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Human retinal organoids have become indispensable tools for retinal disease modeling and drug screening. Despite its versatile applications, the long timeframe for their differentiation and maturation limits the throughput of such research. Here, we successfully shortened this timeframe by accelerating human retinal organoid development using unique pharmacological approaches. Our method comprised three key steps: 1) a modified self-formed ectodermal autonomous multizone (SEAM) method, including dual SMAD inhibition and bone morphogenetic protein 4 treatment, for initial neural retinal induction; 2) the concurrent use of a Sonic hedgehog agonist SAG, activin A, and all-trans retinoic acid for rapid retinal cell specification; and 3) switching to SAG treatment alone for robust retinal maturation and lamination. The generated retinal organoids preserved typical morphological features of mature retinal organoids, including hair-like surface structures and well-organized outer layers. These features were substantiated by the spatial immunostaining patterns of several retinal cell markers, including rhodopsin and L/M opsin expression in the outermost layer, which was accompanied by reduced ectopic cone photoreceptor generation. Importantly, our method required only 90 days for retinal organoid maturation, which is approximately two-thirds the time necessary for other conventional methods. These results indicate that thoroughly optimized pharmacological interventions play a pivotal role in rapid and precise photoreceptor development during human retinal organoid differentiation and maturation. Thus, our present method may expedite human retinal organoid research, eventually contributing to the development of better treatment options for various degenerative retinal diseases., Competing Interests: The study was funded by Santen Pharmaceutical Co. Ltd and Tokiyoshi Matsushita, Takahiro Matsuyama, and Masaaki Kageyama are employed by Santen Pharmaceutical Co., Ltd. Tokiyoshi Matsushita, Akishi Onishi, and Masayo Takahashi are co-inventors of a patent related to this work, filed by RIKEN and Santen Pharmaceutical Co., Ltd [WO2022138803A1]. None of these alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2024 Matsushita et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Natural killer (NK) cells can migrate quickly to the tumor site to exert cytotoxic effects on tumors, and some chemokines, including CXCL8, CXCL10 or and CXCL12, can regulate the migration of NK cells. Activin A, a member of the transforming growth factor β (TGF-β) superfamily, is highly expressed in tumor tissues and involved in tumor development and immune cell activation. In this study, we focus on the effects of activin A on NK cell migration. In vitro, activin A induced NK cell migration and invasion, promoted cell polarization and inhibited cell adhesion. Moreover, activin A increased Ca 2+ , p-SMAD3 and p-AKT levels in NK cells. An AKT inhibitor and Ca 2+ chelator partially blocked activin A-induced NK cell migration. In vivo, exogenous activin A increased tumor-infiltrating NK cells in NS-1 cell solid tumors and inhibited tumor growth, and blocking endogenous activin A with anti-activin A antibody reduced tumor-infiltrating NK cells in 4T-1 cell solid tumors. These results suggest that activin A induces NK cell migration through AKT signaling and calcium signaling and may enhance the antitumor effect of NK cells by increasing tumor-infiltrating NK cells.

Aim: Cell therapies for diabetes rely on differentiation of stem cells into insulin-producing cells, which is complex and expensive. Our goal was to evaluate production costs and test ways to reduce it. Methods: Cost of Goods (COGs) analysis for differentiation was completed and the effects of replacement or reduction of the most expensive item was tested using qRT-PCR, immunohistochemistry, flow cytometry along with glucose-stimulated insulin release. Results: Activin A (AA) was responsible for significant cost. Replacement with small molecules failed to form definitive endoderm (DE). Reducing AA by 50% did not negatively affect expression of beta cell markers. Conclusion: Reduction of AA concentration is feasible without adversely affecting DE and islet-like cell differentiation, leading to significant cost savings in manufacturing.

Human embryonic stem cells (hESCs) cultured in media containing bone morphogenic protein 4 (BMP4; B) differentiate into trophoblast-like cells. Supplementing media with inhibitors of activin/nodal signaling (A83-01) and of fibroblast growth factor 2 (PD173074) suppresses mesoderm and endoderm formation and improves specification of trophoblast-like lineages, but with variable effectiveness. We compared differentiation in four BMP4-containing media: mTeSR1-BMP4 only, mTeSR1-BAP, basal medium with BAP (basal-BAP), and a newly defined medium, E7-BAP. These media variably drive early differentiation towards trophoblast-like lineages with upregulation of early trophoblast markers CDX2 and KRT7 and downregulation of pluripotency markers (OCT4 and NANOG). As expected, based on differences between media in FGF2 and its inhibitors, downregulation of mesendoderm marker EOMES was variable between media. By day 7, only hESCs grown in E7-BAP or basal-BAP expressed HLA-G protein, indicating the presence of cells with extravillous trophoblast characteristics. Expression of HLA-G and other differentiation markers (hCG, KRT7, and GCM1) was highest in basal-BAP, suggesting a faster differentiation in this medium, but those cultures were more inhomogeneous and still expressed some endodermal and pluripotency markers. In E7-BAP, HLA-G expression increased later and was lower. There was also a low but maintained expression of some C19MC miRNAs, with more CpG hypomethylation of the ELF5 promoter, suggesting that E7-BAP cultures differentiate slower along the trophoblast lineage. We conclude that while all protocols drive differentiation into trophoblast lineages with varying efficiency, they have advantages and disadvantages that must be considered when selecting a protocol for specific experiments., (© 2023. The Author(s).)

The adept and systematic differentiation of embryonic stem cells (ESCs) and human-induced pluripotent stem cells (hiPSCs) to diverse lineage-prone cell types involves crucial step-by-step process that mimics the vital strategic commitment phase that is usually observed during the process of embryo development. The development of precise tissue-specific cell types from these stem cells indeed plays an important role in the advancement of imminent stem cell-based therapeutic strategies. Therefore, the usage of hiPSC-derived cell types for subsequent cardiovascular disease modeling, drug screening, and therapeutic drug development undeniably entails an in-depth understanding of each and every step to proficiently stimulate these stem cells into desired cardiomyogenic lineage. Thus, to accomplish this definitive and decisive fate, it is essential to efficiently induce the mesoderm or pre-cardiac mesoderm, succeeded by the division of cells into cardiovascular and ultimately ensuing with the cardiomyogenic lineage outcome. This usually commences from the earliest phases of pluripotent cell induction. In this chapter, we discuss our robust and reproducible step-wise protocol that will describe the subtype controlled, precise lineage targeted standardization of activin/nodal, and BMP signaling molecules/cytokines, for the efficient differentiation of ventricular cardiomyocytes from hiPSCs via the embryoid body method. In addition, we also describe techniques to dissociate hiPSCs, hiPSC-derived early cardiomyocytes for mesoderm and pre-cardiac mesoderm assessment, and hiPSC-derived cardiomyocytes for early and mature markers assessment., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Small molecules such as ROCK inhibitors (Fasudil) and inducer of definitive endoderm 1 (IDE1) can promote differentiation of definitive endoderm, but their effects remain controversial. Therefore, we attempted to verify the effect of these small molecules on promoting definitive endoderm differentiation and found that Fasudil or IDE1 alone could not achieve a similar effect as activin A. On the contrary, CHIR99021 could efficiently promote definitive endoderm differentiation. Nearly 43.4% of experimental cells were SRY-box transcription factor 17 (SOX17)-positive under the synergistic effect of IDE1 and CHIR99021, but its ability to differentiate towards definitive endoderm was still insufficient. Transcriptional analysis and comparison of IDE1 and CHIR99021 synergistic groups (IC) and activin A and CHIR99021 synergistic groups (AC) showed significantly down-regulated definitive endoderm markers in the IC group compared with those in the AC group and the differences between the two groups were mainly due to bone morphogenetic proteins (BMP4) and fibroblast growth factor 17 (FGF17). Further single-cell transcriptome analysis revealed lower expression of BMP4 in SOX17-positive populations, while mothers against decapentaplegic homolog (SMAD) protein translation signal and FGF17 in the AC group were higher than that in the IC group. Western blot analysis showed a significant difference in levels of p-SMAD2/3 between AC and IC groups, which suggests that regulating p-SMAD2/3 may provide a reference to improve the differentiation of definitive endoderm., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Human umbilical cord-derived mesenchymal stem cells (hucMSCs) can differentiate into multiple cell lineages, but few methods have been developed to generate kidney lineage cells. Due to their human origin, pluripotent nature and immunomodulatory properties, these stem cells are attractive candidates for clinical applications such as the repair or regeneration of damaged organs. This study evaluated the renal differentiation potential of hucMSCs, when exposed for 10 days to optimised concentrations of retinoic acid, activin-A and bone morphogenetic protein-7 (BMP-7) in various combinations, with and without the priming of the cells with a Wnt signalling pathway activator (CHIR99021). The hucMSCs were isolated and characterised according to surface marker expression (CD73, CD90, CD44, CD146 and CD8) and tri-lineage differentiation potential. The expression of key marker genes ( OSR1 , TBXT , HOXA13 , SIX2 , PAX2 , KRT18 and ZO1 ) was examined by qRT-PCR. Specific marker protein expression (E-cadherin, cytokeratin-8 and cytokeratin-19) was analysed by immunocytochemistry. CHIR99021-primed cells treated with the retinoic acid, activin-A and BMP-7 cocktail showed epithelial cell-like differentiation - i.e. distinct phenotypic changes, as well as upregulated gene and protein expression, were observed that were consistent with an epithelial cell phenotype. Thus, our results showed that hucMSCs can efficiently differentiate into renal epithelial-like cells. This work may help in the development of focused therapeutic strategies, in which lineage-defined human stem cells can be used for renal regeneration., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Activin A (Act A) has been reported to promote oligodendrocyte progenitor cell (OPC) differentiation in vitro and improve neurological outcomes in adult mice. However, the roles and mechanisms of action of Act A in preterm brain injury are unknown. In the present study, P5 rats were subjected to hypoxia‑ischemia to establish a neonatal white matter injury (WMI) model and Act A was injected via the lateral ventricle. Pathological characteristics, OPC differentiation, myelination, and neurological performance were analyzed. Further, the involvement of the Noggin/BMP4/Id2 signaling pathway in the roles of Act A in WMI was explored. Act A attenuated pathological damage, promoted OPC differentiation, enhanced myelin sheath and myelinated axon formation, and improved neurological performance of WMI rats. Moreover, Act A enhanced noggin expression, which, in turn, inhibited the expression of bone morphogenetic protein 4 (BMP4) and inhibitor of DNA binding 2 (Id2). Furthermore, upregulation of Id2 completely abolished the rescue effects of Act A in WMI rats. In conclusion, the present findings suggested that Act A rescues preterm brain injury via targeting a novel Noggin/BMP4/Id2 signaling pathway.

