Characterization of mesenchymal stem cells isolated from murine bone marrow by negative selection

Abstract

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.

Critical role for Y1 receptors in mesenchymal progenitor cell differentiation and osteoblast activity

“…Stem cell antigen 1 (Sca-1), a marker previously described on hematopoietic stem cells, also has been described on mesenchymal osteoprogenitor cells and is directly required for their self-renewal. (21)(22)(23) In addition, new evidence suggests that the majority of MSCs are not actually located within the bone marrow but rather within the bone tissue itself or, more specifically, within the vascular system of the bone. (24)(25)(26) Therefore, employing fluorescence-activated cell sorting (FACS) for Sca-1 on cells purified from murine bone tissue following a rigorous depletion of hematopoietic cells results in two populations enriched for mesenchymal progenitor cells: highly proliferative Sca-1 þ MSCs, which have been shown to exhibit adipogenic, chondrogenic, and osteogenic potential, and Sca-1 À CD51 þ cells that are more committed toward the osteogenic lineage and hence referred to as osteoprogenitor cells.…”

IFN‐γ activation of mesenchymal stem cells for treatment and prevention of graft versus host disease

“…Several factors are likely to contribute to the variable results. MSC tissue source, (i.e., BM, cord blood, adipose tissue), method of isolation to remove myeloid precursors (several weeks [36] vs. rapid immunodepletion [37]) and timing of MSC administration are potential variables that could explain these differences. Notably, such variation has not been observed clinically, with MSC treatment of ongoing GVHD reported to have significant efficacy.…”

BAC transgenic expression efficiency: bicistronic versus ATG‐fusion strategies

“…Here, we choose a GTP-binding nucleolar protein nucleostemin as the target molecule to address this question. Nucleostemin is preferentially expressed in stem cells, cancer cells, and adult testis (Baddoo et al, 2003;Tsai and McKay, 2002). It is essential for early embryogenesis and capable of binding telomeric repeat-binding factor 1 (Zhu et al, 2006) through a nucleolus-nucleoplasmic shuttling mechanism (Meng et al, 2006;Tsai and McKay, 2005).…”