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.

Niche WNT5A regulates the actin cytoskeleton during regeneration of hematopoietic stem cells.

Here, we show that the Wnt5a-haploinsufficient niche regenerates dysfunctional HSCs, which do not successfully engraft in secondary recipients. RNA sequencing of the regenerated donor Lin- SCA-1+ KIT+ (LSK) cells shows dysregulated expression of ZEB1-associated genes involved in the small GTPase-dependent actin polymerization pathway. Misexpression of DOCK2, WAVE2, and activation of CDC42 results in apolar F-actin localization, leading to defects in adhesion, migration and homing of HSCs regenerated in a Wnt5a-haploinsufficient microenvironment. Moreover, these cells show increased differentiation in vitro, with rapid loss of HSC-enriched LSK cells. Our study further shows that the Wnt5a-haploinsufficient environment similarly affects BCR-ABLp185 leukemia-initiating cells, which fail to generate leukemia in 42% of the studied recipients, or to transfer leukemia to secondary hosts. Thus, we show that WNT5A in the bone marrow niche is required to regenerate HSCs and leukemic cells with functional ability to rearrange the actin cytoskeleton and engraft successfully.

Loss of Sfrp2 in the Niche Amplifies Stress-Induced Cellular Responses, and Impairs the In Vivo Regeneration of the Hematopoietic Stem Cell Pool.

Sfrp2 is overexpressed in stromal cells which maintain hematopoietic stem cells (HSCs) during in vitro culture. We here showed, that coculture of hematopoetic cells with stromal cells with reduced expression of Sfrp2 increases the number lineage-negative Kit(+) Sca-1(+) (LSK) and progenitor cells in vitro. The LSK cells from these cocultures showed activation of canonical Wnt signaling, higher levels of Ki-67, BrdU incorporation, and the number of γH2A.X positive foci. Total repopulating activity of these cultures was, however, diminished, indicating loss of HSC. To extend these in vitro data, we modelled stress in vivo, i.e., by aging, or 5-FU treatment in Sfrp2(-) (/) (-) mice, or replicative stress in regeneration of HSCs in Sfrp2(-) (/) (-) recipients. In all three in vivo stress situations, we noted an increase of LSK cells, characterized by increased levels of ß-catenin and cyclin D1. In the transplantation experiments, the increase in LSK cells in primary recipients was subsequently associated with a progressive loss of HSCs in serial transplantations. Similar to the in vitro coculture stress, in vivo genotoxic stress in 5-FU-treated Sfrp2(-) (/) (-) mice increased cell cycle activity of LSK cells with higher levels of BrdU incorporation, increased expression of Ki-67, and canonical Wnt signaling. Importantly, as noted in vitro, increased cycling of LSKs in vivo was accompanied by a defective γH2A.X-dependent DNA damage response and depolarized localization of acetylated H4K16. Our experiments support the view that Sfrp2 expression in the niche is required to maintain the HSC pool by limiting stress-induced DNA damage and attenuating canonical Wnt-mediated HSC activation. Stem Cells 2016;34:2381-2392.

Stroma-Derived Connective Tissue Growth Factor Maintains Cell Cycle Progression and Repopulation Activity of Hematopoietic Stem Cells In Vitro.

Hematopoietic stem cells (HSCs) are preserved in co-cultures with UG26-1B6 stromal cells or their conditioned medium. We performed a genome-wide study of gene expression changes of UG26-1B6 stromal cells in contact with Lineage⁻ SCA-1⁺ KIT⁺ (LSK) cells. This analysis identified connective tissue growth factor (CTGF) to be upregulated in response to LSK cells. We found that co-culture of HSCs on CTGF knockdown stroma (shCtgf) shows impaired engraftment and long-term quality. Further experiments demonstrated that CD34⁻ CD48⁻ CD150⁺ LSK (CD34⁻ SLAM) cell numbers from shCtgf co-cultures increase in G0 and senescence and show delayed time to first cell division. To understand this observation, a CTGF signaling network model was assembled, which was experimentally validated. In co-culture experiments of CD34⁻ SLAM cells with shCtgf stromal cells, we found that SMAD2/3-dependent signaling was activated, with increasing p27(Kip1) expression and downregulating cyclin D1. Our data support the view that LSK cells modulate gene expression in the niche to maintain repopulating HSC activity.

Regulation of hematopoiesis by activators and inhibitors of Wnt signaling from the niche.

Hematopoietic stem cells (HSCs) are a rare population of somatic stem cells that have the ability to regenerate the entire mature blood system in a hierarchical way for the duration of an adult life. Adult HSCs reside in the bone marrow niche. Different niche cell types and molecules regulate the balance of HSC dormancy and activation as well as HSC behavior in both normal and malignant hematopoiesis. Here, we describe the interplay of HSCs and their niche, in particular the involvement of the Wnt signaling pathway. Although the prevailing notion has been that malignant transformation of HSCs is the main cause of leukemia, evidence is mounting that disruption of niche regulation by transformed hematopoietic cells, which may overexpress Wnt signaling or intrinsic stromal defects in gene expression, is at least a collaborative factor in leukemogenesis. Thus, insights into the normal and altered functions of niche components will help to obtain a better understanding of normal and malignant hematopoiesis and how environmental factors affect these processes.