Background: Neuroinflammation and cytokines play critical roles in neuropathic pain and axon degeneration/regeneration. Cytokines of transforming growth factor-β superfamily have implications in pain and injured nerve repair processing. However, the transcriptional profiles of the transforming growth factor-β superfamily members in dorsal root ganglia under neuropathic pain and axon degeneration/regeneration conditions remain elusive., Objective: We aimed to plot the transcriptional profiles of transforming growth factor-β superfamily components in lumbar dorsal root ganglia of sciatic nerve-axotomized rats and to further verify the profiles by testing the analgesic effect of activin C, a representative cytokine, on neuropathic pain., Methods: Adult male rats were axotomized in sciatic nerves, and lumbar dorsal root ganglia were isolated for total RNA extraction or section. A custom microarray was developed and employed to plot the gene expression profiles of transforming growth factor-β superfamily components. Realtime RT-PCR was used to confirm changes in the expression of activin/inhibin family genes, and then in situ hybridization was performed to determine the cellular locations of inhibin α, activin βC, BMP-5 and GDF-9 mRNAs. The rat spared nerve injury model was performed, and a pain test was employed to determine the effect of activin C on neuropathic pain., Results: The expression of transforming growth factor-β superfamily cytokines and their signaling, including some receptors and signaling adaptors, were robustly upregulated. Activin βC subunit mRNAs were expressed in the small-diameter dorsal root ganglion neurons and upregulated after axotomy. Single intrathecal injection of activin C inhibited neuropathic pain in spared nerve injury model., Conclusion: This is the first report to investigate the transcriptional profiles of members of transforming growth factor-β superfamily in axotomized dorsal root ganglia. The distinct cytokine profiles observed here might provide clues toward further study of the role of transforming growth factor-β superfamily in the pathogenesis of neuropathic pain and axon degeneration/regeneration after peripheral nerve injury., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Cancer stem cells (CSCs) are involved in liver metastasis in colorectal cancer (CRC). Activin and hepatocyte growth factor (HGF) are important regulators of stem cell properties. This study was performed to explore the effect of activin and HGF on CRC invasion and metastasis. The key genes involved in the action of activin and HGF in CRC were identified., Methods: HCT116 CRC cells were sequentially treated with activin and HGF and examined for migration and invasion in vitro and liver metastasis in vivo. RNA sequencing was performed to identify differentially expressed genes in response to activin and HGF., Results: Sequential treatment with activin and HGF-enhanced CRC cell migration, invasion, and metastasis. CXCR4 and AFP expressions were increased by activin and HGF treatment. Knockdown of FOXM1 blocked liver metastasis from HCT116 cells pretreated with activin and HGF and suppressed CXCR4 and AFP expression. Activin alone increased the mRNA and protein expression of FOXM1. In contrast, HGF alone enhanced the phosphorylation of FOXM1, without altering the total protein level of FOXM1. SMAD2 was required for activin-mediated FOXM1 induction. FOXM1 transactivated CXCR4 by directly binding to the promoter of CXCR4 . Additionally, CXCR4 regulated AFP expression through the NF- κ B pathway., Conclusions: Sequential treatment with activin and HGF accelerates CRC invasion and liver metastasis, which involves the upregulation and activation of FOXM1 and induction of CXCR4 and AFP., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this article., (Copyright © 2022 Hong Peng et al.)

The TGF-β superfamily members, activins and inhibins, are mainly involved in cell proliferation, cell survival, invasion, immune surveillance, and lesion growth in endometriosis. Herein, we investigated the modulation of the TGF-β type III receptor (betaglycan or BG) by activin A and inhibin A in endometriosis in vitro. Often, BG undergoes ectodomain shedding releasing soluble BG (sBG) which frequently antagonizes TGF-β signaling. The effects of activin A on BG shedding and signaling pathways involved were evaluated with the inhibitors LY364947 and SIS3, siRNA knockdown in human endometrial cells (12Z, THESC, Ishikawa, and primary stromal cells) and were quantified with BG ELISAs. The effects of activin A and inhibin A on the secretion of MMP2 and MMP3 were analyzed using ELISAs. The effects of activin A on the BG expression were analyzed using RT-qPCR and western blot. The CCK-8 and BrdU assays were used to evaluate the effects of the recombinant BG on cell viability and proliferation. Activin A stimulation resulted in a significant time- and dose-dependent reduction in BG shedding, which was found to be activin A/ALK-4/SMAD3- but not SMAD2-dependent. Activin A increased the BG mRNA expression but had no effect on the protein expression. Likewise, inhibin A was found to block BG shedding. Activin A, but not inhibin A, significantly enhanced the secretion of MMP2 and MMP3. The recombinant BG had no effect on the viability and proliferation of endometriotic cells. Together, these observations support a novel role for activin A with BG in modulating the TGF-β superfamily ligands in endometrial cells in vitro., Competing Interests: The authors declare no conflict of interest.

