ABSTRACT

Adult stem cells from human bone marrow stroma, referred to as mesenchymal stem cells or marrow stromal cells (hMSCs), are attractive candidates for clinical use. The optimal conditions for hMSC expansion require medium supplemented with fetal calf serum (FCS). Some forms of cell therapy will involve multiple doses, raising a concern over immunological reactions caused by medium-derived FCS proteins. By a sensitive fluorescence-based assay we determined that 7 to 30 mg of FCS proteins are associated with a standard preparation of 100 million hMSCs, a dosage that probably will be needed for clinical therapies. Here we present ex vivo growth conditions for hMSCs that reduce the FCS proteins to less than 100 ng per 100 million hMSCs, approximately a 100,000-fold reduction. The cells maintain their proliferative capacity and sustain their ability for multilineage differentiation. Experiments in rats demonstrate that rat MSCs grown in 20% FCS induce a substantial humoral response after repeated administrations, whereas cells grown under the conditions described in this study reduce the immunogenicity in terms of IgG response over 1000-fold to barely detectable levels. Our results have the potential to dramatically improve cellular and genetic therapies using hMSCs and perhaps other cells.

To investigate stem cell differentiation in response to tissue injury, human mesenchymal stem cells (hMSCs) were cocultured with heatshocked small airway epithelial cells. A subset of the hMSCs rapidly differentiated into epithelium-like cells, and they restored the epithelial monolayer. Immunocytochemistry and microarray analyses demonstrated that the cells expressed many genes characteristic of normal small airway epithelial cells. Some hMSCs differentiated directly after incorporation into the epithelial monolayer but other hMSCs fused with epithelial cells. Surprisingly, cell fusion was a frequent rather than rare event, in that up to 1% of the hMSCs added to the coculture system were recovered as binucleated cells expressing an epithelial surface epitope. Some of the fused cells also underwent nuclear fusion.

Attempts have been made to develop cell and gene therapies using the adult stem cells from bone marrow referred to as mesenchymal stem cells or marrow stromal cells (MSCs). However, the results have been variable in part because there are no standardized protocols for preparing and characterizing MSCs. In the experiments presented here, we developed a standardized assay by light scattering to measure the content of rapidly self-renewing cells (RS cells) in preparations of MSCs. The assay quickly identifies preparations of MSCs that replicate rapidly in subsequent culture. In addition, the standardized assay enabled us to isolate RS cells that were up to 90% clonogenic and that generated single cell-derived colonies that differentiated into either mineralizing cells or adipocytes with appropriate additions to the medium.

Recently there has been interest in developing cell and gene therapies with adult stem cells from human bone marrow referred to as mesenchymal stem cells or marrow stromal cells (hMSCs). We incubated early-passage hMSCs in serumfree medium without cytokines or other supplements for 2 to 4 weeks. Surprisingly, a subpopulation of the cells sur- IntroductionHuman mesenchymal stem cells or marrow stromal cells (hMSCs) can differentiate into osteoblasts, chondrocytes, adipocytes, myocytes, and other cell types. [1][2][3][4][5][6][7][8][9] They are readily isolated from bone marrow by their adherence to tissue culture plastic and can be expanded extensively in medium containing high concentrations of fetal calf serum (FCS). The cells are readily cloned as single-cellderived colonies, but both the colonies and the cells within a colony are heterogeneous in morphology, rates of proliferation, and efficacy with which they differentiate. 3,10,11 Two morphologically distinct cell phenotypes are seen in early-passage, low-density cultures: small, spindle-shaped cells that are rapidly self renewing (RS cells) and large, flat cells that replicate slowly and appear more mature. 3,10,12 The proportion of RS cells remains high for several passages if the cultures are maintained at low density, but the larger cells predominate in later passages and the cells cease proliferation at about the Hayflick limit of 50 population doublings. 9,10,12,13, Several investigators 10,13-15 identified distinctive surface epitopes on hMSCs but none of the epitopes have been shown to distinguish early progenitors from more mature cells in cultures of hMSCs. Marrow cells that may or may not be related to hMSCs were recently isolated by first selecting Lin Ϫ and glycophorin Ϫ marrow cells and then culturing the cells on fibronectin-coated plated cells in medium containing 2% FCS and a series of growth factors. 16,17 After about 25 population doublings, cultures of multipotent adult precursor cells (MAPCs) were obtained that expressed telomerase, were multipotential for differentiation, and continued to expand for well over 50 population doublings. Recently, we found that a subpopulation of hMSCs with distinctive features can be selected from early-passage cultures of hMSCs by incubation of the cells for 2 to 4 weeks without serum, growth factors, or other supplements. Materials and methodsHuman MSCs were prepared as described previously. 10,12 In brief, nucleated cells were isolated with a density gradient (Ficoll-Paque; Pharmacia, Uppsala, Sweden) from 2 mL human bone marrow aspirated from the iliac crests of healthy volunteers under a protocol approved by the Institutional Review Board of Tulane University Health Sciences Center. All the nucleated cells (30 to 70 million) were plated in a 145-cm 2 dish in 20 mL complete culture medium: ␣-modified minimum essential media (␣MEM; GIBCO BRL, Carlsbad, CA); 17% fetal bovine serum ([FBS] lot-selected for rapid growth of MSCs; Atlanta Biologicals, Norcross, GA); 100 units/mL penicillin; 100 g/mL strepto...