We cultured MSCs on an ECM made by bone marrow cells to attempt to reconstitute the MSC niche. This ECM promoted replication of mesenchymal progenitors and retention of their multipotentiality. We conclude that the marrow ECM facilitates expansion of mesenchymal progenitors and hypothesize that it plays an important role in the maintenance of MSC stemness. Introduction: Mesenchymal colony-forming cells of the bone marrow comprise mesenchymal stem cells (MSCs) and their transit amplifying progeny, which we term mesenchymal colony-forming units (MCFUs). These progenitors undergo self-renewal and can differentiate into many different cell types including osteoblasts. However, they lose their unique properties when cultured on tissue culture plastic. This indicates that a critical feature of the marrow microenvironment that facilitates retention of stem cell properties is missing in such culture systems. In other tissues, the extracellular matrix (ECM) forms part of the specialized niche that controls stem cell behavior. Therefore, we examined whether a marrow cell–derived ECM promotes retention of the stem cell characteristics of MCFUs in vitro. Materials and Methods: A cell-free ECM was prepared from cultured murine marrow adherent cells. The replication and multipotentiality of murine MCFUs maintained on this marrow cell–derived ECM were examined in vitro and in vivo and compared with the behavior of MCFUs maintained on plastic. Results: The marrow cell–derived ECM was made up of collagen types I, III, and V, syndecan-1, perlecan, fibronectin, laminin, biglycan, and decorin, similar to the composition of the marrow ECM. This ECM preparation promoted MCFU replication, restrained their “spontaneous” differentiation toward the osteoblast lineage, and preserved their ability to differentiate into osteoblasts or adipocytes. Moreover, transplantation of MCFUs expanded on the marrow cell–derived ECM into immunocompromised mice generated five times more bone and eight times more hematopoietic marrow compared with MCFUs expanded on plastic. Conclusions: The marrow ECM facilitates expansion of MCFUs in vitro while preserving their stem cell properties. We hypothesize that the ECM made by bone marrow cells plays an important role in the maintenance of MSC function.

This study aimed to determine whether aging negatively affects MSC replication and osteogen‐esis and whether these features could be altered by exposure to an extracellular matrix (ECM) generated by marrow cells from young or old mice. A cell‐free ECM was prepared from cultured femoral marrow cells from either 3‐ or 18‐mo‐old C57BL/6 mice (young‐ECM or old‐ECM, respectively). The replication and osteo‐genesis of young or old MSCs maintained on young‐ECM vs. old‐ECM as well as plastic were examined in vitro and in vivo. We found that the frequency of MSCs in marrow from old mice, measured by colony‐forming cells, was only marginally lower than that of young mice. In contrast, defects in the self‐renewal and bone formation capacity of old MSCs were remarkable. These defects were corrected by provision of a young‐ECM but not old‐ECM. In parallel cultures maintained on a young‐ECM, the intracellular levels of reactive oxygen species from both old and young mice were reduced 30–50% compared to those maintained on old‐ECM or plastic. We concluded that aging negatively affects the formation of an ECM that normally preserves MSC function, and aged MSCs can be rejuvenated by culture on a young‐ECM.—Sun, Y., Li, W., Lu, Z., Chen, Z., Ling, J., Ran, O., Jilka, R. L., Chen, X. D. Rescuing replication and osteogenesis of aged mesenchymal stem cells by exposure to a young extracellular matrix. FASEB J. 25, 1474–1485 (2011). www.fasebj.org

The difficulty in long-term expansion of mesenchymal stem cells (MSCs) using standard culture systems without the loss of their stem cell properties suggests that a critical feature of their microenvironment necessary for retention of stem cell properties is absent in these culture systems. We report here the reconstitution of a native extracellular matrix (ECM) made by human marrow cells ex vivo, which consists of at least collagen types I and III, fibronectin, small leucine-rich proteoglycans such as biglycan and decorin, and major components of basement membrane such as the large molecular weight proteoglycan perlecan and laminin. Expansion of human MSCs on this ECM strongly promoted their proliferation, retained their stem cell properties with a low level of reactive oxygen species (ROS), and substantially increased their response to BMP-2. The quality of the expanded cells following each passage was further tested by an in vivo transplantation assay. The results showed that MSCs expanded on the ECM for multiple passages still retained the same capacity for skeletogenesis. In contrast, the bone formation capacity of cells expanded on plastic was dramatically diminished after 6–7 passages. These findings suggest that the marrow stromal cell-derived ECM is a promising matrix for expanding large-scale highly functional MSCs for eventual use in stem cell-based therapy. Moreover, this system should also be invaluable for establishment of a unique tissue-specific ECM, which will facilitate control of the fate of MSCs for therapeutic applications.

