定绒猴iPS来源的NC细胞的间充质分化潜能。这些研究将提供iPS重新编程、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、动物模型和神经元电生理学方面的培训。这一建议是创新的，因为在使用非人类灵长类多能干细胞的情况下，还没有证明细胞分化。这项拟议的研究意义重大，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细胞和丰富cnc来源的细胞类型，如雪旺细胞前体细胞、成骨细胞和成牙本质细胞，影响iPS来源细胞治疗颅面结构再生的发展。通过评估体内细胞治疗的长期安全性和有效性，未来的绒猴自体细胞治疗将影响iPS衍生治疗的发展。这项研究建议符合申请者的职业目标，即成为一名牙医-科学家，研究再生牙科中的转化研究。 公共卫生相关性：诱导多能干细胞(IPS)是一种新的、令人兴奋的细胞来源，有助于发展干细胞群体，通过细胞或组织移植促进体内组织和结构的再生。由于在脊椎动物发育过程中，脑神经脊干细胞对颅面软骨、骨、牙齿和颅外周神经系统的正常形成具有重要作用，因此是从诱导性多能干细胞发展而来的理想群体。计划中的研究将从普通绒猴(Callithrix Jacchus)的iPS细胞中开发和鉴定cnc干细胞，这是一种非人类灵长类动物模型，可用于开发和评估基于干细胞的治疗方法。

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. PUBLIC HEALTH RELEVANCE: Induced pluripotent stem (iPS) cells are a new and exciting source of cells useful to develop stem cell populations that will promote the regeneration of tissues and structures in the body through cell or tissue transplantation. Cranial neural crest (CNC) stem cells are an ideal population to develop from iPS cells due to their known critical role in contributing to the proper formation of cranial-facial cartilage, bone, teeth, and the cranial peripheral nervous system during vertebrate development. The planned studies will develop and characterize CNC stem cells from iPS cells of the common marmoset (Callithrix jacchus); a non-human primate model useful for developing and evaluating stem cell based therapies.