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

描述（由申请人提供）：我们实验室发现的毛囊表达巢蛋白的干细胞（Proc Natl Acad Sci USA 2003; 100:9658-61[1]），已经被我们的实验室证明能够形成神经元和其他非毛囊细胞类型（Proc Natl Acad Sci USA 2005; 102:5530-4[1]）。我们的实验室已经证明，来自毛囊的表达巢蛋白的干细胞可以修复周围神经（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]）。利用巢蛋白启动子驱动GFP （ND-GFP）的转基因小鼠来表征表达巢蛋白的毛囊。共聚焦显微镜显示，这些细胞具有非常长的突起，并在高频率下分化为神经元细胞，以及在体外分化为多种其他非毛囊细胞。毛囊电镜细胞移植到损伤的外周神经和脊髓后分化为神经元和胶质细胞，促进损伤修复和运动恢复。在最近的一项研究中，从ND-GFP转基因小鼠身上切除毛囊，包括感觉神经残端，并将其置于凝胶泡沫(R)支持的三维组织培养中。- iii微管蛋白阳性纤维，由表达ND-GFP的细胞组成，在组织培养中从须神经残端延伸至500米。生长的纤维顶端有表达f -肌动蛋白的生长锥。这些发现表明，从毛囊感觉神经残端伸长的¿-III微管蛋白阳性纤维是正在生长的轴突。生长中的须感觉神经高度富集ND-GFP细胞，在3D凝胶泡沫(R)组织培养中，ND-GFP细胞似乎在其伸长和与其他神经（包括坐骨神经、三叉神经和三叉神经节）的相互作用中起主要作用。结果表明，毛囊中表达巢蛋白的干细胞的主要功能是促进毛囊感觉神经的生长。中国生物医学工程学报，2013,31(2):551 - 551。坐骨神经巢蛋白表达细胞与毛囊巢蛋白表达细胞一样具有多能性。将表达巢蛋白的小鼠坐骨神经细胞在明胶泡沫塑料上培养，共聚焦显微镜下观察到它们形成了延伸神经的纤维。这些纤维由表达nd - gfp的梭形细胞组成，梭形细胞共表达神经元标志物b-III微管蛋白、未成熟雪旺细胞标志物p75NTR和与神经元相关的TrkB。纤维中还含有表达GFAP（雪旺细胞标志物）的巢蛋白阴性球形细胞。b-III微管蛋白阳性纤维的尖端有表达-actin的生长锥，表明它们是生长轴突。将普遍表达红色荧光蛋白（RFP）的小鼠坐骨神经与ND-GFP转基因小鼠坐骨神经在明胶泡沫上共培养，观察神经间的相互作用。在体内也观察到表达巢蛋白的细胞在损伤的坐骨神经中增殖（PLoS One 8(6), e67153, 2013[8]）。这些结果表明明胶泡沫(R)是一个强大的支架神经生长影响巢表达干细胞。毛囊是巢表达干细胞的完美来源，因为容易获得，供应丰富，缺乏胚胎干细胞和诱导多能干细胞的伦理或肿瘤性问题。在本研究中，将巢蛋白表达细胞毛囊置于凝胶泡沫(R)组织培养中形成生长的神经。明胶泡沫(R)将作为正在生长的神经的生理支架，用于移植和修复被切断的坐骨神经或脊髓损伤的小鼠。第一阶段申请的具体目标是：1。利用表达毛囊巢蛋白的干细胞培养明胶泡沫(R)神经，用于移植和快速有效修复

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.