Degenerative diseases are a major public health concern for the aging population and mesenchymal stem cells (MSCs) have great potential for treating many of these diseases. However, the quantity and quality of MSCs declines with aging, limiting the potential efficacy of autologous MSCs for treating the elderly population. Human bone marrow (BM)-derived MSCs from young and elderly donors were obtained and characterized using standard cell surface marker criteria (CD73, CD90, CD105) as recommended by the International Society for Cellular Therapy (ISCT). The elderly MSC population was isolated into four subpopulations based on size and stage-specific embryonic antigen-4 (SSEA-4) expression using fluorescence-activated cell sorting (FACS), and subpopulations were compared to the unfractionated young and elderly MSCs using assays that evaluate MSC proliferation, quality, morphology, intracellular reactive oxygen species, β-galactosidase expression, and adenosine triphosphate (ATP) content. The ISCT-recommended cell surface markers failed to detect any differences between young and elderly MSCs. Here, we report that elderly MSCs were larger in size and displayed substantially higher concentrations of intracellular reactive oxygen species and β-galactosidase expression and lower amounts of ATP and SSEA-4 expression. Based on these findings, cell size and SSEA-4 expression were used to separate the elderly MSCs into four subpopulations by FACS. The original populations (young and elderly MSCs), as well as the four subpopulations, were then characterized before and after culture on tissue culture plastic and BM-derived extracellular matrix (BM-ECM). The small SSEA-4-positive subpopulation representing ~ 8% of the original elderly MSC population exhibited a “youthful” phenotype that was similar to that of young MSCs. The biological activity of this elderly subpopulation was inhibited by senescence-associated factors produced by the unfractionated parent population. After these “youthful” cells were isolated and expanded (three passages) on a “young microenvironment” (i.e., BM-ECM produced by BM cells from young donors), the number of cells increased ≈ 17,000-fold to 3 × 109 cells and retained their “youthful” phenotype. These results suggest that it is feasible to obtain large numbers of high-quality autologous MSCs from the elderly population and establish personal stem cell banks that will allow serial infusions of “rejuvenated” MSCs for treating age-related diseases.

Introduction: Bone marrow mesenchymal stem cells (BMMSCs) are a heterogeneous population of postnatal precursor cells with the capacity of adhering to culture dishes generating colony-forming unit-fibroblasts (CFU-F). Here we identify a new subset of BMMSCs that fail to adhere to plastic culture dishes and remain in culture suspension (S-BMMSCs).Methods: To catch S-BMMSCs, we used BMMSCs-produced extracellular cell matrix (ECM)-coated dishes. Isolated S-BMMSCs were analyzed byin vitrostem cell analysis approaches, including flow cytometry, inductive multiple differentiation, western blot andin vivoimplantation to assess the bone regeneration ability of S-BMMSCs. Furthermore, we performed systemic S-BMMSCs transplantation to treat systemic lupus erythematosus (SLE)-like MRL/lprmice.Results: S-BMMSCs are capable of adhering to ECM-coated dishes and showing mesenchymal stem cell characteristics with distinction from hematopoietic cells as evidenced by co-expression of CD73 or Oct-4 with CD34, forming a single colony cluster on ECM, and failure to differentiate into hematopoietic cell lineage. Moreover, we found that culture-expanded S-BMMSCs exhibited significantly increased immunomodulatory capacitiesin vitroand an efficacious treatment for SLE-like MRL/lprmice by rebalancing regulatory T cells (Tregs) and T helper 17 cells (Th17) through high NO production.Conclusions: These data suggest that it is feasible to improve immunotherapy by identifying a new subset BMMSCs.