Recently, we reported that gonadotropin-releasing hormone (GnRH) stimulates annexin A1 (Anxa1) and A5 (Anxa5) mRNA expression through the GnRH-receptor-mitogen-activated protein kinase cascade in LβT2 cells. As LβT2 cells respond to activin, we examined the effect of activin A on Anxa1 and a5 expression in LβT2 cells. Activin A (0.4 and 4 ng/mL) treatment decreased Anxa5 mRNA levels in a dose-dependent manner, but did not affect Anxa1 mRNA levels at concentrations up to 40 ng/mL. After activin A treatment (4 ng/mL), Anxa5 mRNA levels significantly decreased at 6 h, gradually declined until 24 h, and remained low until 48 h, whereas Anxa1 mRNA levels did not significantly change following treatment. Pretreatment with activin A for 24 h increased GnRH agonist (GnRHa)-induced Anxa1 increase by approximately 7-fold compared with GnRHa stimulation alone, but Anxa5 was not affected. As previously reported, these activin A treatments increased gonadotropin β subunit and GnRH receptor mRNA levels and slightly decreased common α-glycoprotein subunit mRNA levels. Furthermore, we examined the effect of activin A on Nr4a3, which is repressed by ANXA5 and which reduces Fshb expression, and found that activin A augmented Nr4a3 expression and slightly decreased the GnRHa-induced increase in Nr4a3. These results suggest that in gonadotrope cells, the mechanism regulating Anxa1 and a5 expression is differentially coupled with activin A signal transduction. Activin A suppresses Anxa5 expression under increased Nr4a3 expression, whereas activin A and GnRH synergistically stimulate Anxa1 expression. These GnRH-inducible annexins may have different specific functions in gonadotropes.

Embryogenesis is guided by a limited set of signaling pathways dynamically expressed in different places. How a context-dependent signaling response is generated has been a central question of developmental biology, which can now be addressed with in vitro models of human embryos that are derived from embryonic stem cells (hESCs). Our previous work demonstrated that during early stages of hESC differentiation, cells chronicle signaling hierarchy. Only cells that have been exposed (primed) by WNT signaling can respond to subsequent activin exposure and differentiate to mesendodermal (ME) fates. Here, we show that WNT priming does not alter SMAD2 binding nor its chromatin opening but, instead, acts by inducing the expression of the SMAD2 co-factor EOMES. Expression of EOMES is sufficient to replace WNT upstream of activin-mediated ME differentiation, thus unveiling the mechanistic basis for priming and cellular memory in early development., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Transforming growth factor β (TGF-β) superfamily proteins are potent regulators of cellular development and differentiation. Nodal/Activin/TGF-β and BMP ligands are both present in the intra- and extracellular milieu during early development, and cross-talk between these two branches of developmental signaling is currently the subject of intense research focus. Here, we show that the Nodal induced lncRNA-Smad7 regulates cell fate determination via repression of BMP signaling in mouse embryonic stem cells (mESCs). Depletion of lncRNA-Smad7 dramatically impairs cardiomyocyte differentiation in mESCs. Moreover, lncRNA-Smad7 represses Bmp2 expression through binding with the Bmp2 promoter region via (CA)12-repeats that forms an R-loop. Importantly, Bmp2 knockdown rescues defects in cardiomyocyte differentiation induced by lncRNA-Smad7 knockdown. Hence, lncRNA-Smad7 antagonizes BMP signaling in mESCs, and similarly regulates cell fate determination between osteocyte and myocyte formation in C2C12 mouse myoblasts. Moreover, lncRNA-Smad7 associates with hnRNPK in mESCs and hnRNPK binds at the Bmp2 promoter, potentially contributing to Bmp2 expression repression. The antagonistic effects between Nodal/TGF-β and BMP signaling via lncRNA-Smad7 described in this work provides a framework for understanding cell fate determination in early development., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Fate determination and maintenance of fetal testes in most mammals occur cell autonomously as a result of the action of key transcription factors in Sertoli cells. However, the cases of freemartin, where an XX twin develops testis structures under the influence of an XY twin, imply that hormonal factor(s) from the XY embryo contribute to sex reversal of the XX twin. Here we show that in mouse XY embryos, Sertoli cell-derived anti-Mullerian hormone (AMH) and activin B together maintain Sertoli cell identity. Sertoli cells in the gonadal poles of XY embryos lacking both AMH and activin B transdifferentiate into their female counterpart granulosa cells, leading to ovotestis formation. The ovotestes remain to adulthood and produce both sperm and oocytes, although there are few of the former and the latter fail to mature. Finally, the ability of XY mice to masculinize ovaries is lost in the absence of these two factors. These results provide insight into fate maintenance of fetal testes through the action of putative freemartin factors., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: In our previous study, activin B in combination with ADSCs enhances skin wound healing. However, the underlying molecular mechanisms are not well studied. Cdc42 is recognized to play a critical role in the regulation of stem cells., Methods: Pull-down assay was performed to investigate the activity of Cdc42. The dominant-negative mutant of Cdc42 (Cdc42N17) was used to explore the role of Cdc42 in activin B-induced ADSCs migration, proliferation, and secretion in vitro. Cdc42N17-transfected ADSCs were injected into a full-thickness excisional wound model to explore their efficiency in wound healing in vivo. The wound healing efficacy was evaluated by the wound closure rates and histological examination. The neovascularization and wound contraction were detected by immunohistochemistry staining of CD31 and α-SMA. Finally, the underlying mechanisms were explored by RNA sequencing., Results: Cdc42N17 inhibited ADSCs migration, proliferation, and secretion induced by activin B. Furthermore, Cdc42N17-transfected ADSCs inhibited the wound closure rate and suppressed the expression of CD31 and α-SMA induced by activin B in vivo. The RNA sequencing showed that the differentially expressed genes in Cdc42N17-transfected ADSCs versus ADSCs were associated with cell migration, proliferation, and adhesion. Further study revealed that the Cdc42-Erk-Srf pathway was required for activin B-induced proliferation in ADSCs., Conclusions: Our study indicates that Cdc42 plays a crucial role in ADSCs-mediated skin wound healing induced by activin B., (© 2022. The Author(s).)

Atherosclerosis (AS) is one of the most common vascular diseases. The endothelial injury theory indicates that atherosclerotic plaque is the result of endothelial cell injury. Recent studies have revealed that circRNAs are abnormally expressed in AS cell models, which are implicated in the regulation of various cell behaviors. In this study, we showed the downregulation of circNMD3 in AS, and studied its role in the model of endothelial cell injury by proliferation and apoptosis assay, caspase 3 activity assay, and ELISA. We also identified and validated its downstream targets by luciferase reporter assay, RNA pull-down experiment, Western blot, and RT-qPCR. CircNMD3 overexpression or miR-498 knockdown could inhibit the ox-LDL (oxidatively modified low-density lipoprotein)-induced injury in HUVEC (human umbilical vein endothelial cells), while the co-transfection of miR-498 mimic or siRNA targeting BAMBI (BMP and activin membrane bound inhibitor) attenuated the protective effect of circNMD3 overexpression. Overall, our data suggest that circNMD3 regulates the miR-498/BAMBI axis in endothelial cells to protect ox-LDL-induced damages. As a molecular sponge of miR-498, circNMD3 regulates the level of miR-498, which in turn modulates BAMBI expression and suppresses ox-LDL-induced injury in HUVECs.

Mossy cells (MCs) in the hilus of the dentate gyrus (DG) receive increasing attention as a major player controlling information processing in the DG network. Furthermore, disturbed MC activity has been implicated in widespread neuropsychiatric disorders such as epilepsy and major depression. Using whole-cell patch-clamp recordings from MCs in acute hippocampal slices from wild type and transgenic mice, we demonstrate that activin, a member of the transforming growth factor-β (TGF-β) family, has a strong neuromodulatory effect on MC activity. Disruption of activin receptor signaling reduced MC firing, dampened their excitatory input and augmented their inhibitory input. By contrast, acute application of recombinant activin A strongly increased MC activity and promoted excitatory synaptic drive. Notably, similar changes of MC activity have been observed in a rodent model of depression and after antidepressant drug therapy, respectively. Given that a rise in activin signaling particularly in the DG has been proposed as a mechanism of antidepressant action, our data suggest that the effect of activin on MC excitability might make a considerable contribution in this regard., (© 2022 The Authors. Hippocampus published by Wiley Periodicals LLC.)

