Human amniotic epithelial cell transplantation is safe and well tolerated in patients with compensated cirrhosis: a first-in-human trial.

Placenta-derived human amniotic epithelial cells (hAEC) exhibit anti-inflammatory and anti-fibrotic effects in cirrhosis models. We conducted a first-in-human phase I clinical trial to assess the safety and tolerability of hAEC in adults with compensated cirrhosis. We examined increasing and repeated doses of hAEC in 9 patients in 3 cohorts. Cohort 1 patients received 0.5 × 106/kg hAEC in one IV infusion. Cohort 2 patients received 1 × 106/kg hAEC in one IV infusion. The patients in cohort 3 received 1 × 106/kg hAEC on days 0 and 28. Here, we report follow-up to post-infusion day 56 (D56), during which no serious adverse events occurred. Six patients experienced no study-related adverse events, while 3 patients reported mild (grade 1) headaches that were possibly infusion-related. A transient decrease in serum platelet count occurred in all patients, which returned to baseline screening values by day 5. FIB-4 values to assess fibrosis were significantly lower at D56. Although not statistically significant, serum AST levels and liver stiffness measurements at D56 were lower than those at baseline. The hepatic venous pressure gradient, a measure of portal hypertension, declined in 4 patients, did not change in 3 patients, and increased in 2 patients. In conclusion, intravenous infusion of allogeneic hAEC in patients with compensated cirrhosis at the doses used in this study was safe and well tolerated, with no difference observed between 1 and 2 doses. Decreased hepatic inflammation, liver stiffness, and portal hypertension support larger studies aimed at identifying patients who may benefit from this therapy. Clinical Trial registration: The trial was prospectively entered on the Australian Clinical Trials Registry (ANZCTR12616000437460).

Late-Life Metabolic Dysfunction-Associated Steatotic Liver Disease and its Association With Physical Disability and Dementia.

BACKGROUND: The burden of metabolic dysfunction-associated steatotic liver disease (MASLD) is growing rapidly, including among older adults. The number of older adults is also rising with concomitantly increasing rates of age-related physical and cognitive dysfunction. However, data on whether MASLD affects physical and cognitive function in older adults is limited. As such, we aimed to identify whether prevalent MASLD influences the risk of incident physical disability or dementia in initially healthy older adults. METHODS: A post-hoc analysis of participants from the ASPREE-XT cohort study, which recruited community-dwelling older adults without a history of cardiovascular disease, dementia, or independence-limiting functional impairment. The Fatty Liver Index (to identify MASLD) was calculated in those with complete data. Cox proportional-hazards models were used to investigate the outcomes of dementia and persistent physical disability in participants with MASLD vs those without. RESULTS: Of the 9 097 individuals included (mean age 75.1 ±â€…4.2 years; 45.0% men), 341 (3.7%) developed persistent physical disability and 370 (4.1%) developed dementia over a median follow-up of 6.4 years (IQR 5.3-7.5 years). When adjusting for known contributors including age, gender, education, comorbidity, and functional measures, MASLD was associated with an increased risk of persistent physical disability (HR 1.41 [95% CI: 1.07-1.87]) and reduced risk of incident dementia (HR 0.63 [95% CI: 0.48-0.83]). CONCLUSIONS: Prevalent MASLD is associated with reduced rates of incident dementia but increased risk of persistent physical disability in initially relatively healthy older adults. Understanding the mechanisms underlying these divergent results to allow appropriate risk stratification and counseling is important.

Phase 2, open-label, rollover study of cenicriviroc for liver fibrosis associated with metabolic dysfunction-associated steatohepatitis.

BACKGROUND: Cenicriviroc (CVC) is a novel, orally administered antagonist of chemokine receptor types 2/5 that has demonstrated antifibrotic activity in a phase 2b study of patients with NASH. This phase 2, open-label, rollover study investigated the long-term safety and tolerability of CVC in patients with NASH and stage 0-4 liver fibrosis. METHODS: Eligible patients who completed the phase 2 CENTAUR study or reached a predefined endpoint in the phase 3 AURORA study were rolled over and received open-label CVC 150 mg once daily. Safety assessments were conducted at the start of the study, and patients were seen in the clinic every 3 months until the study sponsor terminated CVC development. Safety endpoints included treatment-emergent adverse events (TEAEs), treatment-related TEAEs, adverse event severity, and clinical laboratory assessments. RESULTS: A total of 167 patients were enrolled, with a median treatment duration of 33.6 months. Before study termination, 36 patients (21.6%) prematurely discontinued the study. Treatment-related TEAEs were reported in 28 patients (16.8%). The most common treatment-related TEAEs were 4 cases of diarrhea (2.4%) and 2 cases each (1.2%) of abdominal pain, nausea, alanine aminotransferase increased, aspartate aminotransferase increased, hypertriglyceridemia, myalgia, pruritus, and rash. The majority of these treatment-related events were mild in intensity, and none were life-threatening. There were no clinically meaningful changes in hepatic function, chemistry, or liver parameters from baseline to the end of the study. CONCLUSIONS: In this rollover study, CVC 150 mg once daily was well tolerated in patients with NASH and stage 0-4 liver fibrosis. No new safety signals were reported, and these data further support the safety and tolerability of CVC.

Metabolic dysfunction-associated steatotic liver disease in older adults is associated with frailty and social disadvantage.

BACKGROUND & AIMS: The burden of metabolic dysfunction-associated steatotic liver disease (MASLD) is growing rapidly, as is the number of older adults globally. However, relatively few studies have been performed evaluating the prevalence and risk factors for MASLD in older adults. As such, we aimed to identify the prevalence of MASLD in older adults, as well as sociodemographic, clinical, functional and biochemical associations. METHODS: The study population included older adults without a history of cardiovascular disease, dementia or independence-limiting functional impairment who had participated in the ASPirin in Reducing Events in the Elderly (ASPREE) randomised trial. MASLD was defined using the Fatty Liver Index (FLI). Associations were identified using Poisson regression with robust variance for FLI ≥ 60 vs FLI < 30. RESULTS: 9097 Australian participants aged ≥70 years had complete biochemical and anthropometric data to identify MASLD. The study population had a mean age of 75.1 ± 4.3 years and was 45.0% male. Almost one-third (33.0%) had prevalent MASLD, and the prevalence decreased with increasing age (adjusted RR [aRR] 0.96, 95% CI: 0.96-0.97). MASLD was also negatively associated with social advantage (aRR 0.94, 95% CI: 0.90-0.99) and exercise tolerance and was positively associated with diabetes mellitus (aRR: 1.22, 95% CI: 1.16-1.29), hypertension (aRR: 1.31, 95% CI: 1.22-1.41), male sex (aRR: 1.66, 95% CI: 1.57-1.74), pre-frailty (aRR: 1.99, 95% CI: 1.82-2.12) and frailty (aRR: 2.36, 95% CI: 2.16-2.56). MASLD and nonalcoholic fatty liver disease (NAFLD) results were 100% concordant. CONCLUSION: This study in a large cohort of relatively healthy community-dwelling older adults shows that MASLD is common, decreases with age and is associated with poorer metabolic health, social disadvantage and frailty.

Time-Restricted Fasting Improves Liver Steatosis in Non-Alcoholic Fatty Liver Disease-A Single Blinded Crossover Trial.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is associated with visceral adiposity. We assessed the effectiveness of time-restricted fasting (TRF) for 16 h daily without calorie restrictions compared to standard care (SC; diet and lifestyle advice) in improving visceral adiposity and steatosis via controlled attenuation parameter (CAP). METHODS: In a prospective single-blind randomized controlled trial, 32 participants with NAFLD were randomly assigned to TRF or SC for 12 weeks. The secondary endpoints were changes in liver stiffness, anthropometry, blood pressure, and other metabolic factors. RESULTS: Twenty-eight participants completed the first arm of the study (TRF = 14, SC = 14), with 23 completing the crossover arm (TRF = 10, SC = 13). The baseline demographics were similar between the groups. Intermittent fasting caused a significant decrease in hepatic steatosis (p = 0.038), weight (p = 0.005), waist circumference (p = 0.001), and BMI (p = 0.005) compared to standard care. Intermittent fasting also resulted in additional within-group changes that were not seen in the standard care intervention. CONCLUSION: TRF offers superior improvements in patients with NAFLD, improving steatosis, weight, and waist circumference despite a lack of change in overall caloric intake. Time-restricted fasting should be considered as a primary weight loss intervention in the context of NAFLD. TRIAL REGISTRATION: ACTRN12613000935730.

