trichostatin-a and Severe-Combined-Immunodeficiency

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.

Topics: Adenosine Deaminase; Agammaglobulinemia; Cells, Cultured; Embryoid Bodies; Fibroblasts; Granulocytes; Humans; Hydroxamic Acids; Hydroxyurea; Induced Pluripotent Stem Cells; Infant; Male; Mutation, Missense; Myelopoiesis; Neutrophils; Niacinamide; Point Mutation; Ribonucleotide Reductases; Severe Combined Immunodeficiency

We investigated whether the addition of two chromatin-modifying agents, 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA), to cord blood (CB) CD34(+) cells in culture results in expansion of the numbers of severe combined immunodeficient (SCID) repopulating cells (SRC).. Human CB CD34(+) cells were cultured with cytokines in the presence or absence of 5azaD/TSA. After 9 days of culture, the fold expansion of CD34(+) and CD34(+)CD90(+) cell numbers, colony-forming unit (CFU)-mix, cobblestone area-forming cell (CAFC), and SRC numbers were determined.. A 12.5-fold expansion of CD34(+)CD90(+) cells was observed in the 5azaD/TSA-treated cultures in comparison to the input cell numbers. Expansion of CD34(+)CD90(+) cells was associated with a 9.8-fold increase in the numbers of CFU-mix and 11.5-fold increase in CAFC. 5azaD/TSA treatment of the CB CD34(+) cells resulted in a 9.6-fold expansion of the absolute number of SRC following 9 days of culture as determined by limiting dilution analysis. Expansion of cells maintaining CD34(+)CD90(+) phenotype was not due to the retention of a quiescent population of cells because all of the CD34(+)CD90(+) cells in the culture had undergone cellular division. 5azaD/TSA-treated CD34(+)CD90(+) cells, but not CD34(+)CD90(-) cells were responsible for in vivo hematopoietic repopulation potential of nonobese diabetic/SCID mice.. Ex vivo expansion strategy using chromatin-modifying agents provides a potential avenue by which to expand the number of hematopoietic stem cells (HSC) with a single CB unit for use as an alternative source of HSC grafts for adult recipients.

Topics: Animals; Antigens, CD34; Azacitidine; Cells, Cultured; Chromatin; Cord Blood Stem Cell Transplantation; Cytokines; Decitabine; Hematopoietic Stem Cells; Humans; Hydroxamic Acids; Mice; Mice, Inbred NOD; Mice, SCID; Severe Combined Immunodeficiency; Thy-1 Antigens; Transplantation, Heterologous