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Remodeling of the bone marrow microenvironment in chronic inflammation and in aging reduces hematopoietic stem cell (HSC) function. To assess the mechanisms of this functional decline of HSC and find strategies to counteract it, we established a model in which the Sfrp1 gene was deleted in Osterix+ osteolineage cells (OS1Δ/Δ mice). HSC from these mice showed severely diminished repopulating activity with associated DNA damage, enriched expression of the reactive oxygen species pathway and reduced single-cell proliferation. Interestingly, not only was the protein level of Catenin beta-1 (bcatenin) elevated, but so was its association with the phosphorylated co-activator p300 in the nucleus. Since these two proteins play a key role in promotion of differentiation and senescence, we inhibited in vivo phosphorylation of p300 through PP2A-PR72/130 by administration of IQ-1 in OS1Δ/Δ mice. This treatment not only reduced the b-catenin/phosphop300 association, but also decreased nuclear p300. More importantly, in vivo IQ-1 treatment fully restored HSC repopulating activity of the OS1Δ/Δ mice. Our findings show that the osteoprogenitor Sfrp1 is essential for maintaining HSC function. Furthermore, pharmacological downregulation of the nuclear b-catenin/phospho-p300 association is a new strategy to restore poor HSC function.

Abstract In this study, we investigated the influence of the loss of cathepsin K (Ctsk) gene on the hematopoietic system in vitro and in vivo. We found that cultures with lineage− SCA1+ KIT+ (LSK) cells on Ctsk deficient stromal cells display reduced colony formation and proliferation, with increased differentiation, giving rise to repopulating cells with reduced ability to repopulate the donor LSKs and T cell compartments in the bone marrow (BM). Subsequent in vivo experiments showed impairment of lymphocyte numbers, but, gross effects on early hematopoiesis or myelopoiesis were not found. Most consistently in in vivo experimental settings, we found a significant reduction of (donor) T cell numbers in the BM. Lymphocyte deregulation is also found in transplantation experiments, which revealed that Ctsk is required for optimal regeneration of small populations of T cells, particularly in the BM, but also of thymic B cells. Interestingly, cell nonautonomous Ctsk regulates both B and T cell numbers, but T cell numbers in the BM require an additional autonomous Ctsk‐dependent process. Thus, we show that Ctsk is required for the maintenance of hematopoietic stem cells in vitro, but in vivo, Ctsk deficiency most strongly affects lymphocyte homeostasis, particularly of T cells in the BM.

Hematopoietic stem cell self-renewal, proliferation, and differentiation are independently regulated by intrinsic as well as extrinsic mechanisms. We previously demonstrated that murine proliferation of hematopoietic stem cells is supported in serum-free medium supplemented with two growth factors, stem cell factor and interleukin 11. The survival of hematopoietic stem cells is additionally improved by supplementing this medium with two more growth factors, neural growth factor and collagen 1 (four growth factors) or serum-free medium conditioned by the hematopoietic stem cell-supportive stromal UG26-1B6 cells1. Here, we describe a robust and versatile alternative source of conditioned medium from mouse embryonic fibroblasts. We found that this conditioned medium supports survival and phenotypical identity of hematopoietic stem cells, as well as cell cycle entry in single cell cultures of CD34- CD48- CD150+ Lineage- SCA1+ KIT+ cells supplemented with two growth factors. Strikingly, in comparison with cultures in serum-free medium with four growth factors, conditioned medium from mouse embryonic fibroblasts increases the numbers of proliferating clones and the number of Lineage- SCA1+ KIT+ cells, both with two and four growth factors. In addition, conditioned medium from mouse embryonic fibroblasts supports self-renewal in culture of cells with short- and long-term hematopoiesis-repopulating ability in vivo. These findings identify conditioned medium from mouse embryonic fibroblasts as a robust alternative serumfree source of factors to maintain self-renewal of in vivo-repopulating hematopoetic stem cells in culture.