CORE RESOURCE: BLOOD AND BONE MARROW COLECTION CORE

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。这些细胞被不同地称为集落形成成纤维细胞、间充质干细胞或骨髓基质细胞（MSC），并且由于其作为多能干细胞样细胞的生物学特性及其用于细胞和基因治疗的潜在用途而引起越来越多的兴趣。然而，该领域的一个主要问题是尚未确定用于分离和扩增培养物中的细胞的可再现条件。本发明的总体目标是基于我们实验室最近的三个观察结果，开发用于分离和扩增培养中的人hMSC的有效条件：1）我们已经开发了培养条件，由此hMSC可以扩增超过108倍而没有显著的复制能力损失; 2）我们发现培养的hMSCs的复制受自分泌/旁分泌因子的调节，这些因子可以从培养液中回收; 3）我们已经在hMSC的培养物中鉴定了小颗粒细胞的亚群，其是高度复制的，并且似乎是培养物中最早的祖细胞。本实验的具体目的是：1）确定我们在hMSCs扩增108倍后获得的高度复制的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性：2）分离和表征在培养中调节hMSCs复制的分泌因子; 3）分离我们在hMSC培养物中鉴定的小的、颗粒状的和高度复制的细胞，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.