Previously, we reported that deficits in self-renewal and bone formation capacity in bone marrow-derived mesenchymal stem cells (BM-MSCs) from elderly could be completely restored by culture on extracellular matrix (ECM) synthesized by BM stromal cells from young donors (young-ECM). However, this restorative effect was substantially diminished when cells from young or old donors were cultured on ECM synthesized by BM stromal cells from old donors (old-ECM). The goal of this renewal application is to dissect out the effective component(s) present in young-ECM that play(s) a critical role in the retention of BM-MSC properties and rejuvenation of BM-MSCs from elderly donors (old MSCs). To identify differences in protein composition between the two young- and old-ECMs, we performed proteomic analyses and discovered that CCN1/Cyr61 was the only protein present in young-ECM that was not in old-ECM. Although CCN1/Cyr61 has been shown to regulate osteoblast differentiation when added exogenously to cell cultures, it is not clear whether it can also function as a structural protein (incorporated during BM-ECM synthesis) and promote the retention of MSC properties. In the renewal application, we hypothesize that the presence of CCN1/Cyr61 in young-ECM is essential for the retention of MSC properties and rejuvenation of elderly BM-MSCs. We are well-positioned to test this hypothesis since we have established a unique 3D native BM-ECM culture system. To test our hypothesis, we will evaluate the role of CCN1/Cyr61 in controlling the behavior of MSCs by decreasing its incorporation into the matrix during synthesis of young-ECM (Specific Aim 1) and increasing its incorporation into the matrix during synthesis of old-ECM (Specific Aim 2). To determine whether CCN1/Cyr61 alone is sufficient to rejuvenate elderly MSCs, recombinant human CCN1/Cyr61 will be added to cell culture media, physisorbed onto tissue culture plastic (TCP) surfaces, or incorporated into self-assembled monolayers (SAMs) (Specific Aim 3). The proposed studies are innovative in that we will for the first time: a) demonstrate the function of CCN1/Cyr61, incorporated into BM-ECM (the BM-MSC microenvironment) as a structural protein during synthesis, in controlling the fate of authentic BM-MSCs, and b) construct a defined ECM, using recombinant CCN1/Cyr61, that mimics the ability of native BM-ECM to produce large-scale expansion of high- quality MSCs and rejuvenation of elderly MSCs. If successful, the results of the proposed studies will be significant because they will overcome challenges related to our established 3D native BM-ECM and be more suitable for clinical applications (e.g: variations in ECM prepared from different donors; safety concerns related to potential contamination with undetected pathogens; and undefined ECM components that may produce variable/unpredictable clinical outcomes). According to the 2015 National Survey of Veteran Enrollees' Health and Reliance on VA, 47% of the US...