Activin-A impedes the establishment of CD4+ T cell exhaustion and enhances anti-tumor immunity in the lung.

BACKGROUND: Although tumor-infiltrating T cells represent a favorable prognostic marker for cancer patients, the majority of these cells are rendered with an exhausted phenotype. Hence, there is an unmet need to identify factors which can reverse this dysfunctional profile and restore their anti-tumorigenic potential. Activin-A is a pleiotropic cytokine, exerting a broad range of pro- or anti-inflammatory functions in different disease contexts, including allergic and autoimmune disorders and cancer. Given that activin-A exhibits a profound effect on CD4+ T cells in the airways and is elevated in lung cancer patients, we hypothesized that activin-A can effectively regulate anti-tumor immunity in lung cancer. METHODS: To evaluate the effects of activin-A in the context of lung cancer, we utilized the OVA-expressing Lewis Lung Carcinoma mouse model as well as the B16F10 melanoma model of pulmonary metastases. The therapeutic potential of activin-A-treated lung tumor-infiltrating CD4+ T cells was evaluated in adoptive transfer experiments, using CD4-/--tumor bearing mice as recipients. In a reverse approach, we disrupted activin-A signaling on CD4+ T cells using an inducible model of CD4+ T cell-specific knockout of activin-A type I receptor. RNA-Sequencing analysis was performed to assess the transcriptional signature of these cells and the molecular mechanisms which mediate activin-A's function. In a translational approach, we validated activin-A's anti-tumorigenic properties using primary human tumor-infiltrating CD4+ T cells from lung cancer patients. RESULTS: Administration of activin-A in lung tumor-bearing mice attenuated disease progression, an effect associated with heightened ratio of infiltrating effector to regulatory CD4+ T cells. Therapeutic transfer of lung tumor-infiltrating activin-A-treated CD4+ T cells, delayed tumor progression in CD4-/- recipients and enhanced T cell-mediated immunity. CD4+ T cells genetically unresponsive to activin-A, failed to elicit effective anti-tumor properties and displayed an exhausted molecular signature governed by the transcription factors Tox and Tox2. Of translational importance, treatment of activin-A on tumor-infiltrating CD4+ T cells from lung cancer patients augmented their immunostimulatory capacity towards autologous CD4+ and CD8+ T cells. CONCLUSIONS: In this study, we introduce activin-A as a novel immunomodulatory factor in the lung tumor microenvironment, which bestows exhausted CD4+ T cells with effector properties.

Involvement of CHOP in activin A­induced myeloma NS­1 cell apoptosis.

Activin A, a multifunctional cytokine, is a member of transforming growth factor­ß (TGF­ß) superfamily. It is associated with a variety of pathophysiological processes, including inflammation, fibrosis, and tumorigenesis. Chronic or prolonged endoplasmic reticulum (ER) stress can lead to cells apoptosis. However, whether ER stress­related proteins, such as CHOP, GADD34 are involved in activin A­induced myeloma cell apoptosis remains unknown. In the present study, it was revealed that activin A inhibited the proliferation of myeloma cell line NS­1 cells and induced NS­1 cell apoptosis. Activin A upregulated the expression of CHOP, GADD34, caspase­3, and caspase­12. Moreover, both Smad3 and p­Smad3 levels were increased with treatment of activin A. Further studies revealed that the overexpression of activin signaling protein Smad3 in NS­1 cells increased the levels of CHOP, caspase­3, and p­Smad3. These data indicated that the CHOP protein of the ER stress pathway may be involved in activin A­induced NS­1 cell apoptosis, and also indicated the potential therapy of activin A­induced apoptosis via CHOP signaling for multiple myeloma.

Intraperitoneal administration of activin A promotes development of endometriotic lesions in a mouse model of endometriosis.

