Pluripotent Adult Spermatogonial Stem Cells: Prospective for Retinal Degeneration

描述（由申请人提供）：视网膜变性是美国和其他第一世界国家失明的主要原因，其特征是视网膜感光细胞的丧失。神经视网膜易发生多种退行性疾病，包括视网膜色素变性、老年性黄斑变性和青光眼。虽然在非哺乳动物的脊椎动物中有许多再生的祖细胞来源，但在成年哺乳动物的视网膜中，这些来源大大减少或缺失。相比之下，最近的报道表明，视网膜祖细胞可以从小鼠胚胎干细胞（ES）中获得，并可能提供一种替代成人视网膜干细胞的方法。在中枢神经系统的其他区域，胚胎干细胞衍生的神经元移植已经取得了一些有希望的结果。然而，使用胚胎干细胞需要破坏胚胎，并产生伦理问题和担忧。最近的报告表明，通过添加逆转录病毒传递的基因，可以诱导小鼠和人类的体细胞皮肤细胞具有多能性。然而，这些方法需要逆转录病毒感染并诱导肿瘤发生，这使得它们不适合用于治疗。最近，两篇发表在《自然》杂志上的文章表明，小鼠精原干细胞（SSCs）可以在不添加基因或逆转录病毒的情况下重编程为胚胎干细胞（ES）细胞。然而，到目前为止，还没有证据表明成人精原干细胞具有多能性。最近，我们已经证明成人ssc可以在适当的培养条件下自发地重编程为多能性，而不需要基因组修饰和逆转录病毒感染。我们首次成功分离和培养了成人精原干细胞，并在规定的培养条件下，我们将ssc重新编程为能够分化为所有三个胚层的多能干细胞。我们已经诱导了各种细胞系的分化，包括表达神经元特异性标记的神经元前体，如巢蛋白、神经丝、突触素和多巴胺能受体2 （Drd2）。此外，我们已经产生了一个Stra8-EGFP转基因小鼠系，在SSCs中特异性表达GFP，这是获得成年小鼠SSCs纯群体的来源。基于我们在人类和老鼠身上的初步结果，我们的目标是：(1)通过优化培养条件，优化人和小鼠SSCs向es样细胞的长期扩增和重编程，验证正常核型、基因表达的稳定性和致瘤性或畸形瘤形成的稳定性；(2)研究SSCs衍生的es样细胞向神经细胞特别是视网膜神经元的分化能力，使其增殖并具有体外繁殖能力，成为功能神经元。经过两年的前期研究，我们的未来目标是将人类和小鼠ssc衍生的神经视网膜祖细胞注射到视网膜变性小鼠模型的视网膜中，并分析这些细胞再生和重新填充视网膜的能力。这种方法是至关重要的，因为人类睾丸活检可以允许基于细胞的自体器官治疗，而不存在与人类胚胎干细胞相关的伦理和免疫问题，不需要基因修饰和逆转录病毒感染。

DESCRIPTION (provided by applicant): Retinal degeneration is the leading cause of blindness in the United States and other first world countries characterized by loss of retinal photoreceptor cells. The neural retina is subject to a number of degenerative conditions, including retinitis pigmentosa, age-related macular degeneration, and glaucoma. Although there are a number of sources of progenitors for regeneration in non-mammalian vertebrates, these are greatly reduced or absent in the adult mammalian retina. By contrast, recent reports show that retinal progenitor cells can be derived from mouse embryonic stem (ES) cells and may provide an alternative to adult derived retinal stem cells. In other regions of the central nervous system, the transplantation of neurons derived from ES cells has led to some promising results. However, the use of ES cells requires the destruction of the embryos and creates ethical issues and concerns. Recent reports have demonstrated that somatic skin cells in mice and in humans can be induced to pluripotency with the addition of genes delivered using retroviruses. Nonetheless, these approaches require the retroviral infections and induce tumorigenecity, which makes them inadequate for therapeutic use. Recently, two publications, both in Nature, have shown that in mice spermatogonial stem cells (SSCs) can reprogram to embryonic stem (ES) cells without the addition of genes or retroviruses. However, to date, there is no evidence showing the pluripotency of adult human spermatogonial stem cells. Lately, we have demonstrated that adult human SSCs can be reprogrammed to pluripotency spontaneously in appropriate culture condition, without genomic modification and retroviral infection. We have successfully isolated and cultured the adult human spermatogonial stem cells for the first time and under defined culture conditions, we have reprogrammed the SSCs to pluripotent stem cells capable of differentiating into all three germ layers. We have induced the differentiation of various cell lineages including neuronal precursors expressing neuronal specific markers, such as, nestin, neurofilament, synaptophysin and dopaminergic receptor 2 (Drd2). Additionally, we have generated a Stra8-EGFP transgenic mouse line that specifically expresses GFP in the SSCs, a source for obtaining a pure population of adult mouse SSCs. Based upon our preliminary results in human and mouse, our goals are to: (1) optimize the long-term expansion and reprogramming of human and mouse SSCs into ES-like cells by optimizing the culture conditions, verify the stability of normal karyotype, gene expression and the tumorigenicity or teratomas formation, (2) investigate differentiation capacity of SSC-derived ES-like cells into neural cells and particularly retinal neurons, make them proliferate and possess the ability to propagate in vitro and to become functional neurons. Our future goal after these two-year pilot studies is to inject the human and mouse SSC-derived neuro-retinal progenitor cells into the retina of a mouse model of retinal degeneration and to analyze the capacity of these cells to regenerate and repopulate the retina. This approach is of paramount importance since human testicular biopsies may allow the cell-based, autologous organ therapy without the ethical and immunological problems associated with human embryonic stem cells, in the absence of genetic modification and retroviral infection. PUBLIC HEALTH RELEVANCE: Retinal degeneration is the leading cause of blindness in the United States and other first world countries characterized by loss of retinal photoreceptor cells. Recent reports show that retinal progenitor cells can be derived from mouse embryonic stem (ES) cells and may provide an alternative to adult derived retinal stem cells. However, the use of ES cells requires the destruction of the embryos and creates ethical issues and concerns. Recently, we have demonstrated that adult human spermatogonial stem cells (SSCs) can be reprogrammed to pluripotency spontaneously, using appropriate culture media, without genomic modification and retroviral infection. We have reprogrammed the SSCs to pluripotent stem cells capable of differentiating into all three germ layers. We have induced the differentiation of various cell lineages including neuronal precursors expressing neuronal specific markers, such as, nestin, neurofilament, synaptophysin and dopaminergic receptor 2 (Drd2).This approach is of paramount importance since human testicular biopsies may allow the cell-based, autologous organ therapy without the ethical and immunological problems associated with human embryonic stem cells, in the absence of genetic modification and retroviral infection. In this proposal we will examine the biology, the plasticity and therapeutic potential of human spermatogonial stem cells.