Increased Level of Fetal Hemoglobin in Umbilical Cord Blood CD34+ Hematopoietic Stem Cell by New Drug GSK-LSD1 for Treatment of Thalassemia

Background and Aim: Switch from fetal γ-globin to β-globin geneexpression occurs at birth. The genetic regulation of this switch has beenstudied for decades, and the molecular basis for γ-globin silencing andchange in molecular mechanisms in the expressed genes have beenelucidated. Elevated γ-globin synthesis can significantly alleviate thesymptoms of β-globin disorders. Several adult-stage γ-globin repressors,such as BCL11A, Ikaros, GATA1, and SOX6 have been identified thatinteract with each other to repress the γ globin genes. In addition, Lysinespecificdemethylase-1 (LSD1) was recently shown to associate with corepressors,including DNA methyltransferase I (DNMT1) and TR2 andTR4, as components of a core heterotetrameric complex. An activatingepigenetic signature. Recent data suggest that CD34+ is involved in themaintenance of the progenitor cells in a phenotypically undifferentiatedstate. Primary human CD34+ cells isolated from umbilical cord bloodmononuclear cells (MNCs) include hematopoietic stem and progenitorcells. Recently, it was shown that lysine-specific demethylase-1 (LSD1)that remove monomethyl and dimethyl residues from the lysine 4 canrepress γ- globin gene expression. In this report, we investigated theinhibition of LSD1 by the GSK-LSD1 inhibitor in human erythroid CD34+cells to increase γ-globin gene expression.Methods: We grew and differentiated the cells ex vivo into the erythroidlineage in 14 d by a two-phase culture method described previously.We examined the effects of the GSK-LSD1 inhibitor on CD34+ cells areisolated from cord blood using positive immunomagnetic separationtechniques, cells ex vivo. Cell number and viability were determined bytrypan blue staining. Cell morphology was examined by Wright-Giemsastaining (Sigma-Aldrich). Flow-cytometric analysis showed that HbF wasinduced in all of the cells in a dose-dependent manner. We treated thecells with 0, 0.5, 1.5, and 5 μM of the GSK-LSD1 inhibitor on days 4 to14 of the differentiation culture. Then we performed an analysis of theexpression of LSD1 and γ-globin genes comparable levels throughoutdifferentiation with real-time PCR using the IQ SYBR Green Master mix.Results: After treatment GSK-LSD1 inhibitor at 0.5, 1.5, and 5 μM didnot alter cell proliferation or viability, but 5 μM GSK-LSD1 reduced cellproliferation and delayed differentiation without affecting cell viability.In 1.5-μM concentration of the GSK-LSD1 inhibitor, the mean of γ-globinmRNA expression was induced up to 33-fold. We observed a decreasein the LSD1 mRNA expression in a 5-μM concentration of the GSK-LSD1inhibitor.Conclusion: Our results indicated that LSD1 played an important rolein γ-globin silencing in adult erythroid cells. Further, the GSK- LSD1inhibitor increase concentration of HbF induction within the therapeuticplasma concentration. Finally, LSD1 is thus a promising therapeutictarget for γ-globin induction, and GSK-LSD1 inhibitor leads compoundfor the development of a new γ-globin inducer.

کلیدواژه ها:

Umbilical Cord Blood ، CD34+ cell ، GSK-LSD1 ، γ- globin ، Lysine-specific demethylase-1