Human definitive hematopoietic specification from pluripotent stem cells is regulated by mesodermal expression of CDX4

Abstract

The generation of hematopoietic stem cells from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine. Achieving this goal is complicated by our incomplete understanding of the mechanism regulating definitive hematopoietic specification. We used our stage-specific hPSC differentiation method to obtain and identify, via CD235a expression, mesoderm harboring exclusively primitive or definitive hematopoietic potential to understand the genetic regulation of definitive hematopoietic specification. Whole-transcriptome gene expression analyses on WNT-dependent KDR⁺CD235a^- definitive hematopoietic mesoderm and WNT-independent KDR⁺CD235a⁺ primitive hematopoietic mesoderm revealed strong CDX gene expression within definitive hematopoietic mesoderm. Temporal expression analyses revealed that <i>CDX4</i> was expressed exclusively within definitive hematopoietic KDR⁺CD235a^- mesoderm in a WNT- and fibroblast growth factor-dependent manner. We found that exogenous <i>CDX4</i> expression exclusively during mesoderm specification resulted in a >90% repression in primitive hematopoietic potential, but conferred fivefold greater definitive hematopoietic potential, similar to that observed following WNT stimulation. In contrast, <i>CDX4</i> knockout hPSCs had intact primitive hematopoietic potential, but exhibited a fivefold decrease in multilineage definitive hematopoietic potential. Taken together, these findings indicate that CDX4 is a critical transcription factor in the regulation of human definitive hematopoietic specification, and provides a mechanistic basis for WNT-mediated definitive hematopoietic specification from hPSCs.