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、丰富的、可再生的、满足有效性和安全性要求的细胞来源。在过去的十年中，基础科学和临床研究人员已经转向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”具有自我更新和沿着多种谱系途径分化的能力。虽然脂肪来源的细胞已被证明在体外显示出这些特征，但关键的“金标准”实验，体内功能性脂肪库的连续移植和重建，从未在文献中报道过。修订后的R21提案验证了我们的假设，即脂肪组织中含有脂肪“干细胞”，包括由脂肪组织胶原酶消化产生的异质基质血管部分（SVF）细胞和相对均匀的、粘附的、培养的扩展脂肪来源基质/干细胞（ASC）。目的1将采用C57BL/6-Tg (UBC-GFP) 30cha/J小鼠模型作为易于追踪的GFP+脂肪源性SVF细胞或ASCs的来源。流式细胞术分选的细胞将被播种到真丝支架上，植入野生型C57BL/6受体。4周后，收获构建体，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、扫描电子显微镜和菌落形成单位测定来监测和量化。连续移植完整的脂肪组织作为阳性对照。Aim 2将使用流式细胞术分选的人SVF细胞和ASC移植到免疫缺陷小鼠中进行比较研究。我们预计，脂肪组织将被证明含有干细胞群能够重建一个完整的脂肪垫系列移植。

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.