The Use of Induced Pluripotent Stem Cells to Study the Effects of Adenosine Deaminase Deficiency on Human Neutrophil Development

Abstract

Inherited defects that abrogate the function of the adenosine deaminase (ADA) enzyme and consequently lead to the accumulation of toxic purine metabolites cause profound lymphopenia and severe combined immune deficiency. Additionally, neutropenia and impaired neutrophil function have been reported among ADA-deficient patients. However, due to the rarity of the disorder, the neutrophil developmental abnormalities and the mechanisms contributing to them have not been characterized. Induced pluripotent stem cells (iPSC) generated from two unrelated ADA-deficient patients and from healthy controls were differentiated through embryoid bodies into neutrophils. ADA deficiency led to a significant reduction in the number of all early multipotent hematopoietic progenitors. At later stages of differentiation, ADA deficiency impeded the formation of granulocyte colonies in methylcellulose cultures, leading to a significant decrease in the number of neutrophils generated from ADA-deficient iPSCs. The viability and apoptosis of ADA-deficient neutrophils isolated from methylcellulose cultures were unaffected, suggesting that the abnormal purine homeostasis in this condition interferes with differentiation or proliferation. Additionally, there was a significant increase in the percentage of hyperlobular ADA-deficient neutrophils, and these neutrophils demonstrated significantly reduced ability to phagocytize fluorescent microspheres. Supplementing iPSCs and methylcellulose cultures with exogenous ADA, which can correct adenosine metabolism, reversed all abnormalities, cementing the critical role of ADA in neutrophil development. Moreover, chemical inhibition of the ribonucleotide reductase (RNR) enzyme, using hydroxyurea or a combination of nicotinamide and trichostatin A in iPSCs from healthy controls, led to abnormal neutrophil differentiation similar to that observed in ADA deficiency, implicating RNR inhibition as a potential mechanism for the neutrophil abnormalities. In conclusion, the findings presented here demonstrate the important role of ADA in the development and function of neutrophils while clarifying the mechanisms responsible for the neutrophil abnormalities in ADA-deficient patients.

Keywords: adenosine deaminase (ADA) deficiency; iPSC (induced pluripotent stem cell); multipotent; neutropenia; ribonucleotide reductase (RNR) inhibitors.

Figure 1

Impaired production of multipotent hematopoietic progenitors from ADA-deficient induced pluripotent stem cells. iPSCs were dissociated to form embryoid bodies (EB) and differentiated for 14 days to generate multipotent hematopoietic progenitors (MHP). (A) The number of CD34⁺/CD45⁺ MHPs generated from 6×10⁶ healthy control (CTL-iPSC-1) or ADA-deficient (ADA-iPSC-1) without or with PEG-ADA was assessed at the indicated days after initiation of early hematopoietic differentiation. (B) The number of MHPs generated from CTL-iPSC-2 and ADA-iPSC-2 ± PEG-ADA in day 14 of differentiation. (C) The viability of CTL-iPSC-1 and ADA-iPSC-1 as well as CTL-iPSC-2 and ADA-iPSC-2 after 14 days of differentiation, determined by exclusion of PI. Data shown as mean + SD of n = 3 replicates.

Figure 2

Impaired production of granulocyte colony-forming units and neutrophils from ADA-deficient induced pluripotent stem cells. iPSCs were differentiated for 14 days to form embryoid bodies (EB). The EBs were then dissociated and differentiated in methylcellulose for an additional 12 days to generate hematopoietic colony-forming units (CFU) and neutrophils. The number of CFU of granulocyte (CFU-G), granulocyte-monocyte (CFU-GM), granulocyte-erythroid-monocyte/macrophage (CFU-GEMM), monocyte CFU (CFU-M), and erythroid (CFU-E), as well as erythroid blast-forming units (BFU-E) visualized following 12-day culture in methylcellulose from healthy control (CTL-iPSC-1) and an ADA-deficient patient (ADA-iPSC-1) (A), as well as another control (CTL-iPSC-2) and ADA-deficient patients (ADA-iPSC-2) (B). The percentage of CD11b⁺/CD45⁺ neutrophils (C), the number of neutrophils obtained per 4×10⁴ cells plated in methylcellulose (D), and the total number of neutrophils harvested from methylcellulose (E) following the differentiation of 6×10⁶ healthy control (CTL-iPSC-1 and CTL-iPSC-2) or ADA-deficient (ADA-iPSC-1 and ADA-iPSC-2). The percentages of viable (PI⁻) neutrophils generated from ADA-iPSC-1 or CTL-iPSC-1 (F). Data shown as mean + SD of n = 9 replicates for CFU experiments, n = 6 replicates for ADA-iPSC-1- and CTL-iPSC-1-derived neutrophils, and n = 3 for CTL-iPSC-2- and ADA-iPSC-2-derived neutrophils.

Figure 3

Improved formation of granulocyte colonies and generation of neutrophils from ADA-deficient induced pluripotent stem cells following supplement with ADA. iPSCs were differentiated for 14 days to form EBs. The EBs were then dissociated and differentiated in methylcellulose for an additional 12 days to generate neutrophils, with ADA-deficient iPSCs treated with PEG-ADA replacement enzyme throughout the entirety of the differentiation. The number of Colony-Forming Units (CFU) of granulocyte (CFU-G), granulocyte-monocyte (CFU-GM), granulocyte-erythroid-monocyte/macrophage (CFU-GEMM), monocyte CFU (CFU-M), and erythroid (CFU-E), as well as erythroid blast-forming units (BFU-E) differentiated per 4×10⁴ cells plated in methylcellulose from healthy control (CTL-iPSC-1 and CTL-iPSC-2) or ADA-deficient (ADA-iPSC-1 or ADA-iPSC-2) (A), the percentage of CD11b⁺/CD45⁺ neutrophils (B), and the number of neutrophils obtained per 4×10⁴ cells plated in methylcellulose (C), as well as the total number of neutrophils obtained from an equal initial seeding 6×10⁶ iPSCs (D), and the percentage of neutrophils that have phagocytized red fluorescent microspheres (E), without or with the supplementation of the media and methylcellulose with PEG-ADA. Data shown as mean + SD of (A) n = 9 replicates for CFU experiments, n = 6 replicates for CTL-iPSC-1 and ADA-iPSC-1 experiments, and n = 3 for CTL-iPSC-2 and ADA-iPSC-2 experiments; *p < 0.05; ***p < 0.001; ns, not significant.

Figure 4

Impaired neutrophil production from induced pluripotent stem cells following inhibition of ribonucleoside reductase. iPSCs were differentiated for 14 days to form EBs. The EBs were then dissociated and differentiated in methylcellulose for an additional 12 days to generate neutrophils, with CTL iPSCs treated with RNR inhibitors HU or NAM + TSA throughout the entirety of the differentiation. The effects of treatment of 6×10⁶ healthy control (CTL-iPSC-1 and CTL-iPSC-2) induced pluripotent stem cells (iPSC) with ribonucleoside reductase inhibitors hydroxyurea (HU) or nicotinamide (NAM) and trichostatin A (TSA) on (A) the number of CD34⁺/CD45⁺ multipotent hematopoietic progenitors (MHPs), (B) the percentage of CD11b⁺/CD45⁺ neutrophils, (C) the number of neutrophils obtained per 4×10⁴ cells plated in methylcellulose, and (D) the total number of neutrophils obtained from the 6×10⁶ seeded iPSCs. Data shown as mean + SD of n = 3 replicates.