Cellular and Molecular Events Underlying the Interaction of Hematopoietic Stem and Progenitor Cells with Mesenchymal Stem Cells.
Authors: Bauer Nicola , Fonseca Ana-Violeta , Freund Daniel , Boxberger Sabine , Oswald Joachim , Werner Carsten , Ehninger Gerhard , Bornhaeuser Martin , Corbeil Denis 
Published: April 2020

Abstract
Understanding the mechanisms underlying the reconstitution of the hematopoietic system after blood stem cell transplantation is of crucial importance for improving therapeutic modalities. In order to address these issues, we have recently established a novel co-culture system consisting of immuno-isolated human CD34+ hematopoietic stem and progenitor cells (HSPCs) growing on primary human mesenchymal stem cells (MSCs) as feeder layer in the presence of relevant cytokines. Here we have investigated i) the morphological modification of HSPCs induced by their interaction with MSCs; ii) the sub-cellular localization of the stem cell markers CD34 and CD133 (prominin-1) as well as other cell surface molecules potentially involved in such intercellular interaction, and iii) the intracellular pathway(s) responsible for the migration as well as the interaction of HSPCs with MSCs. The scanning electron microscopy analysis revealed that the adherent HSPCs display various morphologies; they are either round with, in some cases, the appearance of a microvillar pole or exhibit several distinct types of plasma membrane protrusions such as lamellipodium, filopodium and magnupodium. We could also observe very long and thin plasma membrane processes between adjacent HSPCs suggesting the formation of nanotubes, which have been implicated in intercellular communication. As previously reported (Giebel et al., 2004, Blood, 104:2332), the highly motile HSPCs acquire a polarized cell shape with the formation of a uropod at the rear pole and a leading edge at the front pole. The immunofluorescence analyses revealed that CD34 is randomly distributed over the entire surface of HSPCs, irrespective of their morphological shape, whereas CD133 shows various specific sub-cellular localizations, which are depending on the state of the cell. In migrating cells, CD133 is concentrated in the uropod at the rear pole, which contrasts with the enriched localization of the adhesion receptor β2 integrin in the leading edge. In HSPCs harboring other types of plasma membrane protrusions, which are in close contact with the feeder cell layer, CD133 is concentrated therein. In round adhesive cells, CD133 is located in one pole of the cell suggesting that the HSPCs are highly polarized even in the absence of plasma membrane outgrowths. Interestingly, the formation of uropod and microvillus-like structures is highly reduced in presence of Y-27632 inhibitor suggesting that the Rho GTPase/ROCK pathway is somehow involved in the polarization of HSPCs. Time lapse videomicroscopy revealed that the retraction of filopodia is also affected in Y-27632-treated HSPCs as well as their migration. Taken together, these data provide new insights regarding the molecular cell biology of HSPCs, and increase our understanding of cellular events associated with mobilization and engraftment in the context of blood stem cell transplantation.