Skin Stem Cells: Purification and Characterization

描述（由申请人提供）：干细胞（SCs）是组织修复和体内平衡的自然单位。它们的多功能性为组织再生带来了希望，特别是对于争议较少的成人SCs。成人皮肤上皮SCs位于毛囊间表皮（IFE）的最内层（基底层）以及毛囊（HF）内的凸起处。基础内皮细胞在正常的体内平衡中起作用，形成终末分化细胞的层状上皮屏障，这些细胞不断从体表脱落。HF- scs在体内平衡中发挥作用，促进头发生长和HF的再生和退化的周期性发作。在损伤过程中，HF-SCs与基底IFE -SCs一起修复受损的表皮和皮脂腺。基底内皮细胞和造血干细胞都可以在保留其茎样特征的条件下，在其原生生态位外长期培养和繁殖。角质细胞SCs的这一显著特征使其在一系列临床应用和问题中具有重要意义，从基因治疗到烧伤手术，毛发替代和治疗角膜变性引起的失明。在过去的资助期间，我们开发了一种策略，用于标记和纯化成年鼠SCs的自然生态位中的慢循环种群。在最后一段时间里，我们重点研究了HF标签保留细胞（lrc），表明它们具有SCs的特性，可以自我更新，在循环过程中补充HF，并修复皮脂腺和IFE，以应对其固有祖细胞的损伤/丧失。我们发现，HF-SCs表达一组转录因子——Sox9、Lhx2、NFATc1和Tcf3/4，而基础IFE细胞不表达这些转录因子，我们使用这些标记物来追踪HF-SCs的胚胎发生起源。我们还发现Sox9不仅标记了这些早期sc，而且在体内和体外都是维持其作为hf - sc的必要条件。我们还发现，与成人的HF-SCs只能短暂修复表皮伤口不同，新生儿的HF-SCs可以提供强大的、长期的伤口修复。这是特别有趣的，因为在衰老过程中，HFs的静止阶段增加，IFE变薄并失去增殖潜力。随着时间的推移，我们新发现的跟踪和监测SC的能力，现在为探索SC潜力与年龄相关的下降的分子基础提供了一个突破口。此外，我们的研究结果提出了以下关键问题：(1)。新生儿和成人HF-SCs之间的分子差异是什么？它们如何从机制上解释衰老过程中发生的HF-SCs的生物学差异？(2)内皮基底层的成年SCs是否也像30年前假设的那样缓慢循环，或者这些细胞是衰老过程的反映？基底IFE细胞的分子特征是什么？它们的胚胎起源是什么？基底内皮细胞与造血干细胞有何不同，这些差异是否反映在组织再生和长期自我更新的不同潜力上？(3)定义基础内皮细胞和造血干细胞的差异是内在的还是外在的？两种新生SC群体都可以在体外培养和繁殖——它们是在体外保持分子和组织再生的差异，还是培养的细胞在基因表达和组织再生潜力方面趋同？(4)小鼠基因组中有1%的基因在体内和体外的hf - sc或基础IFE - sc中优先表达上调/下调，哪些基因在功能上对sc的这些特征很重要？什么基因调节自我更新和/或长期潜能？是什么使sc保持未分化状态？我们能否利用优化皮肤上皮SCs在培养中长期维持而不失去其干性特征的能力所获得的信息？通过解决这些问题，我们期望发现新的见解，以了解皮肤SCs如何拥有和维持其增殖/组织再生潜力，并保持在未分化状态，直到动员产生组织。这项研究是确定皮肤SCs在烧伤治疗以外的再生治疗中的潜力的先决条件。

DESCRIPTION (provided by applicant): Stem cells (SCs) are natural units of tissue repair and homeostasis. Their versatility holds promise for tissue regeneration, particularly for less controversial adult SCs. Adult epithelial SCs of the skin reside within the innermost (basal) layer of the interfollicular epidermis (IFE) as well as the bulge, a niche located within the hair follicle (HF). Basal IFE SCs function in normal homeostasis to form the stratified epithelial barrier of terminally differentiated cells that are constantly shed from the body surface. HF-SCs function in homeostasis to fuel hair growth and the cyclical bouts of regeneration and degeneration of the HF. During injury, HF-SCs participate with basal IFE SCs to repair damaged epidermis and sebaceous glands. Both basal IFE SCs and HF-SCs can be cultured and propagated long-term outside their native niches under conditions where their stemlike features are retained. This remarkable feature of keratinocyte SCs renders them fundamentally important to a range of clinical applications and issues, ranging from gene therapy to burn operations, hair replacement and treatment of blindness arising from corneal degeneration. In the past grant period, we developed a strategy for marking & purifying the slow-cycling populations of adult murine SCs from their natural niches. In the last period, we focused on HF label retaining cells (LRCs), showing that they possess properties of SCs and undergo self-renewal, replenish HFs during cycling, and repair both sebaceous glands and IFE in response to injury/loss of their resident progenitors. We showed that HF-SCs express a group of transcription factors-- Sox9, Lhx2, NFATc1 & Tcf3/4--which basal IFE SCs do not, and we used these markers to trace the origins of HF-SCs to embryogenesis. We also showed that Sox9 not only marks these early SCs but also is essential for their maintenance as HF-SCs both in vivo and in vitro. We also showed that in contrast to adult HF-SCs, which only transiently repair epidermal wounds, neonatal HF-SCs provide robust and long-term wound repair. This is particularly intriguing in that during aging, the resting phase of HFs increases, and the IFE becomes thinner and loses proliferative potential. Our newfound ability to track and monitor SCs over time now affords a segue into exploring the molecular basis for the age-related decline in SC potential. In addition, our findings raise the following key questions: (1). What are the molecular differences between neonatal and adult HF-SCs and how can they explain mechanistically the biological differences in HF-SCs that occur during aging? (2) Are the adult SCs within the IFE basal layer also slow-cycling as postulated 3 decades ago, or are these cells a reflection of the aging process? What are the molecular characteristics of basal IFE cells and what are their embryonic origins? How do basal IFE SCs differ from HF-SCs and are these differences reflected in different potentials for tissue regeneration and long-term self-renewal? (3) Are the differences that define basal IFE and HF-SCs intrinsic or extrinsic? Both neonatal SC populations can be cultured and propagated in vitro--do they maintain their molecular and tissue regenerative differences in vitro or do the cultured cells converge upon a common program of gene expression and tissue regenerative potential? (4) Of the 1% of the mouse genome whose expression is preferentially up/downregulated in HF-SCs or basal IFE SCs in vivo and in vitro, which are the genes that are functionally important to these features of SCs? What genes regulate self-renewal and/or long term potential? What maintains SCs in their undifferentiated state? Can we exploit the information gained by optimizing the ability of skin epithelial SCs to be maintained long term in culture without losing features of their stemness? By addressing these issues, we expect to uncover new insights into understanding how skin SCs possess and maintain their proliferative/tissue regenerative potential and are kept in an undifferentiated state until mobilized to generate tissue. This research is prerequisite to ascertaining the potential of skin SCs for regenerative therapies that go beyond burn treatments.