Abstract
Hematopoietic Stem Cell (HSC) self-renewal and multilineage differentiation potential is governed by multiple intrinsic and extrinsic parameters. Collectively, these parameters dictate the fate of HSC and underscore the heterogeneity observed within phenotypically defined groups of stem cells. While cell cycle status and the genetic profile of HSCs are critical intrinsic modulators of cell fate, interactions with cytokines, growth factors, and cellular elements of the hematopoietic niche (HN) are key extrinsic regulators of stem cell function. We examined the impact of cellular elements of the HN on stem cell fate and maintenance by analyzing the combined effect of calvaria-derived osteoblasts (OB) and mesenchymal stromal cells (MSC) on cultured murine HSC. Murine bone marrow-derived KSL cells were co-cultured with OB alone, MSC alone, or with mixtures of OB and MSC at different ratios for one week. Cultures were supplemented with SCF, Fl-3, Tpo, IL-3, IL-6, IGF1 & OPN. OB alone, maintained the functional properties of cultured HSCs significantly better than MSC thus corroborating the importance of OB in the overall competence of the HN. On day 7, the fold-increase in the number of LSK cells was 1473 ± 291 in OB cultures, 561 ± 159 in MSC cultures, and 603 ± 263 in OB+MSC cultures (n= 4 for all 3 groups). During the same 7 day-period, the number of CFU in progeny cells expanded 74 ± 15 fold in OB cultures, 23 ± 2 fold in MSC cultures, and 27 ± 15 in OB+MSC cultures (n=3 for all groups). The substantial increase in KSL progeny in OB cultures on day 7 was accompanied by a high percentage of cells in active phases of cell cycle (% G0/G1 = 72.5 ± 7.0, n=3) compared to their counterparts in MSC or OB+MSC cultures. In addition, co-culture of KSL cells with OB resulted in an unexpected higher maintenance of the Sca-1+Lin- phenotype (26.5% ± 2.8%) relative to MSC cultures (4.6% ± 1.0%) and OB+MSC cultures (11.7% ± 1.8%; n=3 for all). Only some of these results were reproduced when KSL cells were cultured in OB-conditioned medium suggesting that cell-to-cell contact may be essential for the observed activities. To assess the in vivo potential of LSK cells maintained in these cultures, the 10-day expansion equivalent of 1,000 LSK cells were competitively transplanted in lethally irradiated congenic mice and chimerism was monitored for the next 4 months. At 1 and 2 months post-transplantation, the level of chimerism sustained by LSK cells maintained in OB cultures for 10 days surpassed or was slightly lower than that observed with freshly isolated LSK cells (72.7% vs 59.7% and 57.4% vs 74.7%, respectively) suggesting that OB culture conditions effectively expanded short-term repopulating cells. At 4 months post-transplantation, mice receiving freshly isolated LSK cells were 83.6% ± 1.8% chimeric compared to 53.7% ± 16.1% for mice transplanted with cells from OB cultures and 31.9% ± 21.4% for mice receiving cells from OB+MSC cultures. Overall, these data suggest that OB-LSK interactions promote the maintenance of both short-term and long-term repopulating cells while MSC suppress the OB-mediated activity. To investigate the mechanism of OB-mediated maintenance of stem cell phenotype and function, we examined Notch signaling using Real-Time Q-PCR on cells maintained in culture for 7 days. Relative to the expression in KSL cells, expression of Notch 2 was elevated in OB cultures and suppressed over 2-fold in cultures of MSC and OB+MSC. Similarly, the expression of Jagged 1 and 2, Delta 1 and 4, Hes 1 and 5, Deltex, and SKP2 was increased in OB cultures and suppressed in MSC and OB+MSC cultures. Collectively, these data illustrate that cell-to-cell contact between OB and KSL cells promotes the in vitro maintenance of long-term and short-term repopulating cells and suggest that this stem cell function-promoting activity is induced in part by the upregulation of Notch-mediated signaling between HSCs and osteoblasts. The suppressive effect imparted by MSC on stem cell maintenance compared to cultures of OB alone suggest that these two cellular elements of the HN have opposite effects on the fate and function of stem cells.
Long‐term administration of the mitochondria‐targeted antioxidant mitoquinone mesylate fails to attenuate age‐related oxidative damage or rescue the loss of muscle mass and function associated with aging of skeletal muscle