The diffi culty 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, fi bronectin, 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 fi ndings suggest that the marrow stromal cell-derived ECM is a promising matrix for expanding largescale highly functional MSCs for eventual use in stem cell-based therapy. Moreover, this system should also be invaluable for establishment of a unique tissue-specifi c ECM, which will facilitate control of the fate of MSCs for therapeutic applications.

This study aimed to determine whether aging negatively affects MSC replication and osteogenesis 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 osteogenesis 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.

AQP3 (aquaporin-3), known as an integral membrane channel in epidermal keratinocytes, facilitates water and glycerol movement into and out of the skin. Here, we demonstrate that AQP3 is also expressed in cultured human skin fibroblasts, which under normal wound healing processes migrate from surrounding tissues to close the wound. EGF (epidermal growth factor), which induced fibroblast migration, also induced AQP3 expression in a time- and dose-dependent manner. CuSO4 and NiCl2, previously known as AQP3 water transport inhibitors, as well as two other bivalent heavy metals Mn2+ and Co2+, inhibited EGF-induced cell migration in human skin fibroblasts. AQP3 knockdown by small interfering RNA inhibited EGF-induced AQP3 expression and cell migration. Furthermore, an EGFR (EGF receptor) kinase inhibitor, PD153035, blocked EGF-induced AQP3 expression and cell migration. MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK inhibitor U0126 and PI3K (phosphoinositide 3-kinase) inhibitor LY294002 also inhibited EGF-induced AQP3 expression and cell migration. Collectively, our findings show for the first time that AQP3 is expressed in human skin fibroblasts and that EGF induces AQP3 expression via EGFR, PI3K and ERK signal transduction pathways. We have provided evidence for a novel role of AQP3 in human skin fibroblast cell migration, which occurs during normal wound healing.