Abstract
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.
Highlights
Deciphering the cellular and molecular players involved in the homeostasis of the bone marrow (BM) niches is essential to gain insight into processes of hematopoietic stem and progenitor cell (HSPC) mobilization and homing to improve treatment options for patients with several hematological diseases
Flow cytometry and immunofluorescence microscopy showed that cells were positive for nestin, vimentin, CD29, CD44, CD71, CD105, CD140a, CD140b, CD146, CD166, CD325 (N-cadherin) and Sca-1 (Supplementary Fig. S2), and negative for Ter-119, CD11b, CD34, CD45, CD90.1, CD117, CD133, CD135 (FMS-like receptor tyrosine kinase-3) and CD150 (Supplementary Fig. S3) as previously reported[18]
Because the immunophenotypic signature alone might not necessarily correlate with the differentiation status of the cells, we investigated whether the reduced expansion of CD34+CD133+ and CD133+CD34– hHSPCs on mMSCs might be explained by an early release of extracellular membrane vesicles containing CD133, which was demonstrated to occur during the differentiation process of HSPCs6
Summary
Deciphering the cellular and molecular players involved in the homeostasis of the bone marrow (BM) niches is essential to gain insight into processes of hematopoietic stem and progenitor cell (HSPC) mobilization and homing to improve treatment options for patients with several hematological diseases. The interactions between hHSPCs with surrounding cells and/ or matrix molecules, and the binding of growth factors, which are essential for their proliferation and survival, might differ between species To investigate these issues, we set out to compare the behavior of hHSPCs growing on murine (m) versus hMSCs as feeder cell layers. Time-lapse video and scanning electron microscopy (SEM) we found subtle differences in hHSPC expansion, phenotypic profiles, and polarization upon contact with mMSCs by comparison to human ones. These variations prompted us to quantitatively compare hHSPC adhesion strength on MSCs by atomic force microscopy (AFM)-based single-cell force spectroscopy (SCFS). Our data raise some caution as to the interpretation of experimental results when murine models are used to study the primitive properties of human stem and progenitor cells
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