The Effects of Environmental Cues on Hematopoietic Stem Cell Colony Fetal Hemoglobin Production.
Authors: Nichols Jennifer L , Lavelle Donald , DeSimone Joseph 
Published: October 2019

Abstract
Abstract 3860

Objective.
Increased fetal hemoglobin (HbF) levels are associated with increased life span and reduced pain crises in patients with Sickle Cell disease. An increased understanding of the mechanisms controlling HbF expression would be important to develop new therapies to increase HbF. The objective of these experiments was to test the hypothesis that interactions between bone marrow (BM) erythroid progenitor cells and the stromal microenvironment influence HbF expression.

Materials and Methods.
Baboon CD34+ BM cells were harvested and purified by immunomagnetic column chromatography using the 12.8 anti-CD34 mouse monoclonal antibody and immunomagnetic microbeads conjugated to rat anti-mouse IgM (Miltenyi). CD34+ BM Cells were grown in liquid cultures in Iscove's media containing 30% fetal bovine serum, 200ng/ml stem cell factor, 2u/ml erythropoietin, and 1uM dexamethasone in the presence of an AFT024 mouse fetal liver cell line and an OP9 mouse bone marrow cell line as feeder layers, in methylcellulose media, and in liquid media. Globin chain synthesis in cultures was measured on d11 and d14 by biosynthetic radiolabeling. Cells were incubated overnight in leucine-free α-MEM media containing 50uCi/ml [3H leucine]. Globin chains were separated by High Performance Liquid Chromatography (HPLC), and the radioactivity in each fraction was determined by liquid scintillation counting. To correlate changes in γ-globin expression with DNA methylation of the γ-globin gene promoter, the methylation state of 5 CpG sites within the γ-globin promoter region was determined by bisulfite sequencing. Erythroid cells were purified from cultures on d14 by immunomagnetic column chromatography using mouse anti-baboon RBC monoclonal antibody. DNA was isolated from purified erythroid cells using Qiagen kits. Bisulfite modification was performed using Epitect bisulfite kits. Two rounds of PCR amplification were performed using nested primers flanking 5 CpG residues within the baboon γ-globin gene promoter. Minilysate DNA was prepared from at least 10 independent clones for each sample. Sequence analysis of purified DNA samples was performed at the University of Illinois DNA Sequence facility.

Results.
Elevated levels of γ-globin chain synthesis were observed in cells cultured in methylcellulose and liquid media in the absence of a feeder layer when compared to cells grown in the presence of feeder layers. On d11, the γ/γ+β chain synthetic ratio was 0.623 in cells cultured in methylcellulose and 0.324 in cells cultured in liquid media, compared to 0.092 in cells cultured in the presence of AFT024 feeder layers and 0.178 in cells cultured in the presence of OP9 feeder layers. On d14, the γ/γ+β chain synthetic ratio was 0.663 in cells cultured in methylcellulose and 0.349 in cells cultured in liquid media, compared to 0.135 in cells cultured in the presence of AFT024 feeder layers and 0.252 in cells cultured in the presence of OP9 feeder layers. The level of DNA methylation (%dmC) of 5 sites in the γ-globin gene promoter negatively correlated with levels of HbF production. On d14, the level of DNA methylation was 79% in cells cultured in methylcellulose, 72% in cells cultured in liquid media, and 97% in cells grown in the presence of AFT024 feeder layers.

Conclusions.
Cells grown in the presence of AFT024 feeder layers expressed physiologic levels of γ-globin and cells grown in the presence of OP9 feeder layers expressed midrange levels of γ-globin. Significantly increased γ-globin expression was observed in cells cultured in the absence of a feeder layer. The γ-globin promoter in cells grown in the presence of the AFT024 feeder layer exhibited a significantly higher degree of methylation than in cells grown in methylcellulose and liquid media. These results show that interactions between erythroid progenitor cells and the stromal microenvironment can influence both the level of γ-globin expression and the DNA methylation of the γ-globin gene promoter. These results have clinical relevance; if the bone marrow microenvironment could be effectively altered in vivo, methylation of the γ-globin promoter could be decreased and HbF production increased. Further experiments must be performed to determine what is mediating the methylation of the γ-globin promoter when cells are grown on these feeder layers.

Disclosures:
No relevant conflicts of interest to declare.