Carnosine Did Not Affect Vascular and Metabolic Outcomes in Patients with Prediabetes and Type 2 Diabetes: A 14-Week Randomized Controlled Trial.

Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of morbidity and mortality in patients with prediabetes and type 2 diabetes mellitus (T2DM). Carnosine has been suggested as a potential approach to reduce ASCVD risk factors. However, there is a paucity of human data. Hence, we performed a 14-week double-blind randomized placebo-controlled trial to determine whether carnosine compared with placebo improves vascular and metabolic outcomes in individuals with prediabetes and T2DM. In total, 49 patients with prediabetes and T2DM with good glycemic control were randomly assigned either to receive 2 g/day carnosine or matching placebo. We evaluated endothelial dysfunction, arterial stiffness, lipid parameters, blood pressure, heart rate, hepatic and renal outcomes before and after the intervention. Carnosine supplementation had no effect on heart rate, peripheral and central blood pressure, endothelial function (logarithm of reactive hyperemia (LnRHI)), arterial stiffness (carotid femoral pulse wave velocity (CF PWV)), lipid parameters, liver fibroscan indicators, liver transient elastography, liver function tests, and renal outcomes compared to placebo. In conclusion, carnosine supplementation did not improve cardiovascular and cardiometabolic risk factors in adults with prediabetes and T2DM with good glycemic control. Therefore, it is improbable that carnosine supplementation would be a viable approach to mitigating the ASCVD risk in these populations. The trial was registered at clinicaltrials.gov (NCT02917928).

A Randomized, Controlled Trial of the Pan-PPAR Agonist Lanifibranor in NASH.

BACKGROUND: Management of nonalcoholic steatohepatitis (NASH) is an unmet clinical need. Lanifibranor is a pan-PPAR (peroxisome proliferator-activated receptor) agonist that modulates key metabolic, inflammatory, and fibrogenic pathways in the pathogenesis of NASH. METHODS: In this phase 2b, double-blind, randomized, placebo-controlled trial, patients with noncirrhotic, highly active NASH were randomly assigned in a 1:1:1 ratio to receive 1200 mg or 800 mg of lanifibranor or placebo once daily for 24 weeks. The primary end point was a decrease of at least 2 points in the SAF-A score (the activity part of the Steatosis, Activity, Fibrosis [SAF] scoring system that incorporates scores for ballooning and inflammation) without worsening of fibrosis; SAF-A scores range from 0 to 4, with higher scores indicating more-severe disease activity. Secondary end points included resolution of NASH and regression of fibrosis. RESULTS: A total of 247 patients underwent randomization, of whom 103 (42%) had type 2 diabetes mellitus and 188 (76%) had significant (moderate) or advanced fibrosis. The percentage of patients who had a decrease of at least 2 points in the SAF-A score without worsening of fibrosis was significantly higher among those who received the 1200-mg dose, but not among those who received the 800-mg dose, of lanifibranor than among those who received placebo (1200-mg dose vs. placebo, 55% vs. 33%, P = 0.007; 800-mg dose vs. placebo, 48% vs. 33%, P = 0.07). The results favored both the 1200-mg and 800-mg doses of lanifibranor over placebo for resolution of NASH without worsening of fibrosis (49% and 39%, respectively, vs. 22%), improvement in fibrosis stage of at least 1 without worsening of NASH (48% and 34%, respectively, vs. 29%), and resolution of NASH plus improvement in fibrosis stage of at least 1 (35% and 25%, respectively, vs. 9%). Liver enzyme levels decreased and the levels of the majority of lipid, inflammatory, and fibrosis biomarkers improved in the lanifibranor groups. The dropout rate for adverse events was less than 5% and was similar across the trial groups. Diarrhea, nausea, peripheral edema, anemia, and weight gain occurred more frequently with lanifibranor than with placebo. CONCLUSIONS: In this phase 2b trial involving patients with active NASH, the percentage of patients who had a decrease of at least 2 points in the SAF-A score without worsening of fibrosis was significantly higher with the 1200-mg dose of lanifibranor than with placebo. These findings support further assessment of lanifibranor in phase 3 trials. (Funded by Inventiva Pharma; NATIVE ClinicalTrials.gov number, NCT03008070.).

