Hair follicle nestin-expressing stem cells on a scaffold for nerve repair

支架上表达毛囊巢蛋白的干细胞用于神经修复

基本信息

批准号：
8780162
负责人：
Lingna Li
金额：
$ 22.47万
依托单位：
ANTICANCER, INC.
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-15 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8780162
关键词：
3-Dimensional Absorbable Gelatin Sponge Actins Axon Brain Stem Cell Cycle Cells Coculture Techniques Confocal Microscopy Ethics F-Actin Fiber Frequencies Ganglia Glial Fibrillary Acidic Protein Growth Growth Cones Hair follicle structure Human Image In Vitro Laboratories Locomotor Recovery Mus NGFR Protein Nerve Neuroglia Neurons Nude Mice Peripheral Nerves Phase Physiological Play Process Proliferating Regenerative Medicine Role Schwann Cells Source Spinal Cord Spinal cord injury Stem cell transplant Stem cells Transgenic Mice Transplantation Trigeminal nerve structure Tubulin Vibrissae afferent nerve cell type clinical application in vivo induced pluripotent stem cell injured injury and repair mouse model nestin protein promoter public health relevance red fluorescent protein repaired scaffold sciatic nerve spinal cord repair

项目摘要

DESCRIPTION (provided by applicant): Nestin-expressing stem cells of the hair follicle, discovered by our laboratory (Proc Natl Acad Sci USA 2003; 100:9658-61[1]), have been shown by our laboratory to be able to form neurons and other non-follicle cell types (Proc Natl Acad Sci USA 2005; 102:5530-4 [2]). Our laboratory has shown that the nestin-expressing stem cells from the hair follicle can effect repair of the peripheral nerve (Proc Natl Acad Sci USA 2005;102:17734-8 [3]) and spinal cord injury (Cell Cycle 2008; 7:1865-9 [4] and Cell Cycle 10, 830-839, 2011 [5]). Transgenic mice, in which the nestin promoter drives GFP (ND-GFP), were used to characterize the nestin-expressing hair follicle. The cells have very long processes extending from them as shown by confocal microscopy and differentiate into neuronal cells at high frequency as well as into multiple other non-hair follicle cells in vitro. The hair follicle sem cells differentiate into neuronal and glial cells after transplantation to the injured pheripheral nerve and spinal cord and enhance injury repair and locomotor recovery. In a recent study, vibrissa hair follicles, including their sensory nerve stump, were excised from ND-GFP transgenic mice and were placed in 3D histoculture supported by Gelfoam(R). ¿-III tubulin-positive fibers, consisting of ND-GFP- expressing cells extended up to 500 ¿m from the whisker nerve stump in histoculture. The growing fibers had growth cones on their tips expressing F-actin. These findings indicate that ¿-III tubulin-positive fibers elongating from the whisker follile sensory nerve stump were growing axons. The growing whisker sensory nerve was highly enriched in ND-GFP cells which appeared to play a major role in its elongation and interaction with other nerves in 3D Gelfoam(R) histoculture, including the sciatic nerve, the trigeminal nerve, and the trigeminal nerve ganglion. The results suggest a major function of the nestin-expressing stem cells in the hair follicle is for growth of the follicle sensory nerve (J. Cell. Biochem. 114, 1674-1684, 2013 [6], In Vitro Cell Dev Biol Anim 48:301-305, 2012 [7]). The nestin-expressing cells of the sciatic nerve were also found to be multipotent as the nestin-expressing cells in the hair follicle. When the nestin-expressing cells in the mouse sciatic nerve were cultured on Gelfoam and were imaged by confocal microscopy, they were observed forming fibers extending the nerve. The fibers consisted of ND-GFP-expressing spindle cells, which co-expressed the neuron marker b-III tubulin, the immature Schwann-cell marker p75NTR and TrkB which is associated with neurons. The fibers also contain nestin-negative spherical cells expressing GFAP, a Schwann-cell marker. The b-III tubulin-positive fibers had growth cones on their tips expressing ¿-actin, indicating they are growing axons. When the sciatic nerve from mice ubiquitously expressing red fluorescent protein (RFP) was co- cultured on Gelfoam with the sciatic nerve from ND-GFP transgenic mice, the interaction of nerves was observed. Proliferating nestin-expressing cells in the injured sciatic nerve were also observed in vivo (PLoS One 8(6), e67153, 2013 [8]). These results demonstrate that Gelfoam(R) is a powerful scaffolding for nerve growth effected by nestin-expressing stem cells. The hair follicle is the perfect source of the nestin-expressing stem cells due to easy access and a rich supply and lack of ethical or tumorgenicity issues of ES and iPS cells. In the present application, nestin-expressing cells hair follicles will be put in Gelfoam(R) histoculture to form growing nerves. The Gelfoam(R) will serve as a physiological scaffold of the growing nerve for transplantation and repair in mice with a severed sciatic nerve or injured spinal cord. The Specific Aims of the Phase I application are: 1. Develop Gelfoam(R) culture of nerves grown from hair follicle nestin-expressing stem cells for transplantation and rapid and effective repair of the severed sciatic nerve in mouse models. 2. Develop Gelfoam(R) cultures of nerves growth form hair follicle nestin-expressing stem cells for transplantation and rapid and effective spinal cord injury in mouse models. These Aims will be further developed in Phase II using human hair follicle for clinical application in Phase III as a safe, accessible and effective alternative to E and iPS cells for regenerative medicine.

