CD34+ fetal stem cells (FSC) are primitive cells which can be isolated from umbilical cord blood. They have hematopoietic potential and can reconstitute the different cell lineages of the bone marrow similar to adult CD34+ stem cells. There are gaps in knowledge related to mechanisms of FSC differentiation which can be used to develop therapy for genetic diseases such as the hemoglobinopathies. Insights into fetal erythropoiesis can be gained by understanding mechanisms of globin gene regulation in the human β-globin locus where five functional genes (ε, Aγ, Gγ, δ, β-globin) are expressed in a developmentally regulated fashion. Globin gene expression during development is controlled in part by the locus control region however genomic controls during fetal erythropoiesis have not been clearly elucidated. Therefore, we utilized FSC isolated from umbilical cord blood induced to undergo differentiation as a model to characterize the transcriptome associated with the γ/β globin switch during fetal erythropoiesis.
FSC (0.5 million) were grown in the one-phase liquid culture system containing stem cell factor (50ng/ml), interleukin-3 (10ng/ml) and erythropoietin (4U/ml). We observed a 30-fold higher proliferative capacity of FSC compared to adult progenitors reaching 10 billion cells by day-56 in culture. Erythroid differentiation was confirmed by Giemsa staining and increased expression of the differentiation biomarkers CD71 and CD235a and decline of CD34 was observed. Moreover, the γ/β globin switch occurred around day 45 in FSC compared to day 21 for adult stem cells (Li et al. BMC Genomics13:153, 2012). These data suggest that the γ-globin gene is activated for a longer period in the FSC environment in contrast to adult progenitors supporting different mechanisms of gene regulation in the two systems. To gain further insights, microarray analysis was performed on the HumanHT-12 v4 Expression BeadChip containing 47,000 probes, using triplicate RNA samples harvested on day 21, 42, 49 and 56. After data normalization by model-based background correction method, profile-1 genes with decreased expression from day-21 to day-56 (similar to γ-globin silencing) or profile-2 similar to β-globin activation from day-21 to day-56 were defined using Principal Component Analysis. Sixty-five profile-1 genes were subsequently identified by Gene Set Enrichment Analysis (GSEA) for erythroid-specific gene subsets. DAVID (Database for Annotation Visualization and Integrated Discovery) Ontology analysis confirmed 23 out of 65 erythroid genes including known regulators of γ-globin such as KLF4, KLF11, BCL11A and SIRT3. A similar analysis of profile-2 genes demonstrated 155 erythroid genes by GSEA and 28 factors were confirmed by DAVID platform related with β-globin regulation such as KLF1, GATA1, MXl1 and SOX6. To define transcription factor networks related to globin gene expression in FSC, the confirmed gene subsets were analyzed using the Michigan Molecular Interaction plugin for Cytoscape. A transcription factor network centered on KLF4 and GATA2 was defined; KLF4 positively regulates CREB, KLF6, GATA2, POU1F1, JUN and STAT3 while repressing Sp1. We previously demonstrated the ability of KLF4 to trans-activate γ-globin in adult stem cells. How these factors regulate γ-globin expression in FSC will be explored further. Similar studies for profile-2 genes revealed KLF1 and GATA1 as a major network hub. The special characteristics of fetal stem cell including high proliferative capacity and prolonged growth and γ-globin expression combined with a novel transcription factor network will be used to define genomic mechanisms of fetal erythropoiesis.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.