Human Amnion Epithelial Cells Produce Soluble Factors that Enhance Liver Repair by Reducing Fibrosis While Maintaining Regeneration in a Model of Chronic Liver Injury.

Human amnion epithelial cells (hAECs) exert potent antifibrotic and anti-inflammatory effects when transplanted into preclinical models of tissue fibrosis. These effects are mediated in part via the secretion of soluble factors by hAECs which modulate signaling pathways and affect cell types involved in inflammation and fibrosis. Based on these reports, we hypothesized that these soluble factors may also support liver regeneration during chronic liver injury. To test this, we characterized the effect of both hAECs and hAEC-conditioned medium (CM) on liver repair in a mouse model of carbon tetrachloride (CCl4)-induced fibrosis. Liver repair was assessed by liver fibrosis, hepatocyte proliferation, and the liver progenitor cell (LPC) response. We found that the administration of hAECs or hAEC-CM reduced liver injury and fibrosis, sustained hepatocyte proliferation, and reduced LPC numbers during chronic liver injury. Additionally, we undertook in vitro studies to document both the cell-cell and paracrine-mediated effects of hAECs on LPCs by investigating the effects of co-culturing the LPCs and hAECs and hAEC-CM on LPCs. We found little change in LPCs co-cultured with hAECs. In contrast, hAEC-CM enhances LPC proliferation and differentiation. These findings suggest that paracrine factors secreted by hAECs enhance liver repair by reducing fibrosis while promoting regeneration during chronic liver injury.

Emulsion-based encapsulation of pluripotent stem cells in hydrogel microspheres for cardiac differentiation.

Cardiovascular disease is the leading cause of death worldwide, and current treatments are ineffective or unavailable to majority of patients. Engineered cardiac tissue (ECT) is a promising treatment to restore function to the damaged myocardium; however, for these treatments to become a reality, tissue fabrication must be amenable to scalable production and be used in suspension culture. Here, we have developed a low-cost and scalable emulsion-based method for producing ECT microspheres from poly(ethylene glycol) (PEG)-fibrinogen encapsulated mouse embryonic stem cells (mESCs). Cell-laden microspheres were formed via water-in-oil emulsification; encapsulation occurred by suspending the cells in hydrogel precursor solution at cell densities from 5 to 60 million cells/ml, adding to mineral oil and vortexing. Microsphere diameters ranged from 30 to 570 µm; size variability was decreased by the addition of 2% poly(ethylene glycol) diacrylate. Initial cell encapsulation density impacted the ability for mESCs to grow and differentiate, with the greatest success occurring at higher cell densities. Microspheres differentiated into dense spheroidal ECTs with spontaneous contractions occurring as early as Day 10 of cardiac differentiation; furthermore, these ECT microspheres exhibited appropriate temporal changes in gene expression and response to pharmacological stimuli. These results demonstrate the ability to use an emulsion approach to encapsulate pluripotent stem cells for use in microsphere-based cardiac differentiation.

Human amnion epithelial cells and their soluble factors reduce liver fibrosis in murine non-alcoholic steatohepatitis.

BACKGROUND AND AIM: