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

描述(申请人提供)：我们实验室发现的毛囊中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 2005；102：5530-4[2])。我们的实验室已经证明，毛囊中表达巢蛋白的干细胞可以影响周围神经的修复(美国学报，2005年；102：17734-8[3])和脊髓损伤(细胞周期2008；7：1865-9[4]和细胞周期10,830-839,2011年[5])。用Nestin启动子驱动绿色荧光蛋白(ND-GFP)的转基因小鼠来鉴定表达Nestin的毛囊。共聚焦显微镜显示，这些细胞有非常长的突起，并在体外分化为神经细胞和多个其他非毛囊细胞。毛囊sem细胞移植到损伤的周围神经和脊髓后分化为神经元和神经胶质细胞，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了触须毛囊，包括它们的感觉神经残端，并将其置于明胶海绵(R)支持的3D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？m。生长纤维的顶端有表达F-肌动蛋白的生长锥体。这些结果表明，从胡须状叶状感觉神经残端延伸出来的-III微管蛋白阳性纤维正在生长轴突。生长中的胡须感觉神经高度富含ND-GFP细胞，在3D Gelfoam(R)组织培养中，ND-GFP细胞似乎在其伸长和与其他神经的相互作用中发挥主要作用，包括坐骨神经、三叉神经和三叉神经节。结果表明，毛囊中表达巢蛋白的干细胞的主要功能是毛囊感觉神经的生长(J.生物化学。114,1674-1684,2013[6]，体外细胞开发生物动画48：301-305,2012[7])。与毛囊中表达巢蛋白的细胞一样，坐骨神经中表达巢蛋白的细胞也具有多能性。将小鼠坐骨神经中表达Nestin的细胞培养在明胶海绵上，并用共聚焦显微镜成像，观察到它们形成了延伸神经的纤维。这些纤维由表达ND-GFP的纺锤形细胞组成，共同表达神经元标记物b-III微管蛋白、未成熟雪旺细胞标记物p75NTR和与神经元相关的TrkB。这些纤维还含有巢蛋白阴性的球形细胞，表达雪旺细胞标记物GFAP。B-III微管蛋白阳性纤维的顶端有表达肌动蛋白的生长锥体，这表明它们正在生长轴突。将普遍表达红色荧光蛋白(RFP)的小鼠坐骨神经与ND-GFP转基因小鼠的坐骨神经在明胶海绵上共培养，观察神经之间的相互作用。在损伤的坐骨神经中也观察到Nestin表达细胞的增殖(PLoS One 8(6)，e67153,2013[8])。这些结果表明，明胶海绵(注册商标)是一种强大的支架，可以通过表达巢蛋白的干细胞影响神经生长。毛囊是表达巢蛋白的干细胞的完美来源，因为它容易获得，供应丰富，而且缺乏ES和iPS细胞的伦理或致瘤性问题。在目前的应用中，表达巢蛋白的细胞毛囊将被置于明胶海绵(注册商标)组织培养中，以形成生长的神经。明胶海绵(R)将作为生长神经的生理支架，用于移植和修复坐骨神经切断或脊髓损伤的小鼠。第一阶段应用的具体目标是：1.发展从毛囊表达巢蛋白的干细胞长出的神经的明胶海绵(R)培养，用于移植和快速有效的修复 在小鼠模型中观察切断的坐骨神经。2.从毛囊表达巢蛋白的干细胞中培养神经生长的明胶海绵(R)，用于移植和快速有效的脊髓 小鼠脊髓损伤模型。这些目标将在第二阶段进一步发展，在第三阶段将人毛囊用于临床应用，作为再生医学中E和iPS细胞的安全、可获得和有效的替代品。

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.