Human Definitive Hematopoietic Specification from Pluripotent Stem Cells Is Regulated By Mesodermal Expression of CDX4

The generation of hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine, as it will provide an unlimited source of these cells for transplantation, and a unique platform for the study of both normal and disease hematopoietic processes. To reproducibly achieve this goal in all hPSC lines, we must first fully understand hematopoietic ontogeny.

Understanding hematopoietic development is complicated by the existence of at least two distinct programs during development that are difficult to distinguish: a transient "primitive" extra-embryonic hematopoietic program that does not give rise to HSCs, and a "definitive" program that gives rise to HSCs and all hematopoietic lineages. We have recently developed a novel method to obtain and identify, from hPSCs, mesoderm harboring exclusively primitive or exclusively definitive hematopoietic potential, by the differential expression of CD235a within KDR+ mesoderm. With this, we were able to determine that stage-specific canonical Wnt signaling specified the definitive hematopoietic program, while simultaneously repressing the primitive hematopoietic program. Further, we have now found that definitive hematopoietic specification is also dependent on bFGF signaling during this same window of time. We then asked if this tractable system could help us understand the genetic regulation of definitive hematopoietic specification from hPSCs. We isolated Wnt-dependent KDR+CD235a- definitive hematopoietic mesoderm and Wnt-independent KDR+CD235a+ primitive hematopoietic mesoderm and performed whole-transcriptome gene expression analysis, which revealed strong CDX and HOX gene enrichment exclusively within KDR+CD235a- definitive hematopoietic mesoderm. Monitoring CDX expression over time in the differentiation cultures revealed that both CDX1 and CDX2 were expressed in a bFGF-independent manner, prior to Wnt-dependent definitive hematopoietic KDR+CD235a- mesoderm specification. In contrast, CDX4 was expressed exclusively within definitive hematopoietic KDR+CD235a- mesoderm in a Wnt- and bFGF-dependent manner. This expression pattern suggested that CDX4 expression is specific to definitive, but not primitive, hematopoietic specification.

To determine whether CDX4 expression plays a role in definitive hematopoietic specification, we generated an inducible CDX4 expression hPSC line using the "safe-harbor" AAVS1 locus. We manipulated exogenous CDX4 expression during the same stage of the differentiation culture that Wnt signaling is critical for definitive hematopoietic specification. Interestingly, in the absence of Wnt stimulation, exogenous CDX4 expression caused a >90% repression in primitive hematopoietic potential. Critically, CDX4 expression during this same time conferred 10-fold greater definitive hematopoietic potential within CD34+CD73-CD184- hemogenic endothelium, giving rise to definitive erythroid-myeloid-lymphoid multilineage progenitors. This is consistent with CDX4 being the transcriptional effector of Wnt signaling during early hematopoietic specification within mesoderm.

We next generated a CDX4 knockout hPSC line by CRISPR/Cas9, and a CDX4 knockdown hPSC line via shRNA expression from the AAVS1 locus. As expected, the absence of CDX4 expression did not reduce primitive hematopoietic potential in comparison to control lines. However, when Wnt signaling was stimulated to specify definitive hematopoiesis, hPSCs lacking CDX4 expression exhibited a 10-fold decrease in definitive CD34+CD73-CD184- hemogenic endothelium specification, indicating its expression is critical for definitive hematopoietic specification. Taken together, these findings indicate that CDX4 is the earliest identified transcription factor that is a critical regulator of human definitive hematopoietic specification, and provide a mechanistic basis for Wnt-mediated definitive hematopoietic specification from hPSCs. By understanding the genetic regulation of early definitive hematopoietic specification from hPSCs, we can now identify the additional signal pathways required for efficient HSC specification from hPSCs.