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The identification of small molecules that either increase the number and/or enhance the activity of human hematopoietic stem and progenitor cells (hHSPCs) during ex vivo expansion remains challenging. We used an unbiased in vivo chemical screen in a transgenic (c-myb:EGFP) zebrafish embryo model and identified histone deacetylase inhibitors (HDACIs), particularly valproic acid (VPA), as significant enhancers of the number of phenotypic HSPCs, both in vivo and during ex vivo expansion. The long-term functionality of these expanded hHSPCs was verified in a xenotransplantation model with NSG mice. Interestingly, VPA increased CD34 + cell adhesion to primary mesenchymal stromal cells and reduced their in vitro chemokine-mediated migration capacity. In line with this, VPA-treated human CD34 + cells showed reduced homing and early engraftment in a xenograft transplant model, but retained their long-term engraftment potential in vivo, and maintained their differentiation ability both in vitro and in vivo. In summary, our data demonstrate that certain HDACIs lead to a net expansion of hHSPCs with retained long-term engraftment potential and could be further explored as candidate compounds to amplify ex-vivo engineered peripheral blood stem cells.

Adhesion-based cellular interactions involved in breast cancer metastasis to the bone marrow remain elusive. We identified that breast cancer cells directly compete with hematopoietic stem and progenitor cells (HSPCs) for retention in the bone marrow microenvironment. To this end, we established two models of competitive cell adhesion-simultaneous and sequential-to study a potential competition for homing to the niche and displacement of the endogenous HSPCs upon invasion by tumor cells. In both models, breast cancer cells but not non-tumorigenic cells competitively reduced adhesion of HSPCs to bone marrow-derived mesenchymal stromal cells (MSCs) in a tumor cell number-dependent manner. Higher adhesive force between breast cancer cells and MSCs, as compared with HSPCs, assessed by quantitative atomic force microscopy-based single-cell force spectroscopy could partially account for tumor cell mediated reduction in HSPC adhesion to MSCs. Genetic inactivation and blockade studies revealed that homophilic interactions between intercellular adhesion molecule 1 (ICAM-1) expressed on tumor cells and MSCs, respectively, regulate the competition between tumor cells and HSPCs for binding to MSCs. Moreover, tumor cell-secreted soluble ICAM-1(sICAM-1) also impaired HSPC adhesion via blocking CD18-ICAM-1 binding between HSPCs and MSCs. Xenotransplantation studies in NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice revealed reduction of human HSPCs in the bone marrow via metastatic breast cancer cells. These findings point to a direct competitive interaction between disseminated breast cancer cells and HSPCs within the bone marrow micro environment. This interaction might also have implications on niche-based tumor support. Therefore, targeting this cross talk may represent a novel therapeutic strategy., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Xenogenic transplantation models have been developed to study human hematopoiesis in immunocompromised murine recipients. They still have limitations and therefore it is important to delineate all players within the bone marrow that could account for species-specific differences. Here, we evaluated the proliferative capacity, morphological and physical characteristics of human CD34(+) hematopoietic stem and progenitor cells (HSPCs) after co-culture on murine or human bone marrow-derived mesenchymal stromal cells (MSCs). After seven days, human CD34(+)CD133(-) HSPCs expanded to similar extents on both feeder layers while cellular subsets comprising primitive CD34(+)CD133(+) and CD133(+)CD34(-) phenotypes are reduced fivefold on murine MSCs. The number of migrating HSPCs was also reduced on murine cells suggesting that MSC adhesion influences cellular polarization of HSPC. We used atomic force microscopy-based single-cell force spectroscopy to quantify their adhesive interactions. We found threefold higher detachment forces of human HSPCs from murine MSCs compared to human ones. This difference is related to the N-cadherin expression level on murine MSCs since its knockdown abolished their differential adhesion properties with human HSPCs. Our observations highlight phenotypic, morphological and adhesive differences of human HSPCs when cultured on murine or human MSCs, which raise some caution in data interpretation when xenogenic transplantation models are used.

A major obstacle in defining the exact role of extracellular matrix (ECM) in stem cell niches is the lack of suitable in vitro methods that recapitulate complex ECM microenvironments. Here we describe a methodology that permits reliable anchorage of native cell-secreted ECM to culture carriers. We validated our approach by fabricating two types of human bone marrow-specific ECM substrates that were robust enough to support human mesenchymal stem cells (MSCs) and hematopoietic stem and progenitor cells in vitro. We characterized the molecular composition, structural features and nanomechanical properties of the MSC-derived ECM preparations and demonstrated their ability to support expansion and differentiation of bone marrow stem cells. Our methodology enables the deciphering and modulation of native-like multicomponent ECMs of tissue-resident stem cells and will therefore prepare the ground for a more rational design of engineered stem cell niches.