成人从人体皮肤的上皮干细胞的分离和培养
Summary
隔离可行的成人上皮干细胞的人体皮肤快速，可靠的方法是描述。该方法利用酶消化，皮肤胶原质基质，毛囊和隔离的单细胞悬液或细胞培养的组织碎片采摘。
Abstract
所有组织自我更新的动态平衡，是依赖于成人干细胞。由于未分化的干细胞进行不对称分裂，它们所产生的子细胞，保留干细胞表型和过境扩增细胞（助教细胞），移植的干细胞小生境，进行快速增殖和晚期分化重新填充组织。
在表皮，毛囊和肠上皮干细胞已被确定为细胞在体外增殖潜能高，慢骑自行车的标签保留细胞在 体内 1-3 。成年后，组织特异性干细胞再生的组织在其居住，在正常的生理营业额，以及在压力4-5倍。此外，干细胞一般认为是多有力，拥有的能力上升到多种类型的细胞内组织6。例如，啮齿类动物毛囊干细胞可以生成表皮，皮脂腺和毛囊7-9。我们已经表明，从人类毛囊隆起地区干细胞表现出多潜能 10 。
干细胞已经成为在生物医学研究的宝贵工具，由于其作为体外研究发育生物学，分化，肿瘤的发生和其可能的治疗用途的系统实用工具。成人上皮干细胞很可能将在如外胚层发育不良，monilethrix，瑟顿综合征，Menkes病，遗传性大疱性表皮松解症和alopecias 11-13等疾病的治疗非常有用。此外，如烧伤，慢性伤口和溃疡等皮肤问题，将有利于干细胞相关治疗 14,15 。鉴于潜在的成年细胞重新编程为国家多能干（iPS细胞）16,17，可以提供方便和可扩展的成人干细胞在人体皮肤细胞诱导和下游治疗的疾病范围广泛，包括一个非常宝贵的资源糖尿病和帕金森氏病。
Protocol
1。从人体皮肤中提取的上皮干细胞开始之前隔离上皮干细胞的过程，需要准备各自的媒体和试剂（见表1）。 收集新鲜成人换装程序或打孔活检人类头皮，然后孵化的DMEM / 10％FBS / Dispase（4毫克/毫升）一夜之间在4 ° C。潜伏期为2-4小时，在37 ° C也是有效的。皮肤件应为1厘米，最大宽度允许酶渗透。 消毒的培养皿转移到皮肤，从皮肤拉断每个头发抓住附近的皮肤表面发干，?…
Discussion
细胞的提取和培养方法是惊人的轻便和可重复性。我们产生了几十个人在广泛的年龄范围，包括遗传性皮肤缺损18患者的上皮干细胞培养。最好是组织收获的一天开始的过程，但是细胞会继续生存在冰上媒体好几天了，如果需要，促进通宵航运。弃置换装皮肤产生数百个可行的毛囊细胞提取单细胞悬液。对于更有限的组织，如冲床从头皮活检，，外植体的文化可能会更有效，因为细胞胰酶…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由NIH / NCI资助R01CA – 118916
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| DMEM | GIBCO | 11995 | ||
| Hams F12 | GIBCO | 11765 | ||
| Fetal Bovine Serum (FBS) | GIBCO | 16000 | ||
| Insulin | GIBCO | 12585 | ||
| T3 | Sigma | T-2752 | ||
| Transferrin | Roche | 10652202001 | ||
| Hydrocortisone | Sigma | H-4001 | ||
| Cholera Toxin | Sigma | C8052 | ||
| Epidermal growth factor (EGF) | Sigma | E-9644 | ||
| Adenine | Sigma | A9795 | ||
| Trypsin(10X) | GIBCO | 15090 | ||
| VERSENE | GIBCO | 15040 | ||
| G418 Sulfate | Cellgro | 30-234-CR | ||
| Hanks’ Balanced Salt solution | Sigma | H6648 | ||
| 1X PBS | Cellgro | 21-040-CV | ||
| Mitomycin C | Roche | 10107409001 | ||
| Penicillin/streptomycin | Invitrogen | 15140122 | ||
| Dispase | Invitrogen | 17105 | ||
| Crystal Violet | Fisher | C581-25 |
Keratinocyte media (KCM)
[DMEM and Ham s F12 (GIBCO, 3:1), adenine (Sigma, 180 mM), 10% fetal bovine serum (GIBCO), cholera toxin (ICN, 0.1 nM), penicillin/streptomycin (GIBCO, 100 U/ml and 100 mg/ml, respectively), hydrocortisone (Sigma, 0.4 mg/ml, 1.1 mM), T/T3 (transferrin, GIBCO, 5 μg/ml, 649 nM; and triiodo-l-thyronine, Sigma, 2 nM), insulin (Sigma, 5 mg/ml, 862 nM), and EGF (Sigma, 10 ng/ml, 1.6 nM), pH 7.2]
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Tags
IsolationCultureAdult Epithelial Stem CellsHuman SkinHomeostasisAsymmetric DivisionsDaughter CellsStem Cell PhenotypeTransit-amplifying CellsStem Cell NicheRapid ProliferationTerminal DifferentiationEpidermisHair FollicleIntestineIn Vitro Proliferative PotentialLabel-retaining CellsMulti-potent Stem CellsRodent Hair Follicle Stem CellsEpidermisSebaceous GlandsHair FolliclesHuman Hair Follicle Bulge RegionMulti-potentialityBiomedical Research발생학DifferentiationTumorigenesisTherapeutic Utility
Cite This Article
Guo, Z., Draheim, K., Lyle, S. Isolation and Culture of Adult Epithelial Stem Cells from Human Skin. J. Vis. Exp. (49), e2561, doi:10.3791/2561 (2011).