Limited Mitochondrial Activity Coupled With Strong Expression of CD34, CD90 and EPCR Determines the Functional Fitness of ex vivo Expanded Human Hematopoietic Stem Cells

Luena Papa,Christoph Schaniel,Mahtab Zangui,Robert Sebra,Mansour Djedaini,Tiphaine C Martin,Kristin G Beaumont,Ramon Parsons,Ronald Hoffman

doi:10.3389/fcell.2020.592348

Luena Papa, Christoph Schaniel + Show 7 more

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https://doi.org/10.3389/fcell.2020.592348

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Abstract

Ex vivo expansion strategies of human hematopoietic stem cell (HSC) grafts with suboptimal stem cell dose have emerged as promising strategies for improving outcomes of HSC transplantation in patients with hematological malignancies. While exposure of HSCs to ex vivo cultures expands the number of phenotypically identifiable HSCs, it frequently alters the transcriptomic and metabolic profiles, therefore, compromising their long-term (LT) hematopoietic reconstitution capacity. Within the heterogeneous pool of expanded HSCs, the precise phenotypic, transcriptomic and metabolic profile and thus, the identity of HSCs that confer LT repopulation potential remains poorly described. Utilizing valproic acid (VPA) in ex vivo cultures of umbilical cord blood (UCB)-CD34+ cells, we demonstrate that expanded HSCs phenotypically marked by expression of the stem cell markers CD34, CD90 and EPCR (CD201) are highly enriched for LT-HSCs. Furthermore, we report that low mitochondrial membrane potential, and, hence, mitochondrial activity distinguishes LT-HSCs within the expanded pool of phenotypically defined HSCs. Remarkably, such reduced mitochondrial activity is restricted to cells with the highest expression levels of CD34, CD90 and EPCR phenotypic markers. Together, our findings reveal that high expression of CD34, CD90 and EPCR in conjunction with low mitochondrial activity is critical for identification of functional LT-HSCs generated within ex vivo expansion cultures.

Highlights

A long-standing goal in the field of hematopoietic stem cells (HSCs) has been the identification and characterization of functional HSCs with long term (LT)-repopulating capacity upon transplantation
To precisely determine the phenotype of ex vivo expanded HSCs in cultures initiated with umbilical cord blood (UCB)-CD34+ cells, we further assessed expression of the cell surface marker EPCR combined with CD38 and CD45RA
Flow cytometric analysis revealed that during the entire period of culture, valproic acid (VPA) treatment compared to cytokines alone significantly increased both the percentage and the absolute number of HSCs phenotypically defined as CD34+CD90+EPCR+CD38−CD45RA− (Figures 1A,B), or CD34+CD90+CD38−CD45RA− (Supplementary Figures 1A,B)

Summary

Introduction

A long-standing goal in the field of hematopoietic stem cells (HSCs) has been the identification and characterization of functional HSCs with long term (LT)-repopulating capacity upon transplantation. LT-HSCs sustain hematopoiesis throughout the lifespan of an individual by constantly replenishing the hematopoietic system with committed progenitors (HPCs) and differentiated blood cells This LT-repopulating capacity is due to the HSC’s ability to balance self-renewal with commitment decisions (Orkin and Zon, 2008; Seita and Weissman, 2010). Such balance is controlled by complex mechanisms that rely on both the transcriptomic and metabolic properties of LTHSCs (Jang and Sharkis, 2007; Schieke et al, 2008; Takubo et al, 2013; Warr and Passegue, 2013; Kohli and Passegue, 2014; Maryanovich et al, 2015; Mohrin et al, 2015; Vannini et al, 2016; Anso et al, 2017; Papa et al, 2019b; Spurlock et al, 2019). The role of the mitochondrial bioenergetic profile during differentiation has been recently challenged, the impact of mitochondrial metabolism and activity in homeostasis and maintenance of primary HSCs with LT-repopulating potential remains undeniable (Anso et al, 2017; de Almeida et al, 2017; Bonora et al, 2018; Ito et al, 2019; Morganti et al, 2019; Liang et al, 2020)

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