Distinguishing adipose stromal vs. stem cells by serial transplantation

描述(由申请人提供)：再生医学和组织工程需要可获得的、充足的、可重复的细胞来源，以满足疗效和安全性要求。在过去的十年里，基础科学和临床研究人员将脂肪组织作为细胞来源；然而，关于脂肪来源细胞的功能特性仍然存在很大的争议。它们是“基质”细胞还是“干细胞”？这不仅仅是语义上的区别。一个真正的“干细胞”既有自我更新的能力，也有沿着多条谱系分化的能力。虽然脂肪来源的细胞已被证明在体外表现出这些特征，但关键的金标准实验，即体内功能脂肪库的连续移植和重建，尚未见文献报道。这项修订后的R21提案验证了我们的假设，即脂肪组织中含有成脂“干细胞”，既有由脂肪组织胶原酶消化而来的异质基质血管成分(SVF)细胞，也有相对均一、贴壁、培养扩增的脂肪来源基质/干细胞(ASC)。目的1采用C57BL/6-TG(UBC-GFP)30CHA/J小鼠模型作为易于追踪的GFP+脂肪来源的SVF细胞或ASCs的来源。流式细胞仪分选后的细胞将种植到丝质支架上，并植入野生型C57BL/6受体体内。4周后，取材，连续重复移植过程。结果参数将通过流式细胞术、组织化学、共聚焦显微镜、扫描电子显微镜和克隆形成单位分析进行监测和量化。连续移植完整的脂肪组织将作为阳性对照。目的2将使用流式细胞术分选的人SVF细胞和移植到免疫缺陷小鼠的ASC进行对比研究。我们预计，脂肪组织将被证明含有干细胞群体，能够在连续移植后重建完整的脂肪垫。 与公共卫生相关：这项提议的结果将通过确定脂肪组织等实体器官是否包含与造血干细胞具有相同特征的干细胞群体，从而加强我们在基础科学水平上对干细胞生物学的理解。造血干细胞是公认的成人干细胞的“黄金标准”。此外，研究结果将在转化/临床层面对再生医疗战略、方法和最佳实践产生深远影响。

DESCRIPTION (provided by applicant): Regenerative medicine and tissue engineering require an accessible, abundant, and reproducible source of cells that fulfill efficacy and safety requirements. During the past decade, basic science and clinical investigators have turned to adipose tissue as a cell source; however, there remains substantial controversy concerning the functional identity of the adipose-derived cells. Are they "stromal" or "stem" cells? This is not simply a semantic distinction. A true "stem" cell has the ability both to self-renew and to differentiate along multiple lineage pathways. While adipose-derived cells have been demonstrated to display these feature in vitro, the critical "gold standard" experiment, serial transplantation and reconstitution of a functional adipose depot in vivo, has never been reported in the literature. This revised R21 proposal tests our hypothesis that adipose tissue contains adipogenic "stem" cells in both the heterogeneous stromal vascular fraction (SVF) cells derived by collagenase digest of the adipose tissue and the relatively homogeneous, adherent, culture expanded adipose-derived stromal/stem cells (ASC). Aim 1 will employ the C57BL/6-Tg (UBC-GFP) 30cha/J mouse model as a source of easily trackable GFP+ adipose derived SVF cells or ASCs. Flow cytometry sorted cells will be seeded onto silk scaffolds and implanted into wild type C57BL/6 recipients. After a 4 week period, the constructs will be harvested and the process of transplantation repeated serially. Outcome parameters will be monitored and quantified by flow cytometry, histochemistry, confocal microscopy, scanning electron microscopy, and colony forming unit assays. Serial transplantation of intact adipose tissue explants will serve as a positive control. Aim 2 will perform comparable studies using flow cytometry sorted human SVF cells and ASC transplanted into immunodeficient mice. We anticipate that adipose tissue will prove to contain a stem cell population capable of reconstituting an intact fat pad upon serial transplantation. PUBLIC HEALTH RELEVANCE: The outcomes of this proposal will enhance our understanding of stem cell biology at the basic science level by determining if a solid organ such as adipose tissue contains a stem cell population with equivalent features to those exhibited by hematopoietic stem cells, the recognized "gold standard" for adult stem cells. In addition, the outcomes will have far-reaching implications at the translational/clinical level with respect to regenerative medical strategies, approaches, and best-practices.