Allocation of cells to an endodermal fate in the gastrulating embryo is driven by Nodal signaling and consequent activation of TGFβ pathway. In vitro methodologies striving to recapitulate the process of endoderm differentiation, however, use TGFβ family member Activin in place of Nodal. This is despite Activin not known to have an in vivo role in endoderm differentiation. In this study, five epiblast stem cell lines were subjected to directed differentiation using both Activin A and Nodal to induce endodermal fate. A reporter line harboring endoderm markers FoxA2 and Sox17 was further analyzed for TGFβ pathway activation and WNT response. We demonstrated that Activin A-treated cells remain more primitive streak-like when compared to Nodal-treated cells that have a molecular profile suggestive of more advanced differentiation. Activin A elicited a robust TGFβ/SMAD activity, enhanced WNT signaling activity and promoted the generation of DE precursors. Nodal treatment resulted in lower TGFβ/SMAD activity, and a weaker, sustained WNT response, and ultimately failed to upregulate endoderm markers. This is despite signaling response resembling more closely the activity seen in vivo. These findings emphasize the importance of understanding the downstream activities of Activin A and Nodal signaling in directing in vitro endoderm differentiation of primed-state epiblast stem cells., (© 2022 Wiley Periodicals LLC.)

This study explored the roles of activins and follistatin in colorectal cancers. Paired malignant and normal colonic tissues were collected from archived paraffin-embedded ( n = 90 patients) alongside fresh ( n = 40 patients) specimen cohorts. Activin β-subunits, follistatin and Smad4 mRNAs and proteins were measured by real-time PCR and immunohistochemistry (IHC). Mature activin-A, -B, -AB and follistatin proteins were measured by ELISA. Cancer tissues having ≤ the 20th percentile of the Smad4 IHC score were considered as low (L-S4) group. The Smad4-intact SW480 and Smad4-null HT29 colon cancer cell lines were treated with activins and follistatin, and cell cycle was analysed by flow cytometry. The cell cycle inducing (CCND1/CCND3) and inhibitory (p21/p27) proteins alongside the survival (survivin/BCL2) and pro-apoptosis (Casp-8/Casp-3) markers were measured by immunofluorescence. Thirty-nine patients had right-sided cancers (30%) and showed higher rates of L-S4 tumours ( n = 17; 13.1%) alongside worse clinicopathological characteristics relative to left-sided cancers. The βA-subunit and activin-A increased, whilst βB-subunit and activin-AB decreased, in malignant sites and the late-stage cancers revealed the greatest abnormalities. Interestingly, follistatin declined markedly in early-stage malignant tissues, whilst increased significantly in the advanced stages. All activin molecules were comparable between the early stage right- and left-sided tumours, whereas the late-stage right-sided cancers and L-S4 tumours showed more profound deregulations. In vitro , activin-A increased the numbers of the SW480 cells in sub-G1 and G0/G1-phases, whereas reduced the HT29 cell numbers in the sub-G1 phase with simultaneous increases in the G0/G1 and S phases. The p21/p27/Casp-8/Casp-3 proteins escalated, whilst CCND1/CCND3/BCL2/survivin declined in the SW480 cells following activin-A, whereas activin-A only promoted p21 and p27 alongside reduced CCND3 in the HT29 cells. By contrast, activin-AB increased the numbers of SW480 and HT29 cells in Sub-G1 and G0/G1-phases and promoted the anti-cancer and reduced the oncogenic proteins in both cell lines. In conclusion, activins and follistatin displayed stage-dependent dysregulations and were markedly altered during the advanced stages of right-sided and L-S4 cancers. Moreover, the activin-A actions in CRC could be Smad4-dependent, whereas activin-AB may act as a Smad4-independent tumour suppressor protein., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Refaat, Zekri, Aslam, Ahmad, Baghdadi, Meliti, Idris, Sultan, Alardati, Saimeh, Alsaegh, Alhadrami, Hamid, Naeem and Elsamany.)

Background: Activin A has been reported to play important roles in the pathogenesis of atherosclerosis. The purpose of this study is to investigate the effects of activin A on oxidized low-density lipoprotein (ox-LDL)-induced foam cell formation and explore the underlying molecular mechanisms in murine macrophage-like cell line RAW 264.7., Methods: The effects of activin A on Dil-labeled ox-LDL uptake were examined by confocal microscopy and flow cytometry analysis. The mRNA and protein levels of cholesterol receptors were analyzed by RT-qPCR and western blot analysis, respectively. To investigate whether activin receptor-like kinase 4 (Alk4) is required for activin A-mediated cellular effects, cells were pre-treated with SB-431542. The involvement of Smad2, Smad3 and Smad4 was confirmed by transfection with specific small interfering RNAs (siRNAs)., Results: Activin A inhibits ox-ldl-induced foam cell formation and class A scavenger receptors (SR-A) expression, while up-regulates ATP-binding cassette transporter A1 (ABCA1) and ABCG1 expression in RAW 264.7 macrophages. Pre-treatment with SB-431542 abolished activin A-mediated anti-atherogenic effect. Knockdown of Smad2 reversed activin A-induced inhibition of ox-LDL uptake and SR-A expression. However, knockdown of Smad3 or Smad4 did not have such effect. Meanwhile, knockdown of either Smad2, Smad3 or Smad4 reversed the activin A-induced up-regulation of ABCA1 and ABCG1., Conclusions: Our study provides novel evidence that activin A may exert anti-atherogenic effects through Alk4-Smad signaling pathway in RAW 264.7 macrophages., (Copyright © 2021. Published by Elsevier Inc.)

Activin, a member of the transforming growth factor-β (TGF-β) superfamily of proteins, induces various tissues from the amphibian presumptive ectoderm, called animal cap explants (ACs) in vitro. However, it remains unclear how and to what extent the resulting cells recapitulate in vivo development. To comprehensively understand whether the molecular dynamics during activin-induced ACs differentiation reflect the normal development, we performed time-course transcriptome profiling of Xenopus ACs treated with 50 ng/mL of activin A, which predominantly induced dorsal mesoderm. The number of differentially expressed genes (DEGs) in response to activin A increased over time, and totally 9857 upregulated and 6663 downregulated DEGs were detected. 1861 common upregulated DEGs among all Post_activin samples included several Spemann's organizer genes. In addition, the temporal transcriptomes were clearly classified into four distinct groups in correspondence with specific features, reflecting stepwise differentiation into mesoderm derivatives, and a decline in the regulation of nuclear envelop and golgi. From the set of early responsive genes, we also identified the suppressor of cytokine signaling 3 (socs3) as a novel activin A-inducible gene. Our transcriptome data provide a framework to elucidate the transcriptional dynamics of activin-driven AC differentiation, reflecting the molecular characteristics of early normal embryogenesis., (© 2021. The Author(s).)

Background: Naïve and primed pluripotent stem cells (PSCs) represent two different pluripotent states. Primed PSCs following in vitro culture exhibit lower developmental potency as evidenced by failure in germline chimera assays, unlike mouse naïve PSCs. However, the molecular mechanisms underlying the lower developmental competency of primed PSCs remain elusive., Results: We examine the regulation of telomere maintenance, retrotransposon activity, and genomic stability of primed PSCs and compare them with naïve PSCs. Surprisingly, primed PSCs only minimally maintain telomeres and show fragile telomeres, associated with declined DNA recombination and repair activity, in contrast to naïve PSCs that robustly elongate telomeres. Also, we identify LINE1 family integrant L1Md_T as naïve-specific retrotransposon and ERVK family integrant IAPEz to define primed PSCs, and their transcription is differentially regulated by heterochromatic histones and Dnmt3b. Notably, genomic instability of primed PSCs is increased, in association with aberrant retrotransposon activity., Conclusions: Our data suggest that fragile telomere, retrotransposon-associated genomic instability, and declined DNA recombination repair, together with reduced function of cell cycle and mitochondria, increased apoptosis, and differentiation properties may link to compromised developmental potency of primed PSCs, noticeably distinguishable from naïve PSCs., (© 2021. The Author(s).)

FSH is critical for fertility. Transcription of FSHB, the gene encoding the beta subunit, is rate-limiting in FSH production and is regulated by both GnRH and activin. Activin signals through SMAD transcription factors. Although the mechanisms and importance of activin signaling in mouse Fshb transcription are well-established, activin regulation of human FSHB is less well understood. We previously reported a novel enhancer of FSHB that contains a fertility-associated single nucleotide polymorphism (rs10031006) and requires a region resembling a full (8 base-pair) SMAD binding element (SBE). Here, we investigated the role of the putative SBE within the enhancer in activin and GnRH regulation of FSHB. In mouse gonadotrope-derived LβT2 cells, the upstream enhancer potentiated activin induction of both the human and mouse FSHB proximal promoters and conferred activin responsiveness to a minimal promoter. Activin induction of the enhancer required the SBE and was blocked by the inhibitory SMAD7, confirming involvement of the classical SMAD signaling pathway. GnRH induction of FSHB was also potentiated by the enhancer and dependent on the SBE, consistent with known activin/GnRH synergy regulating FSHB transcription. In DNA pull-down, the enhancer SBE bound SMAD4, and chromatin immunoprecipitation demonstrated SMAD4 enrichment at the enhancer in native chromatin. Combined activin/GnRH treatment elevated levels of the active transcriptional histone marker, histone 3 lysine 27 acetylation, at the enhancer. Overall, this study indicates that the enhancer is directly targeted by activin signaling and identifies a novel, evolutionarily conserved mechanism by which activin and GnRH can regulate FSHB transcription., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Naive human embryonic stem cells (hESCs) have been isolated that more closely resemble the pre-implantation epiblast compared to conventional "primed" hESCs, but the signaling principles underlying these discrete stem cell states remain incompletely understood. Here, we describe the results from a high-throughput screen using ∼3,000 well-annotated compounds to identify essential signaling requirements for naive human pluripotency. We report that MEK1/2 inhibitors can be replaced during maintenance of naive human pluripotency by inhibitors targeting either upstream (FGFR, RAF) or downstream (ERK1/2) kinases. Naive hESCs maintained under these alternative conditions display elevated levels of ERK phosphorylation but retain genome-wide DNA hypomethylation and a transcriptional identity of the pre-implantation epiblast. In contrast, dual inhibition of MEK and ERK promotes efficient primed-to-naive resetting in combination with PKC, ROCK, and TNKS inhibitors and activin A. This work demonstrates that induction and maintenance of naive human pluripotency are governed by distinct signaling requirements., Competing Interests: Declaration of interests R.J. is a co-founder of Fate Therapeutics, Fulcrum Therapeutics, and Omega Therapeutics and an advisor to Dewpoint Therapeutics. T.W.T., S.A.K., and R.J. are co-inventors on a patent application related to naive hPSCs., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The endoderm, differentiated from human induced pluripotent stem cells (iPSCs), can differentiate into the small intestine and liver, which are vital for drug absorption and metabolism. The development of human iPSC-derived enterocytes (HiEnts) and hepatocytes (HiHeps) has been reported. However, pharmacokinetic function-deficiency of these cells remains to be elucidated. Here, we aimed to develop an efficient differentiation method to induce endoderm formation from human iPSCs. Cells treated with activin A for 168 h expressed higher levels of endodermal genes than those treated for 72 h. Using activin A (days 0-7), CHIR99021 and PI-103 (days 0-2), and FGF2 (days 3-7), the hiPSC-derived endoderm (HiEnd) showed 97.97% CD-117 and CD-184 double-positive cells. Moreover, HiEnts derived from the human iPSC line Windy had similar or higher expression of small intestine-specific genes than adult human small intestine. Activities of the drug transporter P-glycoprotein and drug-metabolizing enzyme cytochrome P450 (CYP) 3A4/5 were confirmed. Additionally, Windy-derived HiHeps expressed higher levels of hepatocyte- and pharmacokinetics-related genes and proteins and showed higher CYP3A4/5 activity than those derived through the conventional differentiation method. Thus, using this novel method, the differentiated HiEnts and HiHeps with pharmacokinetic functions could be used for drug development.

Background: Kawasaki disease is an autoimmune disease characterized by systemic vasculitis of unknown aetiology and most commonly occurs in children under 5 years old. Previous studies have found that the over-activation of lymphocytes is an important mechanism of Kawasaki disease. Activin A, also known as immunosuppressive factor P, is a multifunctional growth and transforming factor. However, whether activin A is involved in the regulation of peripheral lymphocytes activity in Kawasaki disease is unclear. Thus, we aimed to investigate the effect of activin A on the activity of peripheral lymphocytes in acute-phase Kawasaki disease., Methods: Seven patients with Kawasaki disease and seven healthy controls were studied. Peripheral blood lymphocytes were isolated by Ficoll density gradient centrifugation. The activation of CD4 + and CD8 + T cells and CD19 + B cells was investigated by flow cytometry. The expression of activin type IIA receptors was investigated by flow cytometry., Results: Immune imbalance in CD4 and CD8 lymphocytes were detected in acute-phase Kawasaki disease. The expression of activin type IIA receptors on CD8 + T cells and CD19 + B cells was increased in acute-phase Kawasaki disease and decreased following treatment with activin A. Activin A suppressed the expression of CD25 and CD69 on CD8 + T cells and the expression of CD69 on CD19 + B cells., Conclusions: The expression of activin type IIA receptor was increased on CD8 + T cells and CD19 + B cells in Kawasaki disease. Activin A suppressed the expression of CD25, CD69 and activin type IIA receptors on peripheral CD8 + T lymphocyte. Activin A plays different roles in different lymphocyte subsets and suppresses peripheral CD8 + T lymphocyte activity in acute-phase Kawasaki disease.

Aims: We investigated the in vitro differentiation of adult rat PDESCs into β-like cells through supplementation of different combinations of GABA, BMP7, and Activin A in basic culture media., Materials and Methods: The PDESCs were cultured using different inducement combinations for 28 days and microscopy, dithizone (DTZ) staining, immunohistochemical staining, real-time PCR, and glucose-stimulated insulin secretion (GSIS) assay were used to delineate the differentiation inducement potential of these combinations., Key Findings: The results show that after 28 days, the PDESCs were differentiated into ICCs containing insulin-secreting β-like cells in different groups treated with A + B, A + G, B + G, and A + B + G but not in the control group. Upon DTZ staining the cells in ICCs were stained crimson red, demonstrating the presence of β-like cells in ICCs and the immunohistochemistry showed the expression of Pdx1 and insulin in ICCs. Further, on 28 d the expression of Pdx1 and insulin mRNA was high in inducement groups as compared to the control group and β-like cells in ICCs also secreted insulin and C-peptide upon glucose stimulation. Thus, the supplementation of GABA, BMP7, and Activin A in different combinations in basic culture media can induce the in vitro differentiation of PDESCs into ICCs containing β-like cells., Significance: The in vitro development of β-like cells is a herald for cell therapy of diabetic patients and our results are a step closer towards finding the cure for diabetes., (Copyright © 2020. Published by Elsevier Inc.)

Fibrotic diseases account for more than 8 million deaths worldwide annually. Reactive oxygen species (ROS) has been shown to activate pyroptosis and promote the production of interleukin (IL)-1β and IL-18, leading to fibrosis development. However, the role of dual oxidase 1 (DUOX1)-induced ROS production and pyroptosis in cardiac fibrosis remains largely unknown. Activin A was used to induce ROS and pyroptosis in cardiomyocytes. ROS level, pyroptosis, and cytokine production were detected using Active Oxygen Detection Kit, flow cytometry, and enzyme-linked immunosorbent assay, respectively. Western blotting analysis was used to measure expression changes of proteins. DUOX1 was silenced or overexpressed to investigate its role in fibrosis. We found that activin A induced ROS production and pyroptosis in cardiomyocytes, which was blocked by the ROS scavenger, N-acetyl-L-cysteine (NAC). Knockdown of DUOX1 reversed activin A-induced ROS production, pyroptosis, cytokine release, and the upregulation of proinflammatory proteins. Overexpression of DUOX1 resulted in opposite effects of knockdown DUOX1. Administration of an ROS scavenger blocked the effect of DUOX1 overexpression. Supplementation of IL-1β and IL-18 caused significant fibrosis in human cardiac fibroblasts (hCFs). The knockdown of DUOX1 protected cardiomyocytes against activin A-induced fibrosis via the inhibition of ROS, cytokine release, and pyroptosis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Activin A promotes human trophoblast invasion during the first trimester of pregnancy and is associated with preeclampsia and pregnancy-induced hypertension (PE/PIH) in naturally conceived pregnancies. However, whether integrin β1 mediates activin A-increased trophoblast invasion remains unknown and the evidence is limited regarding the predictive value of activin A for PE/PIH in women receiving in vitro fertilization (IVF) treatment. Here, we studied the role and underlying molecular mechanisms of integrin β1 in activin A-promoted invasion in immortalized (HTR8/SVneo) and primary human extravillous trophoblast (EVT) cells. A nest case-control study was designed to investigate the predictive/diagnostic value of activin A in IVF pregnancies. Results showed that integrin β1 expression increased after activin A treatment and knockdown of integrin β1 significantly decreased both basal and activin A-increased HTR8/SVneo cell invasion. SB431542 (TGF-β type I receptors inhibitor) abolished activin A-induced SMAD2/SMAD3 phosphorylation and integrin β1 overexpression. Activin A-upregulated integrin β1 expression was attenuated after the depletion of ALK4 or SMAD4 in both HTR8/SVneo and primary EVT cells. Furthermore, we found similar first-trimester activin A levels in IVF patients with or without subsequent PE/PIH. These results reveal that integrin β1 mediates activin A-promoted trophoblast invasion through ALK4-activated SMAD2/3-SMAD4 pathway, and the predictive/diagnostic value of first-trimester maternal serum activin A for hypertensive disorders of pregnancy might be different in IVF population., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

To investigate the role of maternal Activin-like factors in the preservation of stemness and mesendoderm induction, their effects were promoted and inhibited using synthetic human Activin A or SB-505124 treatments, respectively, before the maternal to zygotic transition (MZT). To study the role of zygotic Activin-like factors, SB-505124 treatment was also used after the MZT. Promoting the signaling intensity of maternal Activin-like factors led to premature differentiation, loss of stemness, and no mesendoderm malformation, while its alleviation delayed the differentiation and caused various malformations. Inhibition of the zygotic Activin-like factors was associated with suppressing the ndr1 , ndr2 , oct4 ( pou5f3 ), mycb and notail transcription as well as differentiation retardation at the oblong stage, and a broad spectrum of anomalies in a dose-dependent manner. Together, promoting the signal intensity of maternal Activin-like factors drove development along with mesendodermal differentiation, while suppression of the maternal or zygotic ones maintained the pluripotent state and delayed differentiation.

Fibroblasts play an important role in inflammation and tissue fibrosis. Both activin A and TNF-α can activate immune cells, however, the roles and relationship of them in activating fibroblasts in inflammation remain unclear. Here, this study revealed that TNF-α promoted the release of NO and IL-6 by L929 fibroblast cells, but co-treatment with activin A attenuated these effects. In contrast, activin A induced cell migration and increased the production of tissue fibrosis-related TGF-β1 and fibronectin, while TNF-α inhibited these function changes of L929 cells induced by activin A. Moreover, this study revealed that activin A and TNF-α regulated the activities of L929 cells via ERK1/2/MAPK pathway, rather than Smad3-dependent signaling pathway. Taken together, these data indicate that activin A and TNF-α exert mutually antagonistic effects on regulating fibroblasts activities, and the balance between their action may determine the process and outcome of fibroblasts-mediated inflammation.

We recently reported that epiblast stem cells (EpiSCs)-like cells could be derived from preimplantation embryos (named as AFSCs). Here, we established AFSCs from pre-implantation embryos of multiple mouse strains and showed that unlike EpiSCs, the derivation efficiency of AFSCs was affected by the genetic background. We then used AFSCs lines to dissect the roles of Activin A (Act A) and basic fibroblast growth factor and reported that Act A alone was capable of maintaining self-renewal but not developmental potential in vivo. Finally, we established a novel experimental system, in which AFSCs were efficiently converted to multipotent progenitor stem cells using Act A and bone morphogenetic protein 4 (named as ABSCs). Importantly, these ABSCs contributed to neural mesodermal progenitors and lateral plate mesoderm in postimplantation chimeras. Taken together, our study established a robust experimental system for the generation of specific multipotent progenitor stem cells that was self-renewable and capable of contributing to embryonic and extra-embryonic tissues., (© 2020 Wiley Periodicals, Inc.)

Objective: Cancer cachexia and muscle loss are associated with increased morbidity and mortality. In preclinical animal models, blocking activin receptor (ACVR) ligands has improved survival and prevented muscle wasting in cancer cachexia without an effect on tumour growth. However, the underlying mechanisms are poorly understood. This study aimed to identify cancer cachexia and soluble ACVR (sACVR) administration-evoked changes in muscle proteome., Methods: Healthy and C26 tumour-bearing (TB) mice were treated with recombinant sACVR. The sACVR or PBS control were administered either prior to the tumour formation or by continued administration before and after tumour formation. Muscles were analysed by quantitative proteomics with further examination of mitochondria and nicotinamide adenine dinucleotide (NAD + ) metabolism. To complement the first prophylactic experiment, sACVR (or PBS) was injected as a treatment after tumour cell inoculation., Results: Muscle proteomics in TB cachectic mice revealed downregulated signatures for mitochondrial oxidative phosphorylation (OXPHOS) and increased acute phase response (APR). These were accompanied by muscle NAD + deficiency, alterations in NAD + biosynthesis including downregulation of nicotinamide riboside kinase 2 (Nrk2), and decreased muscle protein synthesis. The disturbances in NAD + metabolism and protein synthesis were rescued by treatment with sACVR. Across the whole proteome and APR, in particular, Serpina3n represented the most upregulated protein and the strongest predictor of cachexia. However, the increase in Serpina3n expression was associated with increased inflammation rather than decreased muscle mass and/or protein synthesis., Conclusions: We present evidence implicating disturbed muscle mitochondrial OXPHOS proteome and NAD + homeostasis in experimental cancer cachexia. Treatment of TB mice with a blocker of activin receptor ligands restores depleted muscle NAD + and Nrk2, as well as decreased muscle protein synthesis. These results indicate putative new treatment therapies for cachexia and that although acute phase protein Serpina3n may serve as a predictor of cachexia, it more likely reflects a condition of elevated inflammation., (Copyright © 2020 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The efficient genesis of pluripotent cells or therapeutic cells for regenerative medicine involves several external manipulations and conditioning protocols, which drives down clinical applicability. Automated programming of the genesis by microscale physical forces and chronological biochemistry can increase clinical success. The design and fabrication of nested polysaccharide droplets (millimeter-sized) with cell sustaining properties of natural tissues and intrinsic properties for time and space evolution of cell transformation signals between somatic cells, pluripotent cells and differentiated therapeutic cells in a swift and efficient manner without the need for laborious external manipulation are reported. Cells transform between phenotypic states by having single and double nested droplets constituted with extracellular matrix proteins and reprogramming, and differentiation factors infused chronologically across the droplet space. The cell transformation into germ layer cells and bone cells is successfully tested in vitro and in vivo and promotes the formation of new bone tissues. Thus, nested droplets with BMP-2 loaded guests synthesize mineralized bone tissue plates along the length of a cranial non-union bone defect at 4 weeks. The advantages of sequenced somatic cell reprogramming and differentiation inside an individual hydrogel module without external manipulation, promoted by formulating tissue mimetic physical, mechanical, and chemical microenvironments are shown., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Disrupted fetal germline development underpins testicular germ cell neoplasia, which is increasing worldwide. The complex signaling milieu during normal testis development includes TGFβ superfamily ligands; this study tests the hypothesis that, activin A, a TGFβ superfamily member, can influence gonocyte development. The human seminoma-derived cell line, TCam-2, a model of fetal gonocytes, was cultured with activin A (1.25-25 ng/mL) for 48 h, or with 5 ng/mL activin A for short- (6, 24, and 48 h) and long-term (13 days) exposures, and downstream targets measured by qRT-PCR. Transcripts that exhibited significant dose-dependent responses to activin A included the early germ cell markers KIT, NODAL, and CRIPTO (NODALl co-receptor and activin inhibitor) which all increased and the differentiation marker DNMT3L which decreased. After 48 h, KIT, NODAL, and CRIPTO levels were significantly higher, while the differentiation marker NANOS2 was significantly lower. Interestingly, activin A exposure also significantly reduced both transcript and protein levels of the PIWI/piRNA pathway component DNMT3L. Because TCam-2 cells produce the activin inhibitor CRIPTO, CRIPTO was reduced using siRNA prior to activin A exposure. This selectively increased KIT in response to activin A. Other ligands present in the fetal testis (BMP4, FGF9, TGFβ1, and TGFβ2) induced distinct effects on germline marker expression. This study showed that activin A can directly modulate germline markers in this human gonocyte-like cell, promoting a less-differentiated phenotype. Additional findings indicate evidence of signaling crosstalk between activin A and NODAL, leading to target-specific effects on gonocyte differentiation.

Objective: This study aimed to investigate the effects of combined activin A and Wnt3a treatment on definitive endoderm (DE) differentiation from human parthenogenetic embryonic stem cells (hPESCs)., Methods: hPESCs on human foreskin fibroblast feeder layers were induced to differentiate into DE using a combination of 50 ng/ml activin A and 25 ng/ml Wnt3a. Expression of the DE markers CXCR4, E-cadherin (ECD), Sox17, and Goosecoid (Gsc) were examined using flow cytometry and real-time quantitative PCR., Results: The combination of activin A and Wnt3a significantly enhanced the percentages of CXCR4 + , ECD + , Sox17 + , and Gsc + cells, culminating on day 2 of induction. This combined use promoted DE differentiation from hPESCs in vitro ., Conclusions: Through the combination treatment using activin A and Wnt3a, DE differentiation from hPESCs culminated at 48 h, which can be regarded as the optimal time-point to induce differentiation of endodermal cells such as pancreatic, liver, and intestinal cells., (© 2020 by the Association of Clinical Scientists, Inc.)

In multiple sclerosis (MS), Th17 cells are critical drivers of autoimmune central nervous system (CNS) inflammation and demyelination. Th17 cells exhibit functional heterogeneity fostering both pathogenic and nonpathogenic, tissue-protective functions. Still, the factors that control Th17 pathogenicity remain incompletely defined. Here, using experimental autoimmune encephalomyelitis, an established mouse MS model, we report that therapeutic administration of activin-A ameliorates disease severity and alleviates CNS immunopathology and demyelination, associated with decreased activation of Th17 cells. In fact, activin-A signaling through activin-like kinase-4 receptor represses pathogenic transcriptional programs in Th17-polarized cells, while it enhances antiinflammatory gene modules. Whole-genome profiling and in vivo functional studies revealed that activation of the ATP-depleting CD39 and CD73 ectonucleotidases is essential for activin-A-induced suppression of the pathogenic signature and the encephalitogenic functions of Th17 cells. Mechanistically, the aryl hydrocarbon receptor, along with STAT3 and c-Maf, are recruited to promoter elements on Entpd1 and Nt5e (encoding CD39 and CD73, respectively) and other antiinflammatory genes, and control their expression in Th17 cells in response to activin-A. Notably, we show that activin-A negatively regulates the metabolic sensor, hypoxia-inducible factor-1α, and key inflammatory proteins linked to pathogenic Th17 cell states. Of translational relevance, we demonstrate that activin-A is induced in the CNS of individuals with MS and restrains human Th17 cell responses. These findings uncover activin-A as a critical controller of Th17 cell pathogenicity that can be targeted for the suppression of autoimmune CNS inflammation., Competing Interests: The authors declare no competing interest.

Background: Members of the T-box family of DNA-binding proteins play a prominent role in the differentiation of the three primary germ layers. VegT, Brachyury, and Eomesodermin function as transcriptional activators and, in addition to directly activating the transcription of endoderm- and mesoderm-specific genes, serve as regulators of growth factor signaling during induction of these germ layers. In contrast, the T-box gene, tbx2, is expressed in the embryonic ectoderm, where Tbx2 functions as a transcriptional repressor and inhibits mesendodermal differentiation by the TGFβ ligand Activin. Tbx2 misexpression also promotes dorsal ectodermal fate via inhibition of the BMP branch of the TGFβ signaling network., Results: Here, we report a physical association between Tbx2 and both Smad1 and Smad2, mediators of BMP and Activin/Nodal signaling, respectively. We perform structure/function analysis of Tbx2 to elucidate the roles of both Tbx2-Smad interaction and Tbx2 DNA-binding in germ layer suppression., Conclusion: Our studies demonstrate that Tbx2 associates with intracellular mediators of the Activin/Nodal and BMP/GDF pathways. We identify a novel repressor domain within Tbx2, and have determined that Tbx2 DNA-binding activity is required for repression of TGFβ signaling. Finally, our data also point to overlapping yet distinct mechanisms for Tbx2-mediated repression of Activin/Nodal and BMP/GDF signaling.

Human breast tumors are not fully autonomous. They are dependent on nutrients and growth-promoting signals provided by the supporting stromal cells. Within the tumor microenvironment, one of the secreted macromolecules by tumor cells is activin A, where we show to downregulate CD36 in fibroblasts. Downregulation of CD36 in fibroblasts also increases the secretion of activin A by fibroblasts. We hypothesize that overexpression of CD36 in fibroblasts inhibits the formation of solid tumors in subtypes of breast cancer models. For the first time, we show that co-culturing organoid models of breast cancer cell lines of MDA-MB-231 (e.g., a triple-negative line) or MCF7 (e.g., a luminal-A line) with CD36 + fibroblasts inhibit the growth and normalizes basal and lateral polarities, respectively. In the long-term anchorage-independent growth assay, the rate of colony formation is also reduced for MDA-MB-231. These observations are consistent with the mechanism of tumor suppression involving the downregulation of pSMAD2/3 and YY1 expression levels. Our integrated analytical methods leverage and extend quantitative assays at cell- and colony-scales in both short- and long-term cultures using brightfield or immunofluorescent microscopy and robust image analysis. Conditioned media are profiled with the ELISA assay., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Activins regulate bone formation by controlling osteoclasts and osteoblasts. We investigated Activin-A mechanism of action on human osteoblast mineralization, RNA and microRNA (miRNA) expression profile. A single 2-day treatment of Activin-A at Day 5 of osteoblast differentiation significantly reduced matrix mineralization. Activin A-treated osteoblasts responded with transient change in gene expression, in a 2-wave-fashion. The 38 genes differentially regulated during the first wave (within 8 hr after Activin A start) were involved in transcription regulation. In the second wave (1-2 days after Activin A start), 65 genes were differentially regulated and related to extracellular matrix. Differentially expressed genes in both waves were associated to transforming growth factor beta signaling. We identified which microRNAs modulating osteoblast differentiation were regulated by Activin-A. In summary, 2-day treatment with Activin-A in premineralization period of osteoblast cultures influenced miRNAs, gene transcription, and reduced matrix mineralization. Modulation of Activin A signaling might be useful to control bone quality for therapeutic purposes., (© 2019 The Authors. Journal of Cellular Physiology published by Wiley Periodicals, Inc.)

Follicle-stimulating hormone (FSH), an essential regulator of mammalian fertility, is synthesized by pituitary gonadotrope cells in response to activins. In mice, activins signal via SMAD3, SMAD4, and FOXL2 to regulate transcription of the FSHβ subunit (Fshb) gene. Gonadotrope-specific deletion of Foxl2, alone or in combination with Smad4, renders mice FSH-deficient. Whether human FSHB expression is similarly regulated is not known. Here, we used a combination of transgenic and conditional knockout mouse strains to assess the roles of activins, FOXL2, and SMAD4 in regulation of the human FSHB gene. First, we cultured pituitaries from mice harboring a human FSHB transgene (hFSHB mice) and measured both murine Fshb and human FSHB messenger ribonucleic acid (mRNA) expression in response to exogenous activins or two antagonists of endogenous activin-like signaling (follistatin-288 and SB431542). Both murine Fshb and human FSHB expression were stimulated by activins and reduced by the inhibitors. Next, we analyzed human FSHB expression in hFSHB mice carrying floxed Foxl2 and Smad4 alleles. Cre-mediated ablation of FOXL2 and SMAD4 strongly reduced basal and activin-stimulated murine Fshb and human FSHB expression in cultured pituitaries. Finally, the hFSHB transgene was previously shown to rescue FSH production and fertility in Fshb knockout mice. However, gonadotrope-specific Foxl2/Smad4 knockout females carrying the hFSHB transgene have significantly reduced murine Fshb and human FSHB pituitary mRNA levels and are hypogonadal. Collectively, these data suggest that similar to Fshb regulation in mice, FOXL2 and SMAD4 play essential roles in human FSHB expression., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Alongside in vivo models, a simpler and more mechanistic approach is required to study the effects of myostatin on skeletal muscle because myostatin is an important negative regulator of muscle size. In this study, myostatin was administered to murine (C2C12) and human (CHQ) myoblasts and myotubes. Canonical and noncanonical signaling downstream to myostatin, related ligands, and their receptor were analyzed. The effects of tumorkines were analyzed after coculture of C2C12 and colon cancer-C26 cells. The effects of myostatin on canonical and noncanonical signaling were strongly reduced in C2C12 cells after differentiation. This may be explained by increased follistatin, an endogenous blocker of myostatin and altered expression of activin receptor ligands. In contrast, CHQ cells were equally responsive to myostatin, and follistatin remained unaltered. Both myostatin administration and the coculture stimulated pathways associated with inflammation, especially in C2C12 cells. In conclusion, the effects of myostatin on intracellular signaling may be cell line- or organism-specific, and C2C12 myotubes seem to be a nonoptimal in vitro model for investigating the effects of myostatin on canonical and noncanonical signaling in skeletal muscle. This may be due to altered expression of activin receptor ligands and their regulators during muscle cell differentiation.

Establishing appropriate sizes and shapes of dendritic arbors is critical for proper wiring of the central nervous system. Here we report that Insulin-like Peptide 2 (DILP2) locally activates transiently expressed insulin receptors in the central dendrites of Drosophila Dm8 amacrine neurons to positively regulate dendritic field elaboration. We found DILP2 was expressed in L5 lamina neurons, which have axonal terminals abutting Dm8 dendrites. Proper Dm8 dendrite morphogenesis and synapse formation required insulin signaling through TOR (target of rapamycin) and SREBP (sterol regulatory element-binding protein), acting in parallel with previously identified negative regulation by Activin signaling to provide robust control of Dm8 dendrite elaboration. A simulation of dendritic growth revealed trade-offs between dendritic field size and robustness when branching and terminating kinetic parameters were constant, but dynamic modulation of the parameters could mitigate these trade-offs. We suggest that antagonistic DILP2 and Activin signals from different afferents appropriately size Dm8 dendritic fields., Competing Interests: JL, CT, YL, PM, TL, CH, CL No competing interests declared

The transforming growth factor-β (TGF-β) family controls embryogenesis, stem cell differentiation, and tissue homeostasis. However, how post-translation modifications contribute to fine-tuning of TGF-β family signaling responses is not well understood. Inhibitory (I)-Smads can antagonize TGF-β/Smad signaling by recruiting Smurf E3 ubiquitin ligases to target the active TGF-β receptor for proteasomal degradation. A proteomic interaction screen identified Vpr binding protein (VprBP) as novel binding partner of Smad7. Mis-expression studies revealed that VprBP negatively controls Smad2 phosphorylation, Smad2-Smad4 interaction, as well as TGF-β target gene expression. VprBP was found to promote Smad7-Smurf1-TβRI complex formation and induce proteasomal degradation of TGF-β type I receptor (TβRI). Moreover, VprBP appears to stabilize Smurf1 by suppressing Smurf1 poly-ubiquitination. In multiple adult and mouse embryonic stem cells, depletion of VprBP promotes TGF-β or Activin-induced responses. In the mouse embryo VprBP expression negatively correlates with mesoderm marker expression, and VprBP attenuated mesoderm induction during zebrafish embryogenesis. Our findings thereby uncover a novel regulatory mechanism by which Smurf1 controls the TGF-β and Activin cascade and identify VprBP as a critical determinant of embryonic mesoderm induction., (© The Author(s) (2019). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.)

Human pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient, broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple, robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding (SCS) and BMP4 and Activin A (BA) treatment. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation, while fine-tuning of BA conditions facilitated definitive endoderm commitment. After prolonged differentiation in vitro or in vivo, BA pretreatment resulted in higher mesoderm and endoderm levels at the expense of ectoderm formation. These data demonstrate that SCS with BA treatment is a powerful method for induction of mesendoderm that can be adapted for use in mesoderm and endoderm differentiation., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

A definitive endodermal cell lineage is a prerequisite for the efficient generation of mature endoderm derivatives that give rise to organs, such as the pancreas and liver. We previously reported that the induction of mesenchymal definitive endoderm cells depends on autocrine TGF-β signaling and that pharmacological blockage of TGF-β signaling by Repsox disrupts endoderm specification. The definitive endoderm arises from a primitive streak, which depends largely on TGF-β signaling. If the TGF-β pathway is blocked by Repsox, cell fate after the primitive streak induction is so-far unknown. We report here, that an induced primitive streak cell-population contained many T/SOX2 co-expressing cells, and subsequent inhibition of TGF-β signaling by Repsox promoted neuroectodermal cell fate, which was characterized using single-cell qPCR analysis and immunostaining. The process of epithelial-to-mesenchymal transition, which is inherent to the process of definitive endoderm differentiation, was also disrupted upon Repsox treatment. Our findings may provide a new approach to produce neural progenitors., Competing Interests: The authors have declared that no competing interests exist.

Hepatocytes have been successfully generated from human pluripotent stem cells (hPSCs). However, the cost-effective and clinical-grade generation of hepatocytes from hPSCs still need to be improved. In this study, we reported the production of functional hepatocytes from clinical-grade human embryonic stem cells (hESCs) under good manufacturing practice (GMP) requirements. We sequentially generated primitive streak (PS), definitive endoderm (DE), hepatoblasts and hepatocyte-like cells (HLCs) from hESCs in the different stages with completely defined reagents. During hepatoblast differentiation, dimethyl sulfoxide (DMSO), transferrin, L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Vc-Mg), insulin, and sodium selenite were used instead of cytokines and FBS/KOSR. Then, hepatoblasts were differentiated into HLCs that had a typical hepatocyte morphology and possessed characteristics of mature hepatocytes, such as metabolic-related gene expression, albumin secretion, fat accumulation, glycogen storage, and inducible cytochrome P450 activity in vitro. HLCs integrated into the livers of Tet-uPA Rag2 -/- Il2rg -/- (URG) mice, which partially recovered after transplantation. Furthermore, a series of biosafety-related experiments were performed to ensure future clinical applications. In conclusion, we developed a chemically defined system to generate qualified clinical-grade HLCs from hESCs under GMP conditions. HLCs have been proven to be safe and effective for treating liver failure. This efficient platform could facilitate the treatment of liver diseases using hESC-derived HLCs transplantation.

Kidney formation is regulated by the balance between maintenance and differentiation of nephron progenitor cells (NPCs). Now that directed differentiation of NPCs from human induced pluripotent stem cells (iPSCs) can be achieved, maintenance and propagation of NPCs in vitro should be beneficial for regenerative medicine. Although WNT and FGF signals were previously shown to be essential for NPC propagation, the requirement for BMP/TGFβ signaling remains controversial. Here we reveal that activin has superior effects to BMP7 on maintenance efficiency of human iPSC-derived NPCs. Activin expanded ITGA8 + /PDGFRA - /SIX2-GFP + NPCs by 5-fold per week at 80%-90% efficiency, and the propagated cells possessed robust capacity for nephron formation both in vitro and in vivo. The expanded cells also maintained their nephron-forming potential after freezing. Furthermore, the protocol was applicable to multiple non-GFP-tagged iPSC lines. Thus, our activin-based protocol will be applicable to a variety of research fields including disease modeling and drug screening., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)