Baddoo Melody, Hill Katy, Wilkinson Robin, Gaupp Dina, Hughes Catherine, Kopen Gene C, Phinney Donald G
Center for Gene Therapy, Tulane University Health Sciences Center, New Orleans, Louisiana 70112, USA.
J Cell Biochem. 2003 Aug 15;89(6):1235-49. doi: 10.1002/jcb.10594.
Mesenchymal stem cells (MSCs) are typically enriched from bone marrow via isolation of the plastic adherent, fibroblastoid cell fraction. However, plastic adherent cultures elaborated from murine bone marrow are an admixture of fibroblastoid and hematopoietic cell types. Here we report a reliable method based on immunodepletion to fractionate fibroblastoid cells from hematopoietic cells within plastic adherent murine marrow cultures. The immunodepleted cells expressed the antigens Sca-1, CD29, CD44, CD81, CD106, and the stem cell marker nucleostemin (NST) but not CD11b, CD31, CD34, CD45, CD48, CD90, CD117, CD135, or the transcription factor Oct-4. They were also capable of differentiating into adipocytes, chondrocytes, and osteoblasts in vitro as well as osteoblasts/osteocytes in vivo. Therefore, immunodepletion yields a cell population devoid of hematopoietic and endothelial cells that is phenotypically and functionally equivalent to MSCs. The immunodepleted cells exhibited a population doubling time of approximately 5-7 days in culture. Poor growth was due to the dramatic down regulation of many genes involved in cell proliferation and cell cycle progression as a result of immunodepletion. Exposure of immunodepleted cells to fibroblast growth factor 2 (FGF2) but not insulin-like growth factor (IGF), murine stem cell factor, or leukemia inhibitory factor (LIF) significantly increased their growth rate. Moreover, 82% of the transcripts down regulated by immunodepletion remain unaltered in the presence of FGF2. Exposure to the later also reversibly inhibited the ability of the immunodepleted cells to differentiate into adipocytes, chondrocytes, and osteoblasts in vitro. Therefore, FGF2 appears to function as a mitogen and self-maintenance factor for murine MSCs enriched from bone marrow by negative selection.
间充质干细胞（MSCs）通常通过分离贴壁生长的成纤维细胞样细胞组分从骨髓中富集得到。然而，源自小鼠骨髓的贴壁培养物是成纤维细胞样细胞和造血细胞类型的混合物。在此，我们报告一种基于免疫去除法的可靠方法，用于从小鼠骨髓贴壁培养物中的造血细胞中分选成纤维细胞样细胞。免疫去除后的细胞表达抗原Sca-1、CD29、CD44、CD81、CD106以及干细胞标志物核干细胞因子（NST），但不表达CD11b、CD31、CD34、CD45、CD48、CD90、CD117、CD135或转录因子Oct-4。它们在体外还能够分化为脂肪细胞、软骨细胞和成骨细胞，在体内则可分化为成骨细胞/骨细胞。因此，免疫去除法产生了一个不含造血细胞和内皮细胞的细胞群体，其在表型和功能上与间充质干细胞相当。免疫去除后的细胞在培养中的群体倍增时间约为5 - 7天。生长不佳是由于免疫去除导致许多参与细胞增殖和细胞周期进程的基因显著下调。将免疫去除后的细胞暴露于成纤维细胞生长因子2（FGF2）而非胰岛素样生长因子（IGF）、小鼠干细胞因子或白血病抑制因子（LIF），可显著提高其生长速率。此外，在FGF2存在的情况下，82%因免疫去除而下调的转录本保持不变。暴露于FGF2还可逆性地抑制免疫去除后的细胞在体外分化为脂肪细胞、软骨细胞和成骨细胞的能力。因此，FGF2似乎作为一种促有丝分裂剂和自我维持因子，作用于通过阴性选择从骨髓中富集的小鼠间充质干细胞。
