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

Abstract

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...

A cytokine screen using CRISPR-Cas9 knock-in reporter pig iPS cells reveals that Activin A regulates NANOG

“…This is also observed in humans and mice. NANOG is a direct target of the Activin A-SMAD2/3 pathway that maintains human [24,53] and mouse ESC [54] pluripotency. Activin A can also maintain human ESC pluripotency in the absence of feeder layers [55], which is consistent with our results using pig cells.…”

Genetic stability of pluripotent stem cells during anti-cancer therapies

“…Pluripotent SCs are able to self-renew unrestrictedly. They can differentiate in vitro and in vivo in all cell types deriving from the three germ layers ( 1 , 2 ). This ability makes them useful in cell replacement therapy and the treatment of numerous diseases ( 3 ), including diabetes ( 4 ), neurodegenerative ( 5 ), retinal ( 6 ) and cardiac ( 7 ) diseases, as well as muscular dystrophy ( 8 ).…”

Full biological characterization of human pluripotent stem cells will open the door to translational research

“…These signals are basic fibroblast growth factor (FGF2), activin/nodal/transforming growth factor β (TGFβ) and insulin/insulin-like growth factor (IGF) (Beattie et al 2005;Bendall et al 2007;Chen et al 2012;Eiselleova et al 2009;Tomizawa et al 2011;Vallier et al 2005;Wang et al 2007;Xu et al 2005). In the presence of these factors, pluripotency and self-renewal are sustained, while their withdrawal results in interruption of the core transcription regulatory network-leading to subsequent differentiation.…”