ARYL HYDROCARBON RECEPTOR REGULATES HEMATOENDOTHELIAL DIFFERENTIATION FROM
HUMAN PLURIPOTENT STEM CELLS
Mathew G. Angelos and Dan S. Kaufman
University of Minnesota, Department of Medicine and Stem Cell Institute, Minneapolis, MN, USA
The aryl hydrocarbon receptor (AHR) is an evolutionarily conserved transcription factor originally characterized
for its role in mediating biological responses to carcinogenic environmental agents. Recent studies have
elucidated the importance of AHR-mediated signaling for normal physiological function in the absence of
environmental ligands, most notably the development of Th17 cells, regulatory T-cells, and natural killer (NK)
cells. Additionally, AHR is highly expressed in hematopoietic stem/progenitor cells (HSPCs), and antagonism
of AHR using small molecules results in a dramatic expansion of umbilical cord blood derived HSPCs suitable
for transplantation. It remains unclear what role, if any, AHR plays during early human hematoendothelial
development. We hypothesized modification of AHR signaling regulates early human hematopoietic cell
development. To test this hypothesis, we differentiated human embryonic stem cells (hESCs) under defined
hematopoietic-promoting media conditions for 15 days. qRT-PCR analysis demonstrated a significant increase
in AHR transcript (7.161.02, p<0.05) at Day 11 of differentiation relative to undifferentiated hESCs, which
correlated with an enriched CD31+CD34+ hematoendothelial population and the appearance of CD34+CD41+
and CD34+CD43+ hematopoietic progenitor cells. We modified the relative activity of AHR signaling by
differentiating hESCs in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a prototypical AHR
agonist, or StemReginin-1 (SR-1), an AHR antagonist, and assessed hematopoietic progenitor cell production
at Day 12. Interestingly, TCDD treatment increased the number CD41a+, CD43+, and CD45+ hematopoietic
cells relative to controls, while SR-1 treatment caused a reciprocal decrease in these populations. In assessing
hESCs with an integrated RUNX1c-tdTomato reporter, which serves as a marker of definitive hematopoiesis
from hemogenic endothelial cells, TCDD treatment further resulted in ~15% enrichment of tdTom+ cells from
CD34+CD41- and CD34+CD43- subsets. Collectively, these results suggest AHR activation can promote
hematoendothelial differentiation and may be used as a potential molecular target to enhance definitive
hematopoietic development from human pluripotent stem cells.
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Hematopoietic stem cell transplantation (HSCT) is an efficacious therapy for several hematological
malignancies, acquired genetic diseases, and bone marrow dysfunctions. Although successful, clinical hurdles
in the treatment of HSCT patients still persist, such as the availability of optimally matched HLA-donors and
immunological rejection of donor cells. Human pluripotent stem cells are an ideal cellular platform for the
derivation of hematopoietic cells with potential for clinical translation. While simplistic in theory, the production
of hematopoietic stem cells with robust long-term, multilineage engraftment in animal models from human
embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) has not been achieved. One
overarching hypothesis is hESCs/hiPSCs differentiate into cells that are intrinsically biased towards primitive
hematopoiesis, which is not capable of producing cells of lymphoid origin. My research focuses on the aryl
hydrocarbon receptor (AHR) and its role in definitive hematopoietic differentiation, the wave of hematopoiesis
that results in permanent, multilineage engraftment. Previous studies have demonstrated AHR signaling
pathway inhibition can support the expansion of umbilical cord blood derived hematopoietic stem cells that are
suitable for long-term transplantation. I seek to investigate whether this pathway can be correlated to
differentiating hESCs/hiPSCs in vitro to enhance definitive hematopoiesis. This work is significant because it
may potentially result in a novel method to improve hESC/hiPSC-derived hematopoietic cell engraftment.