Therapeutic hair follicle-derived neurospheres

描述(由申请人提供)：多能成体干细胞的易得性将有许多重要的治疗应用。我们最近的研究结果表明，毛囊是一种很有希望的干细胞来源。我们发现神经干细胞标记物Nestin表达于毛囊隆起区细胞，即毛囊干细胞的位置(1)，提示毛囊干细胞和神经干细胞具有共同的特征。我们已经证明，毛囊在体内产生血管，血管起源于毛囊隆起细胞(13)。我们还在体外证明了毛囊膨大细胞可以被诱导形成神经球，进而形成神经元(21)。这些结果表明，多能毛囊成体突起细胞可能具有重要的治疗应用，特别是在神经疾病方面。这项应用利用在巢蛋白调控序列(巢蛋白驱动的[ND]-GFP)控制下的绿色荧光蛋白(GFP)转基因小鼠作为标记毛囊隆起细胞的来源。我们分离的毛囊隆起区细胞对干细胞标记CD34呈阳性，角蛋白15呈阴性，β-III微管蛋白呈阴性，表明它们处于相对未分化的状态。这些细胞在体外可以分化为神经胶质细胞、平滑肌细胞、角质形成细胞以及神经元。将ND-GFP细胞接种于C57BL/6免疫活性小鼠的坐骨神经断端的两个断端之间。神经随后又重新连接起来。大部分表达GFP的毛囊膨大细胞在再生的坐骨神经中分化为带髓鞘的GFAP阳性的雪旺细胞。再接的坐骨神经在电刺激下收缩腓肠肌。在断端的胫神经间移植毛囊膨大细胞后，小鼠的行走指印长度和中间趾伸展明显恢复，表明移植的小鼠已恢复正常行走能力。初步结果表明，ND-GFP隆起区细胞能促进小鼠脊髓功能恢复。这些结果表明毛囊突起细胞促进周围神经损伤的恢复(22)。此二期应用的具体目的如下：(1)用流式细胞仪鉴定毛囊隆起区ND-GFP细胞，以鉴定和分类存在的细胞类型。(2)确定毛囊膨大细胞与免疫功能正常小鼠断端周围神经结合的最佳条件。(3)确定毛囊膨大细胞与免疫功能正常小鼠断端脊髓结合并赋予功能的最佳条件。这些目标将检验毛囊隆起细胞可以提供神经治疗性干细胞的现成来源的假设。人类毛囊隆起细胞将在第三阶段进一步表征和开发神经再生的治疗潜力。

DESCRIPTION (provided by applicant): The facile availability of pluripotent adult stem cells would have many important therapeutic applications. Our recent results suggest that the hair follicle is a promising source for such stem cells. Our discovery that the neural stem cell marker nestin is expressed in hair follicle bulge area cells, the site of hair-follicle stem cells (1), suggested that hair-follicle stem cells and neural stem cells have common features. We have demonstrated that the hair follicle gives rise to blood vessels in vivo with the blood vessels originating from the hair-follicle bulge cells (13). We have also demonstrated in vitro that hair-follicle bulge cells can be induced to form neurospheres, which in turn form neurons (21). These results suggest pluripotent hair follicle adult bulge cells could have important therapeutic applications, in particular for neurological diseases. This application utilizes transgenic mice with green fluorescent protein (GFP) under the control of the nestin regulatory sequences (nestin-driven [ND]-GFP) as the source of labeled hair follicle bulge cells. The hair follicle bulge area cells that we have isolated are positive for the stem cell marker CD34, as well as keratin 15-negative and beta-III-tubulin- negative, suggesting their relatively undifferentiated state. These cells can differentiate into glial cells, smooth muscle cells and keratinocytes as well as neurons in vitro. The severed sciatic nerve of C57BL/6 immunocompetent mice was transplanted with ND-GFP cells from the bulge by injection between the two severed regions of the nerve. The nerve was subsequently rejoined. Most of the transplanted GFP-expressing hair follicle bulge cells differentiated into GFAP-positive Schwann cells with myelin sheaths in the rejoined sciatic nerve. The rejoined sciatic nerve contracted the gastrocnemius muscle upon electrical stimulation. Walking print length and intermediate toe spread significantly recovered after transplantation of hair-follicle bulge cells between the severed tibial nerve indicating the transplanted mice recovered the ability to walk normally. Preliminary results indicate that ND-GFP bulge area cells can promote the functional rejoining of the severed spinal cord in mice. These results suggest that hair-follicle bulge cells promote the recovery of peripheral nerve injury (22). The specific aims of this Phase II application are as follows: (1) Characterize the hair follicle bulge area ND-GFP cells by FACS to identify and sort the cell types present. (2) Determine optimal conditions for hair-follicle bulge cells to rejoin and confer function to the severed peripheral nerves in immunocompetent mice. (3) Determine the optimal conditions of hair-follicle bulge cells to rejoin and confer function to the severed spinal cord in immunocompetent mice. These aims will test the hypothesis that hair follicle bulge cells can provide a readily available source of neurologically therapeutic stem cells. Human hair- follicle bulge cells will be further characterized and developed for therapeutic potential for nerve regeneration in Phase III.