Cryopreservation of the Hair Follicle Maintains Pluripotency of Nestin-Expressing Hair Follicle-Associated Pluripotent Stem Cells
Cryopreservation of the Hair Follicle Maintains Pluripotency of Nestin-Expressing Hair Follicle-Associated Pluripotent Stem Cells
复制标题
DOI：
10.1089/ten.tec.2014.0500
复制
发表时间：
2015-08-01
影响因子：
3
通讯作者：
Amoh, Yasuyuki
中科院分区：
文献类型：
--
作者：
Kajiura, Satoshi;Mii, Sumiyuki;Amoh, Yasuyuki
毛囊含有表达巢蛋白的多能干细胞，其起源在隆突区域上方，皮脂腺下方。我们将这些细胞称为毛囊相关多能干细胞（HAP）。在本研究中，我们建立了高效的毛囊冷冻保存方法，以保持HAP干细胞的多能性。我们将绿色荧光蛋白转基因小鼠的整个毛囊在TC-Protector培养基中缓慢冷却并储存在液氮中进行冷冻保存。解冻后，分离毛囊的上部并在含有胎牛血清（FBS）的Dulbecco改良Eagle培养基（DMEM）中培养。培养4周后，毛囊上部的细胞长出。将生长的细胞转移到不含FBS的DMEM/F12中。培养1周后，生长中的细胞形成毛球，每个毛球含有约1x 10（2）个HAP干细胞。毛球中含有分化为神经元、神经胶质细胞和其他细胞类型的细胞。解冻和培养的毛囊上部产生的多能性毛球几乎与新鲜毛囊一样多。从缓慢冷却的冷冻保存的毛囊中提取的毛球与从新鲜毛囊中提取的毛球一样具有多能性。相比之下，快速冷却（玻璃化）冷冻保存毛囊干细胞的多能性较差。干细胞标记基因（巢蛋白，Sox 2和SSEA-1）在慢速冷却冷冻保存的卵泡中高度表达，解冻后，在新鲜卵泡中。而在玻璃化冷冻保存的卵泡中，干细胞标志基因的表达量明显降低。通过快速冷却或缓慢冷却方法直接冷冻保存毛球导致多能性丧失。结果表明，整个毛囊的慢速冷冻保存是有效的保存HAP干细胞。储存的HAP干细胞将在个性化再生医学中非常有用，使任何个人都能够维持一个多能干细胞库以供未来临床使用。
Hair follicles contain nestin-expressing pluripotent stem cells, the origin of which is above the bulge area, below the sebaceous gland. We have termed these cells hair follicle-associated pluripotent (HAP) stem cells. In the present study, we established efficient cryopreservation methods of the hair follicle that maintained the pluripotency of HAP stem cells. We cryopreserved the whole hair follicle from green fluorescent protein transgenic mice by slow-rate cooling in TC-Protector medium and storage in liquid nitrogen. After thawing, the upper part of the hair follicle was isolated and cultured in Dulbecco's Modified Eagle's Medium (DMEM) with fetal bovine serum (FBS). After 4 weeks of culture, cells from the upper part of the hair follicle grew out. The growing cells were transferred to DMEM/F12 without FBS. After 1 week of culture, the growing cells formed hair spheres, each containing approximate to 1x10(2) HAP stem cells. The hair spheres contained cells that differentiated to neurons, glial cells, and other cell types. The thawed and cultured upper part of the hair follicle produced almost as many pluripotent hair spheres as fresh follicles. The hair spheres derived from slow-cooling cryopreserved hair follicles were as pluripotent as hair spheres from fresh hair follicles. In contrast, rapid-cooling (vitrification) cryopreservation poorly preserved the pluripotency of the hair follicle stem cells. Stem cell marker genes (nestin, Sox2, and SSEA-1) were as highly expressed in slow-rate cooled cryopreserved follicles, after thawing, as in fresh follicles. However, in the vitrification cryopreserved follicles, the expression of the stem cell marker genes was greatly reduced. Direct cryopreservation of hair spheres by either the rapid-cooling, or slow-cooling method, resulted in loss of pluripotency. These results suggest that the slow-rate cooling cryopreservation of the whole hair follicle is effective to store HAP stem cells. Stored HAP stem cells would be very useful in personalized regenerative medicine, enabling any individual to maintain a bank of pluripotent stem cells for future clinical use.