Skin Stem Cells: Purification and Characterization

描述（由申请人提供）：本研究的总体目标是阐明哺乳动物皮肤组织稳态和再生的机制，并了解这一过程在人类疾病（包括癌症）中是如何出错的。实现这一目标的核心是皮肤内不同干细胞（SC）群体的纯化和表征以及确定它们对组织稳态和伤口修复的相对贡献。过去的AR 050452研究导致了毛囊（HF）隆突细胞的纯化，并将它们建立为在毛发循环和伤口修复中起作用的自我更新SC。这些HF-SC还显示出比滤泡间表皮（IFE）-SC更稳健的自我更新和更广泛的组织再生潜力。几个不同之处可以解释这一点。一个是像人类IFE-SC一样，小鼠HF-SC长时间处于静止状态。另一个原因是它们独特地表达转录因子，如Nfatc 1，当缺乏时，会导致HF-SC失去静止状态并持续循环HFs。过去的AR 050452研究为解决以下关键问题奠定了基础：（1）。HF-SC静止对它们的长期维护至关重要吗？当HF连续循环时会发生什么？他们是否会随着时间的推移而消耗SC？伤口能更快愈合吗？(2)控制HF-SC生态位静止期的分子机制是什么？Nfatc 1及其相关的转录调节因子如何维持HF-SC的静止？它的直接目标是什么？它们如何调节静止和长期自我更新之间的平衡？(3)什么样的分子变化决定了干性和过渡放大状态之间的区别？干细胞后代会影响这种转变吗？如果会，又是如何影响的？(4)IFE和HF SC对伤口修复的相对贡献是什么？这是否与浅表和深部伤口不同？在幼年小鼠与成年小鼠中。(5)IFE和HF SC如何改变基因表达以响应损伤？这些变化有多快，是什么控制了这个过程？为了回答这些问题，我们将使用FACS，RNA-seq，ChIP-seq，条件基因敲除和RNAi筛选，并采用这些方法在其天然，突变和伤口诱导的环境中探索皮肤干细胞。

DESCRIPTION (provided by applicant): The global objective of this research is to elucidate the mechanisms underlying tissue homeostasis and regeneration in mammalian skin and to understand how this process goes awry in human disorders, including cancers. Central to achieving this goal is the purification and characterization of the different stem cell (SC) populations within skin and determination of their relative contributions to tissue homeostasis and wound- repair. Past AR050452 research led to purification of hair follicle (HF) bulge cells, and established them as self- renewing SCs that function in hair cycling and wound-repair. These HF-SCs also displayed more robust self- renewing and broader tissue regenerative potential than interfollicular epidermal (IFE)-SCs. Several differences might account for this. One is that like human IFE-SCs, mouse HF-SCs exist in a quiescent state for prolonged periods. Another is that they uniquely express transcription factors such as Nfatc1, which when absent, causes HF-SCs to lose quiescence and cycle HFs continuously. Past AR050452 research has set the foundations to tackle the following key questions: (1). Is HF-SC quiescence critical to their long-term maintenance? What happens when HFs cycle continuously? Do they expend their SCs over time? Can they heal wounds faster? (2) What are the molecular mechanisms governing the resting phase of the HF-SC niche? How do Nfatc1 and its associated transcriptional regulators function to maintain HF-SC quiescence? What are its direct targets and how do they regulate the balance between quiescence and long-term self-renewal? (3) What molecular changes define the difference between stemness and the transit amplifying state? Do stem cell progeny influence this transition and if so how? (4) What are the relative contributions of IFE and HF SCs to wound-repair? Does this differ in superficial vs deep wounds? In young vs adult mice. (5) How do IFE and HF SCs alter gene expression in response to injury? How fast these changes and what are controls the process? To answer these questions, we'll use FACS, RNA-seq, ChIP-seq, conditional gene knockout and RNAi screens in vivo and employ these methods to explore skin stem cells in their native, mutant and wound-induced environments.