Autophagy in mesenchymal progenitors protects mice against bone marrow failure after severe intermittent stress.

The cellular mechanisms required to ensure homeostasis of the hematopoietic niche and the ability of this niche to support hematopoiesis upon stress remain elusive. We here identify Wnt5a in Osterix+ mesenchymal progenitor and stem cells (MSPCs) as a critical factor for niche-dependent hematopoiesis. Mice lacking Wnt5a in MSPCs suffer from stress-related bone marrow (BM) failure and increased mortality. Niche cells devoid of Wnt5a show defective actin stress fiber orientation due to an elevated activity of the small GTPase CDC42. This results in incorrect positioning of autophagosomes and lysosomes, thus reducing autophagy and increasing oxidative stress. In MSPCs from patients from BM failure states which share features of peripheral cytopenia and hypocellular BM, we find similar defects in actin stress fiber orientation, reduced and incorrect colocalization of autophagosomes and lysosomes, and CDC42 activation. Strikingly, a short pharmacological intervention to attenuate elevated CDC42 activation in vivo in mice prevents defective actin-anchored autophagy in MSPCs, salvages hematopoiesis and protects against lethal cytopenia upon stress. In summary, our study identifies Wnt5a as a restriction factor for niche homeostasis by affecting CDC42-regulated actin stress-fiber orientation and autophagy upon stress. Our data further imply a critical role for autophagy in MSPCs for adequate support of hematopoiesis by the niche upon stress and in human diseases characterized by peripheral cytopenias and hypocellular BM.

Secreted factors from mouse embryonic fibroblasts maintain repopulating function of single cultured hematopoietic stem cells.

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.

Cathepsin K maintains the compartment of bone marrow T lymphocytes in vivo.

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.