Skin Stem Cells: Purification and Characterization

皮肤干细胞：纯化和表征

• 批准号: 8461613
• 负责人: ELAINE FUCHS
• 金额: $ 34.33万
• 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/8461613
• 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 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.

描述（由申请人提供）：干细胞（SC）是组织修复和体内平衡的天然单位。它们的多功能性为组织再生提供了希望，特别是对于争议较少的成体SC。皮肤的成体上皮SC位于毛囊间表皮（IFE）的最内层（基底层）以及凸起（位于毛囊（HF）内的小生境）内。基础IFE SC在正常稳态中起作用，以形成终末分化细胞的分层上皮屏障，所述终末分化细胞不断地从身体表面脱落。HF-SC在体内平衡中起作用以促进毛发生长以及HF的再生和退化的周期性发作。在损伤期间，HF-SC与基底IFE SC一起参与修复受损的表皮和皮脂腺。基础IFE SC和HF-SC都可以在保留其茎样特征的条件下在其天然小生境外长期培养和繁殖。角质形成细胞SC的这一显著特征使得它们对于一系列临床应用和问题具有根本重要性，所述临床应用和问题的范围从基因治疗到烧伤手术、毛发替换和治疗由角膜变性引起的失明。在过去的资助期内，我们开发了一种标记和纯化成年小鼠干细胞的慢循环群体的策略。在最后一个时期，我们专注于HF标记保留细胞（LRC），表明它们具有SC的特性并进行自我更新，在循环期间补充HF，并修复皮脂腺和IFE以应对其常驻祖细胞的损伤/损失。我们发现HF-SC表达一组转录因子-Sox 9，Lhx 2，NFATc 1和Tcf 3/4-而基础IFE SC不表达，并且我们使用这些标记来追踪HF-SC的起源到胚胎发生。我们还表明，Sox 9不仅标记这些早期SC，而且对于它们在体内和体外维持HF-SC是必不可少的。我们还表明，与仅短暂修复表皮伤口的成人HF-SC相比，新生儿HF-SC提供了稳健和长期的伤口修复。这是特别有趣的，因为在衰老过程中，HF的静息期增加，IFE变得更薄并失去增殖潜力。我们新发现的跟踪和监测SC随时间推移的能力，现在提供了一个探索与年龄相关的SC潜力下降的分子基础。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HF-SCs在衰老过程中的生物学差异？(2)IFE基底层内的成体SC是否也像30年前假设的那样缓慢循环，或者这些细胞是否反映了衰老过程？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 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上？(3)定义基础IFE和HF-SC的差异是内在的还是外在的？这两个新生SC群体都可以在体外培养和繁殖--它们在体外保持其分子和组织再生差异，还是培养的细胞在基因表达和组织再生潜力的共同程序上聚集？(4)在体内和体外HF-SC或基础IFE SC中表达优先上调/下调的1%小鼠基因组中，哪些基因对SC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？是什么维持了SC的未分化状态？我们能否利用通过优化皮肤上皮干细胞在培养中长期维持而不丧失其干性特征的能力所获得的信息？通过解决这些问题，我们希望揭示新的见解，了解皮肤SC如何拥有和保持其增殖/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干细胞在烧伤治疗之外的再生治疗中的潜力的先决条件。