directed-differentiation-primitive-definitive-hematopoietic

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Washington University in St. Louis

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 CDX4 was expressed exclusively within definitive hematopoietic KDR(+)CD235a(-) mesoderm in a WNT- and fibroblast growth factor-dependent manner. We found that exogenous CDX4 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, CDX4 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.

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

Dyskeratosis congenita (DC) is a bone marrow failure syndrome associated with telomere dysfunction. The progression and molecular determinants of hematopoietic failure in DC remain poorly understood. Here, we use the directed differentiation of human embryonic stem cells harboring clinically relevant mutations in telomerase to understand the consequences of DC-associated mutations on the primitive and definitive hematopoietic programs. Interestingly, telomere shortening does not broadly impair hematopoiesis, as primitive hematopoiesis is not impaired in DC cells. In contrast, while phenotypic definitive hemogenic endothelium is specified, the endothelial-to-hematopoietic transition is impaired in cells with shortened telomeres. This failure is caused by DNA damage accrual and is mediated by p53 stabilization. These observations indicate that detrimental effects of telomere shortening in the hematopoietic system are specific to the definitive hematopoietic lineages. This work illustrates how telomere dysfunction impairs hematopoietic development and creates a robust platform for therapeutic discovery for treatment of DC patients.

Article	Year
Human definitive hematopoietic specification from pluripotent stem cells is regulated by mesodermal expression of CDX4. Blood\| PubMed ID: 28408465	2017
p53 Mediates Failure of Human Definitive Hematopoiesis in Dyskeratosis Congenita. Stem cell reports\| PubMed ID: 28757166	2017

Carissa Dege

Biography

JoVE Journal Publications

Other Publications