Nitric Oxide in the Control of the in vitro Proliferation and Differentiation of Human Hematopoietic Stem and Progenitor Cells.

Julia Hümmer,Saskia Kraus,Cornelia Lee-Thedieck,Katharina Brändle

doi:10.3389/fcell.2020.610369

Abstract

Hematopoietic stem and progenitor cell (HSPC) transplantation is the best-studied cellular therapy and successful in vitro control of HSPCs has wide clinical implications. Nitric oxide (NO) is a central signaling molecule in vivo and has been implicated in HSPC mobilization to the blood stream in mice. The influence of NO on HSPC behavior in vitro is, however, largely obscure due to the variety of employed cell types, NO administration systems, and used concentration ranges in the literature. Additionally, most studies are based on murine cells, which do not necessarily mimic human HSPC behavior. Thus, the aim of the present study was the systematic, concentration-dependent evaluation of NO-mediated effects on human HSPC behavior in vitro. By culture in the presence of the long-term NO donor diethylenetriamine/nitric oxide adduct (DETA/NO) in a nontoxic concentration window, a biphasic role of NO in the regulation of HSPC behavior was identified: Low DETA/NO concentrations activated classical NO signaling, identified via increased intracellular cyclic guanosine monophosphate (cGMP) levels and proteinkinases G (PKG)-dependent vasodilator-stimulated phosphoprotein (VASP) phosphorylation and mediated a pro-proliferative response of HSPCs. In contrast, elevated NO concentrations slowed cell proliferation and induced HSPC differentiation. At high concentrations, s-nitrosylation levels were elevated, and myeloid differentiation was increased at the expense of lymphoid progenitors. Together, these findings hint at a central role of NO in regulating human HSPC behavior and stress the importance and the potential of the use of adequate NO concentrations for in vitro cultures of HSPCs, with possible implications for clinical application of in vitro expanded or differentiated HSPCs for cellular therapies.

Highlights

Tight regulation of proliferation and differentiation is central to blood homeostasis, as its balance secures the life-long regeneration of the blood system
To systematically monitor the concentration-dependent influence of Nitric oxide (NO) on Hematopoietic stem and progenitor cell (HSPC) proliferation, cells were stained with CellTraceTM Violet (CTV) after isolation, cultured in the presence of diethylenetriamine/nitric oxide adduct (DETA/NO) for 5 days and subsequently analyzed via flow cytometry
As concentrations up to 20 μM of DETA/NO were well tolerated by HSPCs (Supplementary Figure 1), they were used for cell stimulation to cover a wide concentration range of the gasotransmitter

Summary

Introduction

Tight regulation of proliferation and differentiation is central to blood homeostasis, as its balance secures the life-long regeneration of the blood system. NO Controls Human HSPC Behavior progenitors (MPPs) that undergo differentiation and proliferation events to produce all types of mature blood cells but lack the ability to self-renew indefinitely (Beckmann et al, 2007). Hematopoietic stem and progenitor cells (HSPCs) lose their multipotency and are committed to one blood lineage, which are categorized as lymphoid and myeloid (Goardon et al, 2011). Human HSPCs are commonly identified by expression of the surface marker CD34, which is lost on mature blood cells (Civin et al, 1984). As expression of CD38 and CD45RA marks more mature progenitor cells, the lack of these antigens can be used to identify HSCs and MPPs (Lansdorp et al, 1990; Bhatia et al, 1997). The transition from CMP to GMP stage is correlated with the expression of CD45RA

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