CD1d expression demarcates CDX4+ hemogenic mesoderm with definitive hematopoietic potential

Area of Science:

• Stem cell biology
• Developmental biology
• Hematopoiesis

Background:

• Efficient differentiation of human pluripotent stem cells (hPSCs) into specific hematopoietic cell types requires understanding signaling pathways and developmental trajectories.
• Previous research identified markers for extra-embryonic-like hemogenic endothelium (HE) but lacked a specific marker for intra-embryonic-like HE.
• CDX4 expression in early mesoderm was linked to definitive HE specification, suggesting a cell-autonomous role.

Purpose of the Study:

• To identify a specific cell surface marker for mesodermal progenitors with intra-embryonic-like hemogenic potential.
• To investigate the role of CDX4 in definitive HE specification.
• To improve hematopoietic differentiation protocols for regenerative medicine.

Main Methods:

• Single-cell RNA sequencing (scRNAseq) of hPSC-derived mesodermal cultures to identify CDX4+ mesodermal populations.
• Flow cytometry to validate the expression of CD1D and CDX4 in specific mesodermal subpopulations.
• Analysis of hemogenic endothelium (HE) markers (CD34, HOXA) and multilineage potential in CD1D+ mesoderm.

Main Results:

• scRNAseq revealed CDX4-high mesodermal populations were uniquely enriched for the non-classical MHC-Class-1 receptor CD1D.
• Flow cytometry confirmed CD1D expression in a significant portion of KDR+CD34-CD235a- mesoderm, with robust CDX4 enrichment within CD1D+ cells.
• Only CD1D+ mesoderm contained CD34+ HOXA+ HE with multilineage erythroid-myeloid-lymphoid potential.

Conclusions:

• CDX4+CD1D+ expression in early mesoderm identifies a specific progenitor population for intra-embryonic-like hemogenic endothelium (HE).
• CD1D serves as a novel positive cell surface marker for HE with multilineage potential.
• These findings enhance the understanding of human hematopoietic development and offer potential for improved regenerative medicine strategies.