In vitro human embryonic stem cell hematopoiesis mimics MYB-independent yolk sac hematopoiesis.
Although hematopoietic precursor activity can be generated in vitro from human embryonic stem cells, there is no solid evidence for the appearance of multipotent, self-renewing and transplantable hematopoietic stem cells. This could be due to short half-life of hematopoietic stem cells in culture or, alternatively, human embryonic stem cell-initiated hematopoiesis may be hematopoietic stem cell-independent, similar to yolk sac hematopoiesis, generating multipotent progenitors with limited expansion capacity. Since a MYB was reported to be an excellent marker for hematopoietic stem cell-dependent hematopoiesis, we generated a MYB-eGFP reporter human embryonic stem cell line to study formation of hematopoietic progenitor cells in vitro. We found CD34(+) hemogenic endothelial cells rounding up and developing into CD43(+) hematopoietic cells without expression of MYB-eGFP. MYB-eGFP(+) cells appeared relatively late in embryoid body cultures as CD34(+)CD43(+)CD45(-/lo) cells. These MYB-eGFP(+) cells were CD33 positive, proliferated in IL-3 containing media and hematopoietic differentiation was restricted to the granulocytic lineage. In agreement with data obtained on murine Myb(-/-) embryonic stem cells, bright eGFP expression was observed in a subpopulation of cells, during directed myeloid differentiation, which again belonged to the granulocytic lineage. In contrast, CD14(+) macrophage cells were consistently eGFP(-) and were derived from eGFP-precursors only. In summary, no evidence was obtained for in vitro generation of MYB(+) hematopoietic stem cells during embryoid body cultures. The observed MYB expression appeared late in culture and was confined to the granulocytic lineage.
Laboratory for Experimental Immunology, Ghent University, Belgium.
Stijn
S
Vanhee
Laboratory for Experimental Immunology, Ghent University, Belgium.
Katrien
K
De Mulder
Laboratory for Experimental Immunology, Ghent University, Belgium.
Yasmine
Y
Van Caeneghem
Laboratory for Experimental Immunology, Ghent University, Belgium.
Greet
G
Verstichel
Center for Medical Genetics, Ghent University, Belgium.
Nadine
N
Van Roy
Center for Medical Genetics, Ghent University, Belgium.
Björn
B
Menten
Laboratory for Experimental Immunology, Ghent University, Belgium.
Imke
I
Velghe
Department of Clinical Biology, Microbiology and Immunology, Ghent University Hospital, Belgium.
Jan
J
Philippé
Red Cross Flanders, Ghent, Belgium.
Dominique
D
De Bleser
Laboratory of Immunoregulation and Mucosal Immunology, Department of Pulmonary Medicine, Ghent University Hospital, Belgium Flanders Institute for Biotechnology (VIB) Inflammation Research Center, Ghent University, Belgium.
Bart N
BN
Lambrecht
Laboratory for Experimental Immunology, Ghent University, Belgium.
Tom
T
Taghon
Laboratory for Experimental Immunology, Ghent University, Belgium.
Georges
G
Leclercq
Laboratory for Experimental Immunology, Ghent University, Belgium.
Tessa
T
Kerre
Laboratory for Experimental Immunology, Ghent University, Belgium bart.vandekerckhove@ugent.be.
Bart
B
Vandekerckhove
D002454:Cell Differentiation; D019070:Cell Lineage; D002478:Cells, Cultured; D058732:Embryoid Bodies; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D006098:Granulocytes / Q000166:cytology / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006410:Hematopoiesis / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D020598:Proto-Oncogene Proteins c-myb / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019076:Transgenes / Q000502:physiology; D015017:Yolk Sac / Q000166:cytology* / Q000378:metabolism
D020598:Proto-Oncogene Proteins c-myb; D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins