Skin Stem Cells: Purification and Characterization

皮肤干细胞：纯化和表征

• 批准号: 8257868
• 负责人: ELAINE FUCHS
• 金额: $ 36.14万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-26 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8257868
• 关键词: Address; Adult; Advanced Malignant Neoplasm; Age; Aging; Aging-Related Process; Basal Cell; Behavior; Biological; Biological Assay; Blindness; Body Surface; Burn injury; Cancerous; Cell Culture Techniques; Cell Cycle; Cell Lineage; Cell Survival; Cell physiology; Cells; Characteristics; Clinical; Cornea; Cultured Cells; Data; Embryo; Embryonic Development; Engineering; Epidermis; Epithelial; Epithelium; Exhibits; Funding; Gene Expression; Genes; Genetic; Grant; Growth; Hair; Hair follicle structure; Health; Homeostasis; Human; In Situ Hybridization; In Vitro; Injury; Label; LacZ Genes; Learning; Maintenance; Measures; Modeling; Molecular; Monitor; Morphogenesis; Mus; Natural regeneration; Neonatal; Pathway interactions; Physiologic pulse; Physiological; Population; Production; Property; Regenerative Medicine; Relative (related person); Research; Resting Phase; Rodent; Sebaceous Glands; Skin; Skin Transplantation; Skin graft; Staging; Stem Cell Development; Stem cells; Stratum Basale; Testing; Time; Tissues; Undifferentiated; Validation; Wound Healing; adult stem cell; age related; base; cancer therapy; clinical application; comparative; epidermis cell; gene therapy; in vitro Assay; in vivo; insight; keratinocyte; mouse genome; operation; progenitor; programs; regenerative; regenerative therapy; repaired; response to injury; self-renewal; small hairpin RNA; stem cell niche; stem cell population; stemness; tissue regeneration; tissue repair; transcription factor

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. PUBLIC HEALTH RELEVANCE: Stem cells are natural units of tissue repair and homeostasis, and their versatility holds promise for tissue regeneration. This research focuses on deciphering the properties of embryonic and adult skin stem cells that exist within the basal layer of the epidermis and the bulge of the hair follicle. We will focus on delineating the intrinsic mechanisms that render skin SCs their ability to self-renew in vitro and in vivo and to select one of three differentiation pathways: epidermis, hair follicle and sebaceous gland. This study is a fundamental prerequisite to ascertaining the potential of skin stem cells for regenerative therapies that go beyond burn treatments.

描述（由申请人提供）：干细胞（SCS）是组织修复和稳态的天然单位。它们的多功能性对组织再生有望，特别是对于有争议的成人SC。皮肤的成年上皮SC居住在毛囊毛囊（HF）内的一个小裂（HF）的膨胀表皮（IFE）的最内向（基底）层中。基础IFE SC在正常体内平衡中的功能，形成了经常从体面脱落的终末分化细胞的分层上皮屏障。 HF-SCS在体内平衡中的功能可以促进头发的生长以及HF再生和退化的周期性回合。在受伤期间，HF-SC与基础IFE SC一起修复受损的表皮和皮脂腺。在保留其干扰性特征的条件下，基础IFE SC和HF-SC都可以在本地壁nike以外培养和繁殖。角质形成细胞SC的这一非凡特征使它们对一系列临床应用和问题至关重要，从基因治疗到燃烧手术，替代头发以及因角膜变性引起的失明的治疗。在过去的赠款期间，我们制定了一项策略，以标记和净化其自然壁ni的成年鼠SC的慢循环种群。在最后一个时期，我们专注于HF标签固定细胞（LRC），表明它们具有SC的特性并在循环过程中进行自我更新，补充HF，并修复皮脂腺，并响应其居住祖细胞的损伤/丧失。我们表明，HF-SC表达一组转录因子-SOX9，LHX2，NFATC1和TCF3/4-基础IFE SC没有，我们使用这些标记来追踪HF-SCS的起源到胚胎发生。我们还表明，Sox9不仅标记了这些早期SC，而且对于它们的维持至关重要，因为它们是体内和体外的HF-SCS。我们还表明，与成年HF-SC相比，新生儿HF-SC可提供健壮且长期的伤口修复。这尤其引人入胜，因为在衰老期间，HF的静息阶段增加，IFE变得更薄并失去了增殖潜力。随着时间的推移，我们追踪和监视SC的新发现的能力现在为探索与年龄相关的SC潜力下降的分子基础提供了帮助。此外，我们的发现提出了以下关键问题：（1）。新生儿和成人HF-SC之间的分子差异是什么？如何从机械上解释衰老过程中HF-SC的生物学差异？ （2）IFE基础层中的成年SC是否也像3年前那样慢循环，还是这些细胞反映了衰老过程？基础IFE细胞的分子特征是什么？它们的胚胎起源是什么？基础IFE SC与HF-SC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中？ （3）定义基础IFE和HF-SC的差异是固有的还是外在的？可以在体外培养和传播两个新生儿SC种群 - 它们在体外保持分子和组织再生差异，还是培养的细胞会在基因表达和组织再生潜力的共同程序上融合？ （4）在1％的小鼠基因组中，其表达在体内和体外优先向上/下调的表达优先向上/下调了，这些基因对SCS的这些特征在功能上很重要？哪些基因调节自我更新和/或长期潜力？什么使SC保持在其未分化状态？我们可以通过优化皮肤上皮SC在培养中长期保持长期而不会失去其干性特征的能力来利用获得的信息？通过解决这些问题，我们希望发现新的见解，以了解皮肤SC如何拥有并保持其增殖/组织再生潜力，并保持在未分化的状态，直到动员起来生成组织。这项研究是确定皮肤SC对再生疗法的潜力的先决条件，这些疗法超出了燃烧治疗。公共卫生相关性：干细胞是组织修复和稳态的天然单位，它们的多功能性有望有望组织再生。这项研究的重点是破译表皮基底层和毛囊凸起的胚胎和成人皮肤干细胞的特性。我们将重点介绍使皮肤SC在体外和体内自我更新的能力以及选择三种分化途径之一的固有机制：表皮，毛囊和皮脂腺。这项研究是确定皮肤干细胞对再生疗法的潜力的基本先决条件。