ABSTRACT
Notch signaling is essential for definitive hematopoiesis, but its role in human embryonic hematopoiesis is largely unknown. We show that in hESCs the expression of the Notch ligand DLL4 is induced during hematopoietic differentiation. We found that DLL4 is only expressed in a sub-population of bipotent hematoendothelial progenitors (HEPs) and segregates their hematopoietic versus endothelial potential. We demonstrate at the clonal level and through transcriptome analyses that DLL4(high) HEPs are enriched in endothelial potential, whereas DLL4(low/-) HEPs are committed to the hematopoietic lineage, albeit both populations still contain bipotent cells. Moreover, DLL4 stimulation enhances hematopoietic differentiation of HEPs and increases the amount of clonogenic hematopoietic progenitors. Confocal microscopy analysis of whole differentiating embryoid bodies revealed that DLL4(high) HEPs are located close to DLL4(low/-) HEPs, and at the base of clusters of CD45+ cells, resembling intra-aortic hematopoietic clusters found in mouse embryos. We propose a model for human embryonic hematopoiesis in which DLL4(low/-) cells within hemogenic endothelium receive Notch-activating signals from DLL4(high) cells, resulting in an endothelial-to-hematopoietic transition and their differentiation into CD45+ hematopoietic cells.
Subject(s)
Biomarkers/metabolism , Cell Differentiation , Embryonic Stem Cells/cytology , Endothelium/cytology , Hematopoiesis/physiology , Hematopoietic Stem Cells/cytology , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Animals , Cell Proliferation , Cells, Cultured , Embryoid Bodies , Embryonic Stem Cells/metabolism , Endothelium/metabolism , Female , Flow Cytometry , Gene Expression Profiling , Hematopoietic Stem Cells/metabolism , Humans , Immunoenzyme Techniques , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins/genetics , Mice , Mice, Inbred NOD , Mice, SCID , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND: Human embryonic stem cells (hESCs) have opened up a new area of research in biomedicine. The efficiency of hESC derivation from frozen poor-quality embryos is low and normally achieved by plating embryos on mouse or human foreskin feeders (HFFs). We attempted to optimize embryo survival and hESC derivation. METHODS: Three conditions were tested on frozen poor-quality embryos: (i) embryo treatment with the Rho-associated kinase (ROCK) inhibitor, Y-27632; (ii) use of human mesenchymal stem cells (hMSCs) as feeders; and (iii) laser drilling (LD) for inner cell mass (ICM) isolation. Two hundred and nineteen thawed embryos were randomly treated with (n = 110) or without (n = 109) 10 microM Y-27632. Surviving embryos that developed to blastocyst stage (n = 50) were randomly co-cultured on HFFs (n = 21) or hMSCs (n = 29). ICM isolation was either by whole-blastocyst culture (WBC) or WBC plus LD. RESULTS: Embryo survival was 52% higher with Y-27632. hMSCs appeared to facilitate ICM outgrowth and hESC derivation: three hESC lines were derived on hMSCs (10.3% efficiency) whereas no hESC line was derived on HFFs. ROCK inhibition and ICM isolation method did not affect hESC efficiency. The lines derived on hMSCs (AND-1, -2, -3) were characterized and showed typical hESC morphology, euploidy, surface marker and transcription factor expression and multilineage developmental potential. The hESC lines have been stable for over 38 passages on hMSCs. CONCLUSION: Our data suggest that Y-27632 increases post-thaw embryo survival and that hMSCs may facilitate the efficiency of hESC derivation from frozen poor-quality embryos.
