CORE RESOURCE: BLOOD AND BONE MARROW COLECTION CORE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。这些细胞被称为集落形成成纤维细胞、间充质干细胞或骨髓基质细胞(MSCs)，由于其作为多潜能干细胞的生物学特性以及在细胞和基因治疗方面的潜在用途而受到越来越多的关注。然而，该领域的一个主要问题是，在培养中分离和扩增细胞的可重复条件尚未定义。本建议的总体目的是根据我们实验室最近的三项观察结果，为其在培养中分离和扩增hMSCs创造有效的条件：1)我们已经开发出hMSCs可以扩增108倍以上而不会显著丧失复制能力的培养条件；2)我们发现hMSCs在培养物中的复制是受调控的自分泌/旁分泌因子；3)我们已经在hMSCs的培养物中鉴定出具有高度复制能力的小颗粒细胞亚群，并且似乎是该培养物中最早的祖细胞。该方案的具体目标是：L)确定我们在108倍扩增的hMSCs获得的高复制细胞是否保持其向成骨细胞、软骨细胞和脂肪细胞分化的多潜能；2)分离和鉴定在培养中调节hMSCs复制的分泌因子；3)分离我们在hMSCs培养中鉴定的小的、颗粒状的和高复制的细胞并确定它们的表面表位。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The cells have been variously referred to as colony-forming fibroblasts, mesenchymal stem cells, or marrow stromal cells (MSCs), and have been attracting increasing interest both for their biological properties as multipotential stem-like cells and their potential use for cell and gene therapy. However, a major problem in the field has been that reproducible conditions for isolation and expansion of the cells in culture has not been defined. The overall aim of the present proposal is to develop effective conditions for their isolation and expansion of human hMSCs in culture based on three recent observations made in our laboratory: 1) we have developed culture conditions whereby hMSCs can be expanded over 108-fold without significant loss in replicative capacity; 2) we have found that the replication of hMSCs in culture is regulated autocrine/paracrine factors that can be recovered from the culture medium; 3) we have identified a sub-population of small granular cells in cultures of hMSCs that are highly replicative and appear to be the earliest progenitors in the cultures. The specific aims of this protocol are: l) to determine whether the highly replicative cells we have obtained after 108-fold expansion of hMSCs retain their multiopotentiality to differentiate into osteoblasts, chondrocytes, and adipocytes; 2) isolate and characterize the secreted factors that regulate replication of hMSCs in culture; 3) isolate the small, granular, and highly replicative cells we have identified in cultures of hMSCs and define their surface epitopes.