Activin A-Induced Differentiation of Embryonic Stem Cells into Endoderm and Pancreatic Progenitors—The Influence of Differentiation Factors and Culture Conditions

Sulzbacher, Sabine; Schroeder, Insa S.; Truong, Thanh; Wobus, Anna M.

doi:10.1007/s12015-009-9061-5

Cited by 92 publications

(63 citation statements)

References 144 publications

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“…In hESCs, however, this factor has been used to direct differentiation toward endoderm [22][23][24][25][26] and its potential action on the specification of hematopoietic mesoderm has yet to be reported. The inability of Activin A to stimulate the development of CD45 neg PFV hemogenic precursors combined with the reduction in frequency of hematopoietic output Bars for all panels indicate mean -SEM (*P < 0.05).…”

Section: Cerdan Et Almentioning

confidence: 99%

“…However, in instances where the activity of AF have been examined in hESCs, these factors have been shown to either maintain pluripotency [8,20,21] or induce endoderm specification [22][23][24][25][26] with one recent article examining their roles in early mesodermal differentiation in conjunction with bone morphogenetic protein 4 (BMP4) [27], but not hematopoiesis. Consequently, despite the documented role of AF in the regulation of human adult hematopoiesis [28,29], little focus has been directed to addressing the role of these factors in the progression of hESC development from mesoderm to blood.…”

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confidence: 99%

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Activin A Promotes Hematopoietic Fated Mesoderm Development Through Upregulation of Brachyury in Human Embryonic Stem Cells

Cerdan

McIntyre

Mechael

et al. 2012

Stem Cells and Development

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The development of the hematopoietic system involves multiple cellular steps beginning with the formation of the mesoderm from the primitive streak, followed by emergence of precursor populations that become committed to either the endothelial or hematopoietic lineages. A number of growth factors such as activins and fibroblast growth factors (FGFs) are known to regulate the early specification of hematopoietic fated mesoderm, notably in amphibians. However, the potential roles of these factors in the development of mesoderm and subsequent hematopoiesis in the human have yet to be delineated. Defining the cellular and molecular mechanisms by which combinations of mesoderm-inducing factors regulate this stepwise process in human cells in vitro is central to effectively directing human embryonic stem cell (hESC) hematopoietic differentiation. Herein, using hESCderived embryoid bodies (EBs), we show that Activin A, but not basic FGF/FGF2 (bFGF), promotes hematopoietic fated mesodermal specification from pluripotent human cells. The effect of Activin A treatment relies on the presence of bone morphogenetic protein 4 (BMP4) and both of the hematopoietic cytokines stem cell factor and fms-like tyrosine kinase receptor-3 ligand, and is the consequence of 2 separate mechanisms occurring at 2 different stages of human EB development from mesoderm to blood. While Activin A promotes the induction of mesoderm, as indicated by the upregulation of Brachyury expression, which represents the mesodermal precursor required for hematopoietic development, it also contributes to the expansion of cells already committed to a hematopoietic fate. As hematopoietic development requires the transition through a Brachyury + intermediate, we demonstrate that hematopoiesis in hESCs is impaired by the downregulation of Brachyury, but is unaffected by its overexpression. These results demonstrate, for the first time, the functional significance of Brachyury in the developmental program of hematopoietic differentiation from hESCs and provide an in-depth understanding of the molecular cues that orchestrate stepwise development of hematopoiesis in a human system.

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Section: Cerdan Et Almentioning

confidence: 99%

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confidence: 99%

Activin A Promotes Hematopoietic Fated Mesoderm Development Through Upregulation of Brachyury in Human Embryonic Stem Cells

Cerdan

McIntyre

Mechael

et al. 2012

Stem Cells and Development

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“…Sulzbacher et al hypothesized that the pluripotency or differentiation of cells is dependent and occurs in a concentration-and stage-dependent manner (1). activin a (5-50 ng/ml) was applied to maintain the pluripotency of human ES and ipS cells alone or with other growth factors (6,10,11,15).…”

Section: Discussionmentioning

confidence: 99%

“…Introduction activin a, a member of the transforming growth factor-β superfamily, mimics nodal, binds activin receptors and phosphorylates Smad2, thus activating it (1). Once activated, Smad2 associates with Smad4, translocates to the nucleus and regulates gene expression in conjuction with other transcription factors.…”

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confidence: 99%

Activin A maintains pluripotency markers and proliferative potential of human induced pluripotent stem cells

Tomizawa¹,

Shinozaki²,

Sugiyama³

et al. 2011

Experimental and Therapeutic Medicine

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Abstract. to investigate the role of activin a in the embryoid bodies (EBs) of human induced pluripotent stem (ipS) cells, EBs were transferred onto dishes coated with matrigel after 4 days of incubation with activin a and observed under a microscope. alkaline phosphatase staining and immunostaining were performed to analyze the pluripotency of the cells, and the mtS assay was performed to analyze their proliferative potential. Fourteen days after EB formation, cells cultured with activin a (100 ng/ml) showed no morphological alterations. cells cultured with 10-100 ng/ml of activin a were positive for alkaline phosphatase staining, while cells cultured with 0-3 ng/ml showed negative staining. cells cultured with 10 ng/ml of activin a were positive for Oct3/4, nanog, SSEa-4 and tra-1-60, while cells cultured with 0 ng/ml activin a were negative. cells cultured with 3-30 ng/ml of activin a maintained their proliferative potential, while loss of proliferative potential was observed in cells cultured with 100 ng/ml activin a. in conclusion, activin a maintained pluripotency markers in human ipS cells cultured as EBs with activin a.Introduction activin a, a member of the transforming growth factor-β superfamily, mimics nodal, binds activin receptors and phosphorylates Smad2, thus activating it (1). Once activated, Smad2 associates with Smad4, translocates to the nucleus and regulates gene expression in conjuction with other transcription factors. nodal/activin a signals play a crucial role in inducing the development of the mesoderm and endoderm in the xenopus embryo (2,3). activin a (100 ng/ml) promotes the differentiation of human embryonic stem (ES) cells into pancreatic β cells (4), and also into endoderm (5). By contrast, 5 ng/ml of activin a was found to maintain self-renewal and to support the longterm feeder-free culture of human ES cells (6). however, it has not been fully established whether activin a induces the differentiation of human ES cells into endoderm or maintains them in an undifferentiated state. curiously, 100 ng/ml of activin a, FGF-2 and Bmp-4 was found to induce the expression of endoderm markers, while still maintaining pluripotency markers such as Oct3/4 and nanog (7). these data suggest that a mixture of other growth factors or the specific concentration of Activin A may determine whether human ES cells differentiate or whether their pluripotency is maintained. to develop a feeder-free medium of human ES or induced pluripotent stem (ipS) cells, it is necessary to determine the appropriate concentration of activin a. Materials and methodsCell culture and embryoid body formation. the human ipS cell line 201B7 (riken cell Bank, tsukuba, Japan) was cultured in reproFF media (reprocell, Yokohama, Japan) for feeder-free culture (reprocell, tokyo, Japan) on dishes (asahi techno Glass, Funabashi, Japan) coated with matrigel (Becton dickinson, Franklin lakes, nJ, uSa) in 5% carbon dioxide at 37˚C in a humidified chamber, and harvested with accutase (innovative cell technologies, inc., San diego, ca...

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“…Activin A is a ubiquitous regulator of myriad cellular functions during development and in the adult animal. It plays roles in cell proliferation (Boitani et al 1995, Ota et al 2003, Mendis et al 2011, differentiation (Seishima et al 1999, Sulzbacher et al 2009), apoptosis (Zhang et al 1997, Chen et al 2000, immune responses (Jones et al 2007, Robson et al 2009, and many other cellular activities. Activin A is a homodimer of bA subunits and signals by binding to types 1 and 2 transmembrane serine kinase receptors.…”

Section: Introductionmentioning

confidence: 99%

Regulation of FSHβ induction in LβT2 cells by BMP2 and an Activin A/BMP2 chimera, AB215

Jung

Ahn

Shim

et al. 2014

Journal of Endocrinology

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Activins and bone morphogenetic proteins (BMPs) share activin type 2 signaling receptors but utilize different type 1 receptors and Smads. We designed AB215, a potent BMP2-like Activin A/BMP2 chimera incorporating the high-affinity type 2 receptor-binding epitope of Activin A. In this study, we compare the signaling properties of AB215 and BMP2 in HEK293T cells and gonadotroph LbT2 cells in which Activin A and BMP2 synergistically induce FSHb. In HEK293T cells, AB215 is more potent than BMP2 and competitively blocks Activin A signaling, while BMP2 has a partial blocking activity. Activin A signaling is insensitive to BMP pathway antagonism in HEK293T cells but is strongly inhibited by constitutively active (CA) BMP type 1 receptors. By contrast, the potencies of AB215 and BMP2 are indistinguishable in LbT2 cells and although AB215 blocks Activin A signaling, BMP2 has no inhibitory effect. Unlike HEK293T, Activin A signaling is strongly inhibited by BMP pathway antagonism in LbT2 cells but is largely unaffected by CA BMP type 1 receptors. BMP2 increases phospho-Smad3 levels in LbT2 cells, in both the absence and the presence of Activin A treatment, and augments Activin A-induced FSHb. AB215 has the opposite effect and sharply decreases basal phospho-Smad3 levels and blocks Smad2 phosphorylation and FSHb induction resulting from Activin A treatment. These findings together demonstrate that while AB215 activates the BMP pathway, it has opposing effects to those of BMP2 on FSHb induction in LbT2 cells apparently due to its ability to block Activin A signaling. Key Words" transforming growth factor beta (TGFb)" activin " bone morphogenetic protein (BMP)" protein structure " signal transduction

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Activin A-Induced Differentiation of Embryonic Stem Cells into Endoderm and Pancreatic Progenitors—The Influence of Differentiation Factors and Culture Conditions

Cited by 92 publications

References 144 publications

Activin A Promotes Hematopoietic Fated Mesoderm Development Through Upregulation of Brachyury in Human Embryonic Stem Cells

Activin A Promotes Hematopoietic Fated Mesoderm Development Through Upregulation of Brachyury in Human Embryonic Stem Cells

Activin A maintains pluripotency markers and proliferative potential of human induced pluripotent stem cells

Regulation of FSHβ induction in LβT2 cells by BMP2 and an Activin A/BMP2 chimera, AB215

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