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，毛囊是这种干细胞的一个有希望的来源。我们发现神经干细胞标记物nestin在毛囊膨出区细胞（毛囊干细胞的位置）中表达(1)，这表明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，血管起源于毛囊膨出细胞（13）。我们还在体外证明毛囊膨出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，多能性毛囊成人隆起细胞可能具有重要的治疗应用，特别是对神经系统疾病。本应用利用巢蛋白调控序列（巢蛋白驱动[ND]-GFP）控制下的绿色荧光蛋白（GFP）转基因小鼠作为标记毛囊膨出细胞的来源。我们分离的毛囊膨出区细胞干细胞标记CD34阳性，角蛋白15阴性和β - iii -微管蛋白阴性，表明它们处于相对未分化状态。这些细胞在体外可分化为神经胶质细胞、平滑肌细胞、角化细胞和神经元。在C57BL/6免疫正常小鼠的坐骨神经断块上注射ND-GFP细胞。神经随后被重新连接。移植后表达gfp的毛囊膨出细胞在再接合的坐骨神经中大部分分化为gfp阳性的髓鞘雪旺细胞。再连接的坐骨神经在电刺激下收缩腓肠肌。毛囊膨出细胞移植于断胫神经后，行走印长和中间趾宽明显恢复，表明移植小鼠恢复了正常行走的能力。初步结果表明，ND-GFP膨出区细胞能促进小鼠断脊髓的功能性再连接。这些结果表明毛囊膨出细胞促进周围神经损伤的恢复（22）。该II期应用的具体目的如下：(1)通过FACS表征毛囊膨出区ND-GFP细胞，以鉴定和分类存在的细胞类型。(2)在免疫功能正常的小鼠中，确定毛囊膨出细胞重新连接并赋予其周围神经功能的最佳条件。(3)确定毛囊膨出细胞重新连接并赋予免疫功能小鼠脊髓功能的最佳条件。这些目标将验证毛囊膨出细胞可以提供神经治疗干细胞的现成来源的假设。人类毛囊膨出细胞将在III期进一步表征和开发用于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.