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以视网膜感光细胞丧失为特征的第一世界国家的主要失明原因。神经视网膜会受到多种退行性疾病的影响，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祖细胞再生的来源，但在成年哺乳动物的视网膜中，这些祖细胞的来源大大减少或缺失。相比之下，最近的报道表明，视网膜前体细胞可以从小鼠胚胎干细胞分化而来，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，胚胎干细胞来源的神经元的移植已经带来了一些有希望的结果。然而，使用胚胎干细胞需要破坏胚胎，并引发伦理问题和担忧。最近的报道表明，通过逆转录病毒传递的基因可以诱导小鼠和人类的体细胞分化为多能细胞。尽管如此，这些方法需要逆转录病毒感染和诱导肿瘤形成，这使得它们不足以用于治疗。最近，两篇发表在《自然》杂志上的文章表明，在小鼠体内，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新编程为胚胎干细胞(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，在适当的培养条件下，成年人的SSCs可以自发地重新编程为多能性，而不需要基因组修饰和逆转录病毒感染。我们首次成功地分离和培养了成人精原干细胞，并在确定的培养条件下，将SSCs重新编程为能够分化为所有三个生殖层的多潜能干细胞。我们已经诱导了多种细胞系的分化，包括表达神经元特异性标记物的神经元前体，如巢蛋白、神经丝、突触素和多巴胺能受体2(DRD2)。此外，我们还建立了一个在SSCs中特异表达GFP的Stra8-EGFP转基因小鼠系，这是获得纯种成年小鼠SSCs的来源。基于我们在人和小鼠身上的初步结果，我们的目标是：(1)通过优化培养条件，优化人和小鼠干细胞向ES样细胞的长期扩增和重编程，验证正常核型、基因表达的稳定性以及成瘤性或畸胎瘤的形成；(2)研究SSC来源的ES样细胞向神经细胞，尤其是视网膜神经元分化的能力，使其能够在体外增殖，并成为功能神经元。在这些为期两年的先导研究之后，我们的未来目标是将人类和小鼠SSC来源的视网膜神经前体细胞注射到视网膜变性的小鼠模型中，并分析这些细胞再生和重新填充视网膜的能力。这一方法至关重要，因为人类睾丸活检可以在没有基因修饰和逆转录病毒感染的情况下，实现基于细胞的自体器官治疗，而不会出现与人类胚胎干细胞相关的伦理和免疫学问题。 公共卫生相关性：在美国和其他以视网膜感光细胞丧失为特征的第一世界国家，视网膜变性是导致失明的主要原因。最近的报道表明，视网膜前体细胞可以从小鼠胚胎干细胞分化而来，可能成为成人视网膜干细胞的替代品。然而，使用胚胎干细胞需要破坏胚胎，并引发伦理问题和担忧。最近，我们已经证明，成人精原干细胞(SSCs)可以在不需要基因组修饰和逆转录病毒感染的情况下，使用适当的培养液自发地重新编程为多潜能细胞。我们已经将SSCs重新编程为能够分化为所有三个生殖层的多潜能干细胞。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)。这种方法非常重要，因为人类睾丸活检可能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学问题，没有遗传修饰和逆转录病毒感染。在这项建议中，我们将研究人类精原干细胞的生物学、可塑性和治疗潜力。

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.