PURPOSE: This study aimed to investigate the effect of intraperitoneal administration of activin on the occurrence of endometriosis using a mouse model of endometriosis. METHODS: A mouse model of endometriosis was prepared by intraperitoneally administering endometrial tissue and blood collected from donor mice to C57BL/6J 7-8- week-old recipient mice. A total of 400 µg of activin A was intraperitoneally administered to model mice in the activin group for 5 days. Intraperitoneal endometriotic lesions were confirmed macroscopically and IL-6 and TNF-α levels in washed ascites were measured by ELISA. RESULTS: Endometriotic lesions were observed in all mice. In the activin group, the maximum diameter of endometriotic lesions was significantly larger than that in control group (4.7?1.3 vs 2.9?0.9 mm, p?0.01). The total area of the lesion was also significantly higher in the activin group than in the control group (21.1?9.9 vs 8.8?5.4 mm2,p?0.01). Furthermore, IL-6 and TNF-α levels in ascites were significantly higher in the activin group than in the control group (IL-6 : 85.8?15.3 vs 75.1?19.3 pg/ml, p?0.05 ; TNF-α : 629.8?15.4 vs 605.9?11.4 pg/ml, p?0.05). CONCLUSION: Activin promotes occurrence of endometriosis. Inflammatory cytokines are also elevated by activin administration,suggesting that they may contribute to progression of endometriosis J. Med. Invest. 66 : 123-127, February, 2019.

Activin A improves retinal pigment epithelial cell survival on stiff but not soft substrates.

In several retinal degenerative disease pathologies, such as dry age-related macular degeneration (AMD), the retinal pigment epithelium (RPE) cell monolayer becomes dysfunctional. Promising tissue engineering treatment approaches implant RPE cells on scaffolds into the subretinal space. However, these approaches are not without challenges. Two major challenges that must be addressed are RPE dedifferentiation and the inflammatory response to cell/scaffold implantation. Design and optimization of scaffold cues for the purpose of RPE transplantation remain relatively unexplored, specifically the mechanical properties of the scaffolds. Prior work from our group indicated that by varying substrate moduli significant differences could be induced in cell cytoskeleton structure, cellular activity, and expression of inflammatory markers. We hypothesized that Activin A would provide rescue effects for cells demonstrating dedifferentiated characteristics. Results demonstrated that for cells on low modulus scaffolds, the mechanical environment was the dominating factor and Activin A was unable to rescue these cells. However, Activin A did demonstrate rescue effects for cells on high modulus scaffolds. This finding indicates that when cultured on scaffolds with an appropriate modulus, exogenous factors, such as Activin A, can improve RPE cell expression, morphology, and activity, while an inappropriate scaffold modulus can have devastating effects on RPE survival regardless of chemical stimulation. These findings have broad implications for the design and optimization of scaffolds for long-term successful RPE transplantation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2871-2880, 2018.

Injury of Adult Zebrafish Expressing Acvr1lQ204D Does Not Result in Heterotopic Ossification.

Fibrodysplasia Ossificans Progressiva (FOP) is a rare, autosomal dominant genetic disorder in humans characterized by the gradual ossification of fibrous tissues, including skeletal muscle, tendons, and ligaments. In humans, mutations in the Type I BMP/TGFß family member receptor gene, ACVR1, are associated with FOP. Zebrafish acvr1l, previously known as alk8, is the functional ortholog of human ACVR1. We previously created and characterized the first adult zebrafish model for FOP by generating animals harboring heat shock-inducible mCherry-tagged constitutively active Acvr1l (Q204D). Since injury is a known trigger for heterotopic ossification (HO) development in human FOP patients, in this study, we investigated several injury models in Acvr1lQ204D-expressing zebrafish and the subsequent formation of HO. We performed studies of Activin A injection, cardiotoxin (CTX) injection, and caudal fin clip injury. We found that none of these methods resulted in HO formation at the site of injury. However, some of the cardiotoxin-injected and caudal fin-clipped animals did exhibit HO at distant sites, including the body cavity and along the spine. We describe these results in the context of new and exciting reports on FOP, and discuss future studies to better understand the etiology and progression of this disease.

Post-injury Nose-to-Brain Delivery of Activin A and SerpinB2 Reduces Brain Damage in a Mouse Stroke Model.

Synaptic NMDA receptors activating nuclear calcium-driven adaptogenomics control a potent body-own neuroprotective mechanism, referred to as acquired neuroprotection. Viral vector-mediated gene transfer in conjunction with stereotactic surgery has previously demonstrated the proficiency of several nuclear calcium-regulated genes to protect in vivo against brain damage caused by toxic extrasynaptic NMDA receptor signaling following seizures or stroke. Here we used noninvasive nose-to-brain administration of Activin A and SerpinB2, two secreted nuclear calcium-regulated neuroprotectants, for post-injury treatment of brain damage following middle cerebral artery occlusion (MCAO) in C57BL/6N mice. The observed reduction of the infarct volume was comparable to the protection obtained by intracerebroventricular injection of recombinant Activin A or SerpinB2 or by stereotactic delivery 3 weeks prior to the injury of a recombinant adeno-associated virus containing an expression cassette for the potent neuroprotective transcription factor Npas4. These results establish post-injury, nose-to-brain delivery of Activin A and SerpinB2 as effective and possibly clinically applicable treatments of acute and chronic neurodegenerative conditions.

Luspatercept for the treatment of anaemia in patients with lower-risk myelodysplastic syndromes (PACE-MDS): a multicentre, open-label phase 2 dose-finding study with long-term extension study.

BACKGROUND: Myelodysplastic syndromes are characterised by ineffective erythropoiesis. Luspatercept (ACE-536) is a novel fusion protein that blocks transforming growth factor beta (TGF ß) superfamily inhibitors of erythropoiesis, giving rise to a promising new investigative therapy. We aimed to assess the safety and efficacy of luspatercept in patients with anaemia due to lower-risk myelodysplastic syndromes. METHODS: In this phase 2, multicentre, open-label, dose-finding study (PACE-MDS), with long-term extension, eligible patients were aged 18 years or older, had International Prognostic Scoring System-defined low or intermediate 1 risk myelodysplastic syndromes or non-proliferative chronic myelomonocytic leukaemia (white blood cell count <13 000/µL), and had anaemia with or without red blood cell transfusion support. Enrolled patients were classified as having low transfusion burden, defined as requiring less than 4 red blood cell units in the 8 weeks before treatment (and baseline haemoglobin <10 g/dL), or high transfusion burden, defined as requiring 4 or more red blood cell units in the 8 weeks before treatment. Patients received luspatercept subcutaneously once every 21 days at dose concentrations ranging from 0·125 mg/kg to 1·75 mg/kg bodyweight for five doses (over a maximum of 12 weeks). Patients in the expansion cohort were treated with 1·0 mg/kg luspatercept; dose titration up to 1·75 mg/kg was allowed, and patients could be treated with luspatercept for a maximum of 5 years. Patients in the base study were assessed for response and safety after 12 weeks in order to be considered for enrolment into the extension study. The primary endpoint was the proportion of patients achieving modified International Working Group-defined haematological improvement-erythroid (HI-E), defined as a haemoglobin concentration increase of 1·5 g/dL or higher from baseline for 14 days or longer in low transfusion burden patients, and a reduction in red blood cell transfusion of 4 or more red blood cell units or a 50% or higher reduction in red blood cell units over 8 weeks versus pre-treatment transfusion burden in high transfusion burden patients. Patient data were subcategorised by: luspatercept dose concentrations (0·125-0·5 mg/kg vs 0·75-1·75 mg/kg); pre-study transfusion burden (high transfusion burden vs low transfusion burden, defined as ≥4 vs <4 red blood cell units per 8 weeks); pre-study serum erythropoietin concentration (<200 IU/L, 200-500 IU/L, and >500 IU/L); presence of 15% or more ring sideroblasts; and presence of SF3B1 mutations. Efficacy analyses were carried out on the efficacy evaluable and intention-to-treat populations. This trial is currently ongoing. This study is registered with ClinicalTrials.gov, numbers NCT01749514 and NCT02268383. FINDINGS: Between Jan 21, 2013, and Feb 12, 2015, 58 patients with myelodysplastic syndromes were enrolled in the 12 week base study at nine treatment centres in Germany; 27 patients were enrolled in the dose-escalation cohorts (0·125-1·75 mg/kg) and 31 patients in the expansion cohort (1·0-1·75 mg/kg). 32 (63% [95% CI 48-76]) of 51 patients receiving higher dose luspatercept concentrations (0·75-1·75 mg/kg) achieved HI-E versus two (22% [95% CI 3-60]) of nine receiving lower dose concentrations (0·125-0·5 mg/kg). Three treatment-related grade 3 adverse events occurred in one patient each: myalgia (one [2%]), increased blast cell count (one [2%]), and general physical health deterioration (one [2%]). Two of these treatment-related grade 3 adverse events were reversible serious grade 3 adverse events: one patient (2%) had myalgia and one patient (2%) had general physical health deterioration. INTERPRETATION: Luspatercept was well tolerated and effective for the treatment of anaemia in lower-risk myelodysplastic syndromes and so could therefore provide a novel therapeutic approach for the treatment of anaemia associated with lower-risk myelodysplastic syndromes; further studies are ongoing. FUNDING: Acceleron Pharma.

