Skin Stem Cells: Purification and Characterization

描述(申请人提供)：干细胞(SCs)是组织修复和动态平衡的天然单位。它们的多功能性为组织再生提供了希望，尤其是对争议较小的成年干细胞。成体皮肤上皮干细胞位于毛囊间表皮(IFE)的最内层(基底层)，以及毛囊(HF)内的突起。基本的IFE SCs在正常内稳态下发挥作用，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在动态平衡中起作用，以促进头发生长和HF的周期性再生和退化。在损伤期间，HF-SCs与基础IFE SCs一起参与修复受损的表皮和皮脂腺。无论是基本的IFE干细胞还是HF-SCs，都可以在保留其茎类特征的条件下，在其原生生态位之外长期培养和繁殖。角质形成细胞干细胞的这一显著特征使其在一系列临床应用和问题上具有重要意义，从基因治疗到烧伤手术、毛发置换和治疗角膜退化引起的失明。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小鼠干细胞的慢循环群体，使其脱离其自然生态位。在过去的一段时间里，我们重点研究了HF标签保留细胞(LRC)，表明它们具有干细胞的特性，并经历自我更新，在循环过程中补充HFs，并修复皮脂腺和IFE，以应对其常驻祖细胞的损伤/丧失。我们发现HF-SCs表达一组转录因子--Sox9、LHX2、NFATc1和Tcf3/4，而基本的IFE-SCs不表达这些转录因子，我们使用这些标记来追踪HF-SCs的起源到胚胎发生。我们还发现，Sox9不仅标志着这些早期的干细胞，而且在体内和体外都对它们作为HF-SCs的维持是必不可少的。我们还表明，与成人HF-SCs只能暂时修复表皮伤口不同，新生儿HF-SCs提供了强大和长期的伤口修复。这一点特别耐人寻味，因为在衰老过程中，HFs的静止期增加，IFE变得更薄，失去了增殖能力。我们新发现的随着时间推移跟踪和监测干细胞的能力，现在为探索与年龄相关的SC电位下降的分子基础提供了一个环节。此外，我们的发现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？它们如何从力学上解释衰老过程中发生的HF-SCs的生物学差异？(2)IFE基底层中的成人SCs是否也像30年前假设的那样慢周期，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？基础IFE干细胞与HF-SCs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能中？(3)定义基础IFE和HF-SCs的差异是内在的还是外在的？这两个新生干细胞群体都可以在体外培养和繁殖--它们在体外是保持它们的分子和组织再生差异，还是培养的细胞汇聚到一个共同的基因表达和组织再生潜力程序中？(4)在体内和体外，在HF-SCs或基础IFE SCs中优先上调/下调表达的1%小鼠基因组中，哪些基因对SCs的这些特征具有重要功能？哪些基因调节自我更新和/或长期潜力？是什么使干细胞保持在未分化的状态？我们能否利用通过优化皮肤上皮干细胞在培养中长期保持的能力而获得的信息，而不会失去它们的干性特征？通过解决这些问题，我们希望发现新的见解，以了解皮肤干细胞如何拥有和保持其增殖/组织再生能力，并保持在未分化状态，直到动员起来生成组织。这项研究是确定皮肤干细胞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.