In vitroerythropoiesis from bone marrow-derived progenitors provides a physiological assay for toxic and mutagenic compounds

Abstract

The goal of this study was to create an in vitro cell culture system that captures essential features of the in vivo erythroid micronucleus (MN) genotoxicity assay, thus enabling increased throughput and controlled studies of the hematopoietic DNA damage response. We show that adult bone marrow (BM) cultures respond to erythropoietin, the principal hormone that stimulates erythropoiesis, with physiological erythropoietic proliferation, differentiation, and enucleation. We then show that this in vitro erythropoietic system clearly signals exposure to genotoxicants through erythroid MN formation. Furthermore, we determined that DNA repair-deficient (MGMT ؊/؊ ) BM displayed sensitivity to genotoxic exposure in vivo compared with WT BM and that this phenotypic response was reflected in erythropoietic cultures. These findings suggest that this in vitro erythroid MN assay is capable of screening for genotoxicity on BM in a physiologically reflective manner. Finally, responses to genotoxicants during erythroid differentiation varied with exposure time, demonstrating that this system can be used to study the effect of DNA damage at specific developmental stages.in vitro toxicity screens ͉ genotoxicity ͉ hematopoiesis ͉ DNA damage and repair

The LSD1 inhibitor RN-1 induces fetal hemoglobin synthesis and reduces disease pathology in sickle cell mice

“…40,41 PGC-1a was forcibly expressed in Lin -BM cells by infection with a PGC-1a-expressing adenovirus (Ad-PGC-1a) ( Figure 7C-D). After several days, the cells were examined for mRNA expression of the globin genes.…”

“…40,41 After 1 day of culture, Lin -cells were infected with an adenovirus that expressed PGC-1a (Ad-PGC1a) or GFP (Ad-GFP) as a control. 42 Infected cells were harvested at the indicated times for QRT-PCR, western blot, and flow cytometric analyses.…”

Ineffective erythropoiesis caused by binucleated late-stage erythroblasts in mDia2 hematopoietic specific knockout mice

“…[11][12][13] Compared to the controls, the lineage-negative cells from mDia2 fl/fl Mx-Cre mice showed significantly decreased proliferation, differentiation, and enucleation (Online Supplementary Figure S3A, S3B, and S3C, respectively). Morphologic analysis of the erythroid cells with loss of mDia2 revealed frequent binucleated and enucleating cells (Online Supplementary Figure S3D), further confirming the in vivo assays and the cell-autonomous defects.…”

Compound loss of function of nuclear receptors Tr2 and Tr4 leads to induction of murine embryonic β-type globin genes

“…20 We found that compound depletion of TR2 and TR4 led to a substantial increase of ey-and bh1-globin transcripts (3.6-and 8.2-fold, respectively). Furthermore, the loss of TR2/TR4 resulted in blocking the differentiation, and impairing the maturation, of erythroid cells as anticipated from an earlier RNA sequencing (RNA-seq) analysis showing that the majority of genes affected by TR4 loss of function are intimately linked to vital cellular metabolic functions, 21 indicating that TR2/TR4 are required for erythroid cell survival.…”

“…16,24 Murine Lin 2 BM cells were cultured and induced to undergo terminal erythroid differentiation. 20,25 After 1 day of culture, Lin 2 cells were infected with an adenovirus that expressed Cre recombinase (Ad-Cre). After 48 hours, the cells were reinfected.…”