Defining the Role of a GATA-2 Enhancer during Hematopoietic Differentiation of Human iPSCs

Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, US Food and Drug Administration, USA

Rationale : Differentiation of hematopoietic stem cells from human induced pluripotent stem cells (iPSCs) has the potential to provide novel therapeutic options for a wide range of blood diseases; however, despite significant progress that has been made in driving iPSC differentiation, issues with limited differentiation potential and engraftment capability in vivo still persist. A better understanding of the key regulators of hematopoiesis is essential for the development of improved differentiation protocols. GATA-2, a transcription factor shown to be essential for hematopoiesis, has been examined to determine its role in both hematopoiesis and endothelial cell development. Mouse studies have described an early developmental Gata-2 hematopoietic-specific enhancer located 5′ to its distal first exon. Based on homology within this regulatory domain between mouse and human sequences, we hypothesize that this cis-regulatory region plays an essential role in human hematopoietic expression of GATA-2 and in endothelial-to-hematopoietic transition.

Methods : In this study, we use a genome editing approach and an in vitro human iPSC differentiation system to examine our hypothesis. A pair of CRISPR/Cas9 guide RNA was designed to recognize sequences of a DNase I hypersensitive site (DHS) within the putative enhancer region immediately upstream of the GATA-2 distal first exon. Human iPSCs were treated with CRISPR/Cas9 to create cell lines that have a deletion at this DHS. These genetically modified iPSC lines were compared to their isogenic parental controls, to examine functional consequences of the deletion. Both adherent culture-based and spin-embryoid body formation-based protocols were used for mesodermal differentiation, endothelial specification and hematopoietic induction.

Results : Screening of human iPSC clones after co-transfection of Cas9 and guide RNA-expression vectors has identified clones with ~400 bp deletion of the DNase I hypersensitive site that is conserved among vertebrates. When maintained in iPSC expansion condition, these iPSC clones display typical undifferentiated human iPSC morphology and expression of pluripotency-related markers such as TRA-1-60. Upon treatment with a Wnt signaling activator, BMP4, bFGF and VEGF, the genetically modified cells showed similar differentiation efficiency, compared to parental controls, to mesoderm and endothelial lineages. However, they exhibit a lower proliferation rate in endothelial expansion conditions. Preliminary studies also showed a deficiency of the modified cells in their ability to form capillary-like structures in tube formation assays. In hematopoietic differentiation experiments using the spin-EB method, these cells showed very low viability upon hematopoietic induction and failed to produce hematopoietic progenitor cells. These results suggest that the DNase I hypersensitive site upstream of GATA2 distal first exon plays an important role in human endothelial cell function including transition from hemogenic endothelium to hematopoietic cells upon stimulation.

Currently, experiments are being conducted to characterize the functionality of endothelial-like cells and to confirm previous observations in additional iPSC lines. Differential expression of GATA-2 between parental and genome edited cells at various developmental stages is being determined. Hematopoietic differentiation capacity of these iPSCs by stromal cell co-culture methods is being used to confirm observations from the spin-EB protocol. Defining the functional outcome of this targeted deletion, across several iPSC lines, will provide a system for understanding molecular regulations in the first steps of human hematopoietic stem cell generation.