描述（由申请人提供）：我们的实验室发现了表达巢蛋白的毛囊干细胞（Proc Natl Acad Sci USA 2003; 100:9658-61[1]），我们的实验室已证明能够形成神经元和其他非滤泡细胞类型（Proc Natl Acad Sci USA 2005；102:5530-4 [2]）。表明来自毛囊的表达巢蛋白的干细胞可以影响周围神经的修复（Proc Natl Acad Sci USA 2005;102:17734-8 [3]）和脊髓损伤（Cell Cycle 2008;7:1865-9） [4] 和 Cell Cycle 10, 830-839, 2011 [5]），其中巢蛋白启动子驱动 GFP。 (ND-GFP)，用于表征表达巢蛋白的毛囊，如共聚焦显微镜所示，这些细胞具有从其延伸的非常长的突起，并以高频率分化为神经细胞以及多种其他非毛囊细胞。在最近的一项研究中，毛囊干细胞移植到受伤的周围神经和脊髓后可分化为神经细胞和神经胶质细胞，并增强损伤修复和运动恢复。从 ND-GFP 转基因小鼠中切下毛囊，包括其感觉神经残端，并将其置于由 Gelfoam 支持的 3D 组织培养中。 -III 微管蛋白阳性纤维，由 ND-GFP 表达细胞组成，延伸至 500 ¿ m 来自组织培养中的须神经印记，生长的纤维在其尖端有表达 F-肌动蛋白的生长锥。 -从须毛囊感觉神经残端伸长的 III 微管蛋白阳性纤维正在生长轴突，生长中的须感觉神经在 ND-GFP 细胞中高度富集，这似乎在 3D 明胶海绵中的伸长和与其他神经的相互作用中发挥了重要作用。 R) 组织培养，包括坐骨神经、三叉神经和三叉神经节。结果表明表达巢蛋白的干细胞具有主要功能。毛囊中的巢蛋白用于毛囊感觉神经的生长（J. Cell. Biochem. 114, 1674-1684, 2013 [6], In Vitro Cell Dev Biol Anim 48:301-305, 2012 [7]）。还发现坐骨神经的表达细胞与毛囊中的巢蛋白表达细胞一样具有多能性。将小鼠坐骨神经中表达巢蛋白的细胞在明胶海绵上培养，并通过共聚焦显微镜成像，观察到它们形成延伸神经的纤维，这些纤维由表达ND-GFP的梭形细胞组成，其共表达神经元标记物b-。 III 微管蛋白、未成熟雪旺细胞标记 p75NTR 和 TrkB，与神经元相关。纤维还含有表达 GFAP 的巢蛋白阴性球形细胞。雪旺细胞标记物 b-III 微管蛋白阳性纤维的尖端有生长锥，表达 ¿ -肌动蛋白，表明它们正在生长轴突。当将普遍表达红色荧光蛋白（RFP）的小鼠的坐骨神经与ND-GFP转基因小鼠的坐骨神经共培养时，观察到神经增殖的相互作用。在体内也观察到了受伤坐骨神经中的表达细胞（PLoS One 8(6), e67153, 2013 [8]）。 Gelfoam(R) 是表达巢蛋白的干细胞影响神经生长的强大支架。由于 ES 易于获取、供应丰富且不存在伦理或致瘤性问题，因此毛囊是表达巢蛋白的干细胞的完美来源。在本申请中，表达巢蛋白的细胞毛囊将被放入Gelfoam 组织培养物中以形成生长的神经。用于移植和修复坐骨神经切断或脊髓损伤的小鼠的生长神经支架第一阶段应用的具体目标是： 1. 开发表达毛囊巢蛋白的干细胞生长的神经的 Gelfoam(R) 培养物。用于移植和快速有效的修复 2. 开发表达毛囊巢蛋白的干细胞的 Gelfoam(R) 神经生长培养物，用于移植和快速有效的脊髓。这些目标将在第二阶段进一步开发，在第三阶段使用人类毛囊进行临床应用，作为再生医学中 E 和 iPS 细胞的安全、易获得和有效的替代品。