TY - JOUR
T1 - CD1d expression demarcates CDX4+ hemogenic mesoderm with definitive hematopoietic potential
AU - Philip Creamer, J.
AU - Luff, Stephanie A.
AU - Yu, Hao
AU - Sturgeon, Christopher M.
N1 - Funding Information:
J.P.C. and C.M.S. formulated the initial concept, designed and performed the experiments, and analyzed the data; H.Y. performed experiments; J.P.C. and S.A.L., performed bioinformatics analyses; J.P.C., S.A.L. and C.M.S. wrote the manuscript. J.P.C and S.A.L. received support from the NHLBI T32 Training Grant (HL007088-41). This study was supported by an American Society of Hematology Scholar Award, an American Society of Hematology Bridge Grant, the Bill & Melinda Gates Foundation INV-002414, and NIH R01HL145290 and R01HL151777. Stem cell studies have been approved by the Washington University Embryonic Stem Cell Research Oversight Committee, ESCRO #14-001, and the Icahn School of Medicine Embryonic Stem Cell Research Oversight Committee, ESCRO #20-06.
Funding Information:
J.P.C. and C.M.S. formulated the initial concept, designed and performed the experiments, and analyzed the data; H.Y. performed experiments; J.P.C. and S.A.L. performed bioinformatics analyses; J.P.C. S.A.L. and C.M.S. wrote the manuscript. J.P.C and S.A.L. received support from the NHLBI T32 Training Grant (HL007088-41). This study was supported by an American Society of Hematology Scholar Award, an American Society of Hematology Bridge Grant, the Bill & Melinda Gates Foundation INV-002414, and NIH R01HL145290 and R01HL151777. Stem cell studies have been approved by the Washington University Embryonic Stem Cell Research Oversight Committee, ESCRO #14-001, and the Icahn School of Medicine Embryonic Stem Cell Research Oversight Committee, ESCRO #20-06.
Publisher Copyright:
© 2022 The Author(s)
PY - 2022/7
Y1 - 2022/7
N2 - To achieve efficient, reproducible differentiation of human pluripotent stem cells (hPSCs) towards specific hematopoietic cell-types, a comprehensive understanding of the necessary cell signaling and developmental trajectories involved is required. Previous studies have identified the mesodermal progenitors of extra-embryonic-like and intra-embryonic-like hemogenic endothelium (HE), via stage-specific WNT and ACTIVIN/NODAL, with GYPA/GYPB (CD235a/b) expression serving as a positive selection marker for mesoderm harboring exclusively extra-embryonic-like hemogenic potential. However, a positive mesodermal cell-surface marker with exclusively intra-embryonic-like hemogenic potential has not been identified. Recently, we reported that early mesodermal expression of CDX4 critically regulates definitive HE specification, suggesting that CDX4 may act in a cell-autonomous manner during hematopoietic development. To identify CDX4+ mesoderm, we performed single cell (sc)RNAseq on hPSC-derived mesodermal cultures, revealing CDX4hi expressing mesodermal populations were uniquely enriched in the non-classical MHC-Class-1 receptor CD1D. Flow cytometry demonstrated approximately 60% of KDR+CD34-CD235a- mesoderm was CD1d+, and CDX4 was robustly enriched within CD1d+ mesoderm. Critically, only CD1d+ mesoderm harbored CD34+ HOXA+ HE with multilineage erythroid-myeloid-lymphoid potential. Thus, CDX4+CD1d+ expression within early mesoderm demarcates an early progenitor of HE. These insights may be used for further study of human hematopoietic development and improve hematopoietic differentiation conditions for regenerative medicine applications.
AB - To achieve efficient, reproducible differentiation of human pluripotent stem cells (hPSCs) towards specific hematopoietic cell-types, a comprehensive understanding of the necessary cell signaling and developmental trajectories involved is required. Previous studies have identified the mesodermal progenitors of extra-embryonic-like and intra-embryonic-like hemogenic endothelium (HE), via stage-specific WNT and ACTIVIN/NODAL, with GYPA/GYPB (CD235a/b) expression serving as a positive selection marker for mesoderm harboring exclusively extra-embryonic-like hemogenic potential. However, a positive mesodermal cell-surface marker with exclusively intra-embryonic-like hemogenic potential has not been identified. Recently, we reported that early mesodermal expression of CDX4 critically regulates definitive HE specification, suggesting that CDX4 may act in a cell-autonomous manner during hematopoietic development. To identify CDX4+ mesoderm, we performed single cell (sc)RNAseq on hPSC-derived mesodermal cultures, revealing CDX4hi expressing mesodermal populations were uniquely enriched in the non-classical MHC-Class-1 receptor CD1D. Flow cytometry demonstrated approximately 60% of KDR+CD34-CD235a- mesoderm was CD1d+, and CDX4 was robustly enriched within CD1d+ mesoderm. Critically, only CD1d+ mesoderm harbored CD34+ HOXA+ HE with multilineage erythroid-myeloid-lymphoid potential. Thus, CDX4+CD1d+ expression within early mesoderm demarcates an early progenitor of HE. These insights may be used for further study of human hematopoietic development and improve hematopoietic differentiation conditions for regenerative medicine applications.
KW - CD1d
KW - CDX
KW - Definitive hematopoiesis
KW - Hemogenic endothelium
KW - Hemogenic mesoderm
KW - Human pluripotent stem cells
UR - http://www.scopus.com/inward/record.url?scp=85130125398&partnerID=8YFLogxK
U2 - 10.1016/j.scr.2022.102808
DO - 10.1016/j.scr.2022.102808
M3 - Article
C2 - 35569347
AN - SCOPUS:85130125398
SN - 1873-5061
VL - 62
JO - Stem Cell Research
JF - Stem Cell Research
M1 - 102808
ER -