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一个可获得的，丰富的，可再生的细胞来源，满足疗效和安全性要求。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，仍然存在大量的争议有关的脂肪来源的细胞的功能特性。它们是“基质”细胞还是“干”细胞？这不仅仅是语义上的区别。真正的“干”细胞具有自我更新和沿沿着多个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这些功能，关键的“金标准”实验，连续移植和重建的功能性脂肪库在体内，从来没有在文献中报道。这一修订的R21提案检验了我们的假设，即脂肪组织包含脂肪形成“干”细胞，其来源于脂肪组织的胶原酶消化的异质基质血管部分（SVF）细胞和相对同质的、粘附的、培养扩增的脂肪来源的基质/干细胞（ASC）。目的1将采用C57 BL/6-Tg（UBC-GFP）30 cha/J小鼠模型作为容易追踪的GFP+脂肪来源的SVF细胞或ASC的来源。将流式细胞术分选的细胞接种到丝支架上并植入野生型C57 BL/6受体中。4周后，收获构建体并连续重复移植过程。将通过流式细胞术、组织化学、共聚焦显微镜、扫描电子显微镜和集落形成单位测定监测和定量结局参数。完整脂肪组织外植体的连续移植将用作阳性对照。目的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可比性研究。我们预期脂肪组织将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。

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.