Current and future chemical therapies for treating anaemia in chronic kidney disease.

INTRODUCTION: Erythropoiesis-stimulating agents (ESAs) are not perfect, since they have potential side effects. Iron therapy is also receiving growing attention in recent years. Areas covered: We performed a literature search on PubMed using the following key words: anemia, chronic kidney disease, HIF stabilisers, sotatercept, actin traps, iron, iron-containing phosphate binders, iron dialysate. We reviewed new drugs that are under clinical development to obtain better safety and activity and/or easier and cheaper manufacturing processes in comparison to available ESAs. We also considered new strategies to increase iron stores. Several phase 1 and 2 studies support the beneficial role of increasing Hypoxia Inducible factor (HIF) activity for stimulating endogenous erythropoiesis. Sotatercept and luspatercept, two activin traps, are undergoing clinical development mainly for indications other than CKD. They have the additional effect of improving osteoporosis. Iron-containing phosphate binders have become available recently. Expert opinion: Several medical needs are unmet with ESA. HIF stabilisers are the most appealing drugs undergoing clinical development. They expose patients to lower levels of EPO than ESA, possibly reducing unintended effects. Their long-term safety is still to be demonstrated. One new iron-containing phosphate binders has the potential of combining two indications: hyperphosphoremia and iron deficiency, possibly improving compliance.

A drug delivery hydrogel system based on activin B for Parkinson's disease.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Activins are members of the superfamily of transforming growth factors and have many potential neuroprotective effects. Herein, at the first place, we verified activin B's neuroprotective role in a PD model, and revealed that activin B's fast release has limited function in the PD therapy. To this end, we developed a multi-functional crosslinker based thermosensitive injectable hydrogels to deliver activin B, and stereotactically injected the activin B-loaded hydrogel into the striatum of a mouse model of PD. The histological evaluation showed that activin B can be detected even 5 weeks post-surgery in PD mice implanted with activin B-loaded hydrogels, and activin B-loaded hydrogels can significantly increase the density of tyrosine hydroxylase positive (TH(+)) nerve fibers and reduce inflammatory responses. The behavioral evaluation demonstrated that activin B-loaded hydrogels significantly improved the performance of the mice in the PD model. Meanwhile, we found that hydrogels can slightly induce the activation of microglia cells and astrocytes, while cannot induce apoptosis in the striatum. Overall, our data demonstrated that the developed activin B-loaded hydrogels provide sustained release of activin B for over 5 weeks and contribute to substantial cellular protection and behavioral improvement, suggesting their potential as a therapeutic strategy for PD.

Efficient endodermal induction of human adipose stem cells using various concentrations of Activin A for hepatic differentiation.

UNLABELLED: Activin A, which is a signaling molecule similar to Nodal, rapidly promotes endoderm induction of both embryonic stem (ES) cells and MSCs. Protocols for hepatic induction exhibit differences in efficiency and reproducibility depending on the specific methods or sources of MSCs. We characterized the effects of Activin A concentration on induction efficiency during hepatic differentiation of MSCs. Human MSCs (hMSCs) were differentiated into a hepatic lineage via a three-step protocol. Cells were first cultured in fetal bovine serum-free MSCs conditioned medium supplemented with Activin A (20, 50, or 100 ng/mL) for 3 days followed by treatment with additional agents. RT-PCR analysis, immunocytochemistry assays, periodic acid and Schiff's solution staining, and ELISAs were performed to confirm hepatic induction of hMSCs. Expression of genes related to the primitive foregut endoderm was observed in cells treated with higher concentration of Activin A. Gene expression related to functional primitive hepatocytes was observed in the early phases of hepatic differentiation. During the early period of the differentiation protocol, greater albumin secretion was observed when cells were treated with higher concentrations of Activin A. CONCLUSION: Thus, Activin A concentration affects the rate of endoderm induction of hMSCs, and at higher concentrations in vitro.

The toxicological evaluation of repetitive 2- and 4-week intravenous injection of Activin A/BMP-2 chimera (AB204) into rats.

The subchronic (28-days) toxicity of an Activin A/BMP-2 chimera (AB204) was assessed in rats. Sprague-Dawley rats received repetitive intravenous injection of AB204 in doses of 0, 0.25 and 0.5 mg/kg for two weeks and in doses of 0, 0.08, 0.16 and 0.32 mg/kg/day for four weeks. No animal was dead and no change caused by the AB204 was observed in general symptoms, weight variation, and food and water intake as well as blood test and autopsy findings. In conclusion, the no observed adverse effects level (NOAEL) of the AB204 on rats was determined to be 0.32 mg/kg/day.

Mast cells are dispensable for normal and activin-promoted wound healing and skin carcinogenesis.

The growth and differentiation factor activin A is a key regulator of tissue repair, inflammation, fibrosis, and tumorigenesis. However, the cellular targets, which mediate the different activin functions, are still largely unknown. In this study, we show that activin increases the number of mature mast cells in mouse skin in vivo. To determine the relevance of this finding for wound healing and skin carcinogenesis, we mated activin transgenic mice with CreMaster mice, which are characterized by Cre recombinase-mediated mast cell eradication. Using single- and double-mutant mice, we show that loss of mast cells neither affected the stimulatory effect of overexpressed activin on granulation tissue formation and reepithelialization of skin wounds nor its protumorigenic activity in a model of chemically induced skin carcinogenesis. Furthermore, mast cell deficiency did not alter wounding-induced inflammation and new tissue formation or chemically induced angiogenesis and tumorigenesis in mice with normal activin levels. These findings reveal that mast cells are not major targets of activin during wound healing and skin cancer development and also argue against nonredundant functions of mast cells in wound healing and skin carcinogenesis in general.

Small molecule-assisted, line-independent maintenance of human pluripotent stem cells in defined conditions.

Human pluripotent stem cells (hPSCs) are conventionally grown in a mouse feeder cell-dependent manner. Chemically defined culture conditions are, however, desirable not only for potential medically oriented applications but also for investigating mechanisms of self-renewal and differentiation. In light of the rather high complexity and cost of existing defined hPSC culture systems, we have systematically evaluated over 20 potential media ingredients. Only components that reproducibly gave beneficial effects were ultimately combined to yield a simple and cost-effective formulation termed FTDA. This xeno-free medium is based on mimicking self-renewal factor activities present in mouse embryonic fibroblast-conditioned medium, at minimal dosages. Additionally, small molecule inhibitors of BMP and WNT signaling served to specifically suppress typical types of spontaneous differentiation seen in hPSC cultures. FTDA medium was suitable for the generation of human induced pluripotent stem cells and enabled robust long-term maintenance of diverse hPSC lines including hard-to-grow ones. Comparisons with existing defined media suggested reduced spontaneous differentiation rates in FTDA. Our results imply that using supportive factors at minimal concentrations may still promote robust self-renewal and preserve pluripotency of hPSCs.

Conversion from mouse embryonic to extra-embryonic endoderm stem cells reveals distinct differentiation capacities of pluripotent stem cell states.

The inner cell mass of the mouse pre-implantation blastocyst comprises epiblast progenitor and primitive endoderm cells of which cognate embryonic (mESCs) or extra-embryonic (XEN) stem cell lines can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of their in vivo tissue of origin. Recently, we demonstrated that XEN-like cells arise within mESC cultures. This raises the possibility that mESCs can generate self-renewing XEN cells without the requirement for gene manipulation. We have developed a novel approach to convert mESCs to XEN cells (cXEN) using growth factors. We confirm that the downregulation of the pluripotency transcription factor Nanog and the expression of primitive endoderm-associated genes Gata6, Gata4, Sox17 and Pdgfra are necessary for cXEN cell derivation. This approach highlights an important function for Fgf4 in cXEN cell derivation. Paracrine FGF signalling compensates for the loss of endogenous Fgf4, which is necessary to exit mESC self-renewal, but not for XEN cell maintenance. Our cXEN protocol also reveals that distinct pluripotent stem cells respond uniquely to differentiation promoting signals. cXEN cells can be derived from mESCs cultured with Erk and Gsk3 inhibitors (2i), and LIF, similar to conventional mESCs. However, we find that epiblast stem cells (EpiSCs) derived from the post-implantation embryo are refractory to cXEN cell establishment, consistent with the hypothesis that EpiSCs represent a pluripotent state distinct from mESCs. In all, these findings suggest that the potential of mESCs includes the capacity to give rise to both extra-embryonic and embryonic lineages.

Activin, BMP and FGF pathways cooperate to promote endoderm and pancreatic lineage cell differentiation from human embryonic stem cells.

The study of how human embryonic stem cells (hESCs) differentiate into insulin-producing beta cells has twofold significance: first, it provides an in vitro model system for the study of human pancreatic development, and second, it serves as a platform for the ultimate production of beta cells for transplantation into patients with diabetes. The delineation of growth factor interactions regulating pancreas specification from hESCs in vitro is critical to achieving these goals. In this study, we describe the roles of growth factors bFGF, BMP4 and Activin A in early hESC fate determination. The entire differentiation process is carried out in serum-free chemically-defined media (CDM) and results in reliable and robust induction of pancreatic endoderm cells, marked by PDX1, and cell clusters co-expressing markers characteristic of beta cells, including PDX1 and insulin/C-peptide. Varying the combinations of growth factors, we found that treatment of hESCs with bFGF, Activin A and BMP4 (FAB) together for 3-4days resulted in strong induction of primitive-streak and definitive endoderm-associated genes, including MIXL1, GSC, SOX17 and FOXA2. Early proliferative foregut endoderm and pancreatic lineage cells marked by PDX1, FOXA2 and SOX9 expression are specified in EBs made from FAB-treated hESCs, but not from Activin A alone treated cells. Our results suggest that important tissue interactions occur in EB-based suspension culture that contribute to the complete induction of definitive endoderm and pancreas progenitors. Further differentiation occurs after EBs are embedded in Matrigel and cultured in serum-free media containing insulin, transferrin, selenium, FGF7, nicotinamide, islet neogenesis associated peptide (INGAP) and exendin-4, a long acting GLP-1 agonist. 21-28days after embedding, PDX1 gene expression levels are comparable to those of human islets used for transplantation, and many PDX1(+) clusters are formed. Almost all cells in PDX1(+) clusters co-express FOXA2, HNF1ß, HNF6 and SOX9 proteins, and many cells also express CPA1, NKX6.1 and PTF1a. If cells are then switched to medium containing B27 and nicotinamide for 7-14days, then the number of insulin(+) cells increases markedly. Our study identifies a new chemically defined culture protocol for inducing endoderm- and pancreas-committed cells from hESCs and reveals an interplay between FGF, Activin A and BMP signaling in early hESC fate determination.

In vitro development of bovine embryos cultured with activin A.

The aim of this study was to investigate the effects of activin A on development, differential cell counts and apoptosis/necrosis rates of bovine embryos produced in vitro. Presumptive zygotes were cultured up to Day 8 in synthetic oviduct fluid containing aminoacids, citrate, myo-inositol and BSA. In Experiment 1, activin (10 ng mL(-1)) was added: 1/from Day 1 to Day 3; 2/from Day 1 to Day 8; 3/from Day 3 to Day 8; or 4/absent (control). In Experiment 2, 10 ng mL(-1) activin were added either before (Day 3 to Day 5) or after (Day 5 to Day 8) the early morula stage. In Experiment 1, activin during the first 72 h of culture reduced Day 3 cleavage, 5-8 cell rates and blastocyst development, while hatching rates increased. No changes were observed within differential cell counts. In experiment 2, activin improved blastocyst development after, and had no effect before, the Day 5 morula stage. However, trophectoderm (TE) cell numbers decreased with activin both before and after the Day 5 morula stage, suggesting that activin inhibits TE differentiation. The presence of activin during the whole culture had no effect on TUNEL positive cells, but when added at shorter periods activin increased apoptotic rates. Effects of activin during in vitro bovine embryo development, depends on timing of its addition to the culture medium.

Temporal specification of blood progenitors from mouse embryonic stem cells and induced pluripotent stem cells.

The efficient and reproducible generation of differentiated progenitors from pluripotent stem cells requires the recapitulation of appropriate developmental stages and pathways. Here, we have used the combination of activin A, BMP4 and VEGF under serum-free conditions to induce hematopoietic differentiation from both embryonic and induced pluripotent stem cells, with the aim of modeling the primary sites of embryonic hematopoiesis. We identified two distinct Flk1-positive hematopoietic populations that can be isolated based on temporal patterns of emergence. The earliest arising population displays characteristics of yolk sac hematopoiesis, whereas a late developing Flk1-positive population appears to reflect the para-aortic splanchnopleura hematopoietic program, as it has reduced primitive erythroid capacity and substantially enhanced myeloid and lymphoid potential compared with the earlier wave. These differences between the two populations are accompanied by differences in the expression of Sox17 and Hoxb4, as well as in the cell surface markers AA4.1 and CD41. Together, these findings support the interpretation that the two populations are representative of the early sites of mammalian hematopoiesis.

Activin A and follistatin-like 3 determine the susceptibility of heart to ischemic injury.

BACKGROUND: Transforming growth factor-beta family cytokines have diverse actions in the maintenance of cardiac homeostasis. Activin A is a member of this family whose regulation and function in heart are not well understood at a molecular level. Follistatin-like 3 (Fstl3) is an extracellular regulator of activin A protein, and its function in the heart is also unknown. METHODS AND RESULTS: We analyzed the expression of various transforming growth factor-beta superfamily cytokines and their binding partners in mouse heart. Activin betaA and Fstl3 were upregulated in models of myocardial injury. Overexpression of activin A with an adenoviral vector (Ad-actbetaA) or treatment with recombinant activin A protein protected cultured myocytes from hypoxia/reoxygenation-induced apoptosis. Systemic overexpression of activin A in mice by intravenous injection of Ad-actbetaA protected hearts from ischemia/reperfusion injury. Activin A induced the expression of Bcl-2, and ablation of Bcl-2 by small interfering RNA abrogated its protective action in myocytes. The protective effect of activin A on cultured myocytes was abolished by treatment with Fstl3 or by a pharmacological activin receptor-like kinase inhibitor. Cardiac-specific Fstl3 knockout mice showed significantly smaller infarcts after ischemia/reperfusion injury that was accompanied by reduced apoptosis. CONCLUSIONS: Activin A and Fstl3 are induced in heart by myocardial stress. Activin A protects myocytes from death, and this activity is antagonized by Fstl3. Thus, the relative expression levels of these factors after injury is a determinant of cell survival in the heart.

Delayed activin A administration attenuates tissue death after transient focal cerebral ischemia and is associated with decreased stress-responsive kinase activation.

Focal cerebral ischemia and reperfusion initiates complex cellular and molecular interactions that lead to either cell repair or destruction. In earlier work, we found that activin A is an early gene response to cerebral ischemia and supports cortical neuron survival in vitro. In this study, the ability of exogenous activin A to attenuate injury from transient middle cerebral artery occlusion was tested in adult mice. Intracerebroventricular administration of activin A prior to middle cerebral artery occlusion reduced infarct volume apparent 1 day after experimental stroke. A single activin A administration at 6 h following ischemia/reperfusion reduced lesion volumes at 1 and 3 days and led to improved neurobehavior. Moreover, activin A treatment spared neurons within the ischemic hemisphere and led to a concomitant reduction in microglial activation. Activation of the stress-responsive kinases p38 and c-jun N-terminal kinase implicated in neuronal apoptosis after stroke was reduced following activin A treatment. Together these findings suggest that activin A promotes tissue survival after focal cerebral ischemia/reperfusion with an extended therapeutic window.

Regulation of membrane progestin receptors in the zebrafish ovary by gonadotropin, activin, TGF-beta and BMP-15.

Progestin hormones are vital for inducing oocyte maturation in fish by binding to membrane progestin receptors (mPRs). The aim of this study was to examine the expression and regulation of mPRalpha and mPRbeta in zebrafish follicles. First, defolliculated fully grown oocytes were subjected to immunofluorescent staining using anti-mPRalpha and mPRbeta antibodies, and their expression on the oocyte membrane was confirmed. Second, total protein was collected from zebrafish follicles and Western blotting revealed that the level of mPRalpha and mPRbeta increased with follicle development. We have previously shown that several members of the transforming growth factor-beta (TGF-beta) superfamily, including TGF-beta1, activin-A, and bone morphogenetic protein (BMP)-15, regulate oocyte maturation in zebrafish. Therefore, the third major focus of this study was to test if these growth factors, as well as gonadotropins, regulate the expression of mPRs. Overexpression of BMP-15 significantly reduced, while knockdown of BMP-15 increased, mPRbeta levels. However, mPRalpha expression level remained unchanged with BMP-15 overexpression or knockdown. Treatment of follicles with human chorionic gonadotropin (hCG) resulted in an increased in mPRbeta, but not mPRalpha, expression levels. Activin-A induced the expression of mPRalpha and mPRbeta in a dose- and time-dependent manner. On the other hand, TGF-beta1 treatment suppressed the expression of mPRbeta, but not mPRalpha. Taken together, these findings further support the role of mPRs in oocyte maturation and suggest that gonadotropins, BMP-15, activin-A, and TGF-beta1 exert their regulatory effects on oocyte maturation in part by regulating mPR expression.