Therapeutic hair follicle-derived neurospheres

治疗性毛囊源性神经球

• 批准号: 7275359
• 负责人: MENG YANG
• 金额: $ 37.45万
• 依托单位: ANTICANCER, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-11-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7275359
• 关键词: Adult; Area; Blood Vessels; CD34 gene; Cell Therapy; Cells; Condition; Contracts; Electric Stimulation; Gastrocnemius Muscle; Goals; Grant; Green Fluorescent Proteins; Hair follicle structure; Human; Immunocompetent; In Vitro; Injection of therapeutic agent; Keratin; Label; Length; Mus; Myelin Sheath; Natural regeneration; Nerve; Nerve Regeneration; Neuroglia; Neurologic; Neurons; Patients; Peripheral Nerves; Peripheral nerve injury; Phase; Population; Population Heterogeneity; Printing; Recovery; Schwann Cells; Site; Smooth Muscle Myocytes; Sorting - Cell Movement; Source; Spinal Cord; Stem cells; Structure of tibial nerve; Testing; Therapeutic; Toes; Transgenic Mice; Transgenic Organisms; Transplantation; Tubulin; Undifferentiated; Vascular Diseases; Walking; adult stem cell; angiogenesis; base; cell type; in vivo; keratinocyte; mouse model; nerve stem cell; nervous system disorder; nestin protein; pluripotency; sciatic nerve

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.

描述（由申请人提供）：多能成体干细胞的容易获得性将具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前途的来源。我们发现神经干细胞标记物巢蛋白在毛囊干细胞的部位--毛囊隆突区细胞中表达（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，血管起源于毛囊隆突细胞（13）。我们还在体外证明了毛囊隆突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，多能毛囊成体隆突细胞可能具有重要的治疗应用，特别是对神经系统疾病。本申请利用具有在巢蛋白调控序列（巢蛋白驱动的[ND]-GFP）控制下的绿色荧光蛋白（GFP）的转基因小鼠作为标记的毛囊隆突细胞的来源。我们分离的毛囊隆突区细胞对干细胞标志物CD 34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、角质形成细胞和神经元。通过在神经的两个切断区域之间注射，将来自隆突的ND-GFP细胞移植到C57 BL/6免疫活性小鼠的切断的坐骨神经。神经随后被重新连接。大多数移植的表达GFP的毛囊隆突细胞分化成GFAP阳性的雪旺细胞与髓鞘在重新连接的坐骨神经。电刺激后，重新连接的坐骨神经收缩腓肠肌。移植毛囊隆突细胞后，小鼠的足印长度和中间趾展度明显恢复，表明移植后小鼠恢复了正常行走的能力。初步结果表明，ND-GFP隆突区细胞可以促进小鼠脊髓的功能性重新连接。这些结果表明，毛囊隆突细胞促进周围神经损伤的恢复（22）。该II期申请的具体目的如下：（1）通过FACS表征毛囊隆起区域ND-GFP细胞以鉴定和分选存在的细胞类型。(2)确定毛囊隆突细胞重新连接的最佳条件，并赋予免疫活性小鼠切断的外周神经功能。(3)确定毛囊隆突细胞在免疫活性小鼠中重新连接并赋予切断的脊髓功能的最佳条件。这些目标将测试假设毛囊隆突细胞可以提供一个现成的神经治疗干细胞来源。人类毛囊隆突细胞将在第三阶段进一步表征和开发用于神经再生的治疗潜力。