Elsevier

New Biotechnology

Volume 33, Issue 1, 25 January 2016, Pages 179-186
New Biotechnology

Research paper
Metabolic profiling of hematopoietic stem and progenitor cells during proliferation and differentiation into red blood cells

https://doi.org/10.1016/j.nbt.2015.05.002Get rights and content

Highlights

  • We examine cell metabolism during ex vivo erythroid differentiation from HSPCs.

  • We demonstrate that both glycolytic and OXPHOS metabolisms are involved during proliferation.

  • Cells show greater reliance on OXPHOS in the differentiation phase.

  • We show the importance of the serum for RBC differentiation and maturation.

An understanding of the metabolic profile of cell proliferation and differentiation should support the optimization of culture conditions for hematopoietic stem and progenitor cell (HSPC) proliferation, differentiation, and maturation into red blood cells. We have evaluated the key metabolic parameters during each phase of HSPC culture for red blood cell production in serum-supplemented (SS) and serum-free (SF) conditions. A simultaneous decrease in growth rate, total protein content, cell size, and the percentage of cells in the S/G2 phase of cell cycle, as well as an increase in the percentage of cells with a CD71/GpA+ surface marker profile, indicates HSPC differentiation into red blood cells. Compared with proliferating HSPCs, differentiating HSPCs showed significantly lower glucose and glutamine consumption rates, lactate and ammonia production rates, and amino acid consumption and production rates in both SS and SF conditions. Furthermore, extracellular acidification was associated with late proliferation phase, suggesting a reduced cellular metabolic rate during the transition from proliferation to differentiation. Under both SS and SF conditions, cells demonstrated a high metabolic rate with a mixed metabolism of both glycolysis and oxidative phosphorylation (OXPHOS) in early and late proliferation, an increased dependence on OXPHOS activity during differentiation, and a shift to glycolytic metabolism only during maturation phase. These changes indicate that cell metabolism may have an important impact on the ability of HSPCs to proliferate and differentiate into red blood cells.

Introduction

There has been considerable progress in the development of systems to generate red blood cells from hematopoietic stem and progenitor cells (HSPCs) as an alternative source for blood transfusion. Several groups have attempted to generate significant numbers of erythroid cells from the HSPCs in mobilized peripheral blood, bone marrow (BM), and umbilical cord blood (UCB), as well as from human embryonic stem cells and induced pluripotent stem cells [1], [2], [3], [4], [5]. The most successful of these to date showed enucleation levels of 100 percent with up to 1.95 × 106-fold expansion by using a three-phase culture system that included co-culture with a stromal cell line [6]. Later, a protocol was developed to produce red blood cells (RBCs) from human cord blood in the absence of a feeder layer [7]. A different approach, undertaken by Fujimi and co-workers, used a four-phase system which incorporated stromal cells in the first phase and macrophages in the third phase [8]; this yielded more than 1013 RBCs from one unit of cord blood with a 99.4 percent enucleation efficiency. Nevertheless, an efficient, cost-effective, and simple system for the industrial expansion and differentiation of HSPCs into RBCs remains a challenge and far from realization.

The identification and purification of hematopoietic stem cells is based on the expression of the cell surface marker CD34 (a 105- to 120-kDa transmembrane cell surface glycoprotein) on hematopoietic stem cells and progenitor cells of all hematopoietic lineages [9], [10]. The CD34 antigen is involved in adhesive interactions between HSCs and the stromal environment and in regulation and compartmentalization of stem cells [11], [12]. CD34+ cells are found at frequencies of 0.1–0.5 percent of mononuclear cells (MNCs) in UCB, 0.5–3 percent in BM, and 0.05–0.2 percent in peripheral blood; however, this number can be increased by granulocyte–macrophage colony-stimulating factor mobilization, which results in the release of immature cells from the BM into the blood stream [13], [14]. Substantial research has been conducted in relation to the therapeutic potential of CD34+ cells in areas such as cancer [15], diabetes mellitus [16], allogenic transplantation [17] and ischemic CVD [18].

Elucidating the cellular and metabolic processes involved in the production of RBCs is an important step in addressing these challenges. Understanding the metabolic changes of HSPCs as they expand and differentiate to mature RBCs will allow the manipulation of intra and extracellular environments through improved media formulations. To the best of our knowledge, the metabolism of HSPCs during their expansion and differentiation into RBCs has not received adequate attention.

In this study, we compared key metabolite profiles in HSPCs between serum-supplemented (SS) and serum-free (SF) conditions at the expansion, differentiation, and maturation phases of erythroid development. Our findings will enable the optimization and identification of components for SF media formulation. Development of SF media is critical for future clinical application.

Section snippets

Source and isolation of CD34+ cells

Peripheral blood buffy coats (a donation by-product) from healthy donors were obtained from the Irish Blood Transfusion Service (Dublin, Ireland). MNCs were enriched using density gradient centrifugation with Histopaque-1077 (Sigma–Aldrich, Dublin, Ireland) followed by an additional separation step with 20 percent (w/v) sucrose to obtain a higher purity of MNCs and reduce plasma, erythrocyte, and platelet contamination. CD34+ cells were purified by magnetic bead separation using a human CD34+

Results and discussion

Typically, HSPCs cultures show an initial lag phase of 2–3 days post-isolation, followed by a high proliferation rate from day 3 until approximately day 11–13 (Fig. 1). In this study, cells were taken at four time-points and grown in SS or SF media for 3 days. The selected time-points represent the early proliferation (day 6), late proliferation (day 9), differentiation (day 13), and maturation (day 20) phases of culture. Distinct growth profiles were seen for each phase. The highest

Conclusion

This study examined cell metabolism during ex vivo erythroid differentiation of HSPCs in SS and SF cultures; an important step in the optimization of serum-free media formulations. Both glycolytic and OXPHOS metabolic pathways were active during early and late proliferation phase in both SS and SF culture, followed by a greater reliance on OXPHOS in differentiation phase, before a total shift to glycolysis as cells matured into RBCs. Cells in SF cultures both consumed and produced fewer

Acknowledgements

This work was supported by the Irish Blood Transfusion Service, Dublin, Ireland and the National Science Fellowship, Ministry of Science, Technology and Innovation, Malaysia.

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