Wnt2 Coordinates the Commitment of Mesoderm to Hematopoietic, Endothelial, and Cardiac Lineages in Embryoid Bodies

Abstract

Our recent gene expression profiling analyses demonstrated that Wnt2 is highly expressed in Flk1؉ cells, which serve as common progenitors of endothelial cells, blood cells, and mural cells. In this report, we characterize the role of Wnt2 in mesoderm development during embryonic stem (ES) cell differentiation by creating ES cell lines in which Wnt2 was deleted. Wnt2؊/؊ embryoid bodies (EBs) generated increased numbers of Flk1؉ cells and blast colony-forming cells compared with wild-type EBs, and had higher Flk1 expression at comparable stages of differentiation. Although Flk1 ؉ cells were increased, we found that endothelial cell and terminal cardiomyocyte differentiation was impaired, but hematopoietic cell differentiation was enhanced and smooth muscle cell differentiation was unchanged in Wnt2 ؊/؊ EBs. Later stage Wnt2 ؊/؊ EBs had either lower or undetectable expression of endothelial and cardiac genes compared with wild-type EBs. Consistently, vascular plexi were poorly formed and neither beating cardiomyocytes nor ␣-actinin-staining cells were detectable in later stage Wnt2 ؊/؊ EBs. In contrast, hematopoietic cell gene expression was upregulated, and the number of hematopoietic progenitor colonies was significantly enhanced in Wnt2 ؊/؊ EBs. Our data indicate that Wnt2 functions at multiple stages of development during ES cell differentiation and during the commitment and diversification of mesoderm: as a negative regulator for hemangioblast differentiation and hematopoiesis but alternatively as a positive regulator for endothelial and terminal cardiomyocyte differentiation. Embryonic stem (ES)2 cells have considerable potential for use in cellular therapies for many human diseases or disorders.However, manipulating the differentiation of pluripotent cells into a desired cell type is difficult to control. Therefore, efforts have been directed toward understanding the molecular and cellular mechanisms for maintaining ES cell pluripotency as well as for ES cell fate determination and lineage specification. Under appropriate culture conditions, ES cells will spontaneously differentiate and form embryoid bodies (EBs) that recapitulate the differentiation program of normal embryonic development. This in vitro experimental model is widely used to study mesoderm development, including the hematopoietic system, vascular endothelial cells, and cardiomyocytes (1-4).Murine hematopoietic and endothelial cell development begins in the yolk sac mesoderm at approximately day 7.5 of gestation and is marked by establishment of blood islands (5, 6). The close physical relationship between these two cell populations within blood islands has led to a widely accepted hypothesis that a common precursor for both hematopoietic and endothelial cells exists, the hemangioblast (7, 8). The receptor for vascular endothelial growth factor (VEGF), fetal liver kinase-1 (Flk1), is an early molecular marker for hemangioblasts (9 -12). Blast colony-forming cells (BLCFCs) derived from EBs can generate hematopoietic and endothelial cell lineag...

Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of WNT5a and WNT11

“…This is further supported by other studies that report the differential WNT5a and WNT11 gene expression during embryonic development (37). In our study, we did not observe difference in the WNT2 expression between EBs of different sizes, which has been known to participate in mesoderm formation (38). Another important feature of our studies may be related to the size of the EBs and their influence on regulating the temporal expression of signaling molecules.…”

Osteogenic potency of stem cell-based genetic engineering targeting Wnt3a and Wnt9a

“…In addition, it has previously been reported that Wnt2 plays an important role in the differentiation of embryonic stem cells [30]. However, the present results revealed little, if any, effect of Wnt2 on the cell proliferation and the level of the ALP activity and Runx2 and OC expression, all of which are key markers of osteoblast differentiation.…”

The Multiple Phases and Faces of Wnt Signaling During Cardiac Differentiation and Development

“…67 It might be appropriate here to discuss the requirement of Wnt2b for cardiogenesis. 68 Wnt2b was initially found to be highly expressed in Flk1 ϩ progenitor cells 69 and subsequent generation of Wnt2b Ϫ/Ϫ ES cells indicated a requirement for Wnt2b for the cardiogenic but not for hematopoietic lineage. 68 More detailed analyses of these cells, however, indicated that the loss of Wnt2b is accompanied by an accelerated expression of Bry and Flk1.…”

“…68 Wnt2b was initially found to be highly expressed in Flk1 ϩ progenitor cells 69 and subsequent generation of Wnt2b Ϫ/Ϫ ES cells indicated a requirement for Wnt2b for the cardiogenic but not for hematopoietic lineage. 68 More detailed analyses of these cells, however, indicated that the loss of Wnt2b is accompanied by an accelerated expression of Bry and Flk1. This is an interesting observation given the positive role of Wnt/␤-catenin signaling for Bry expression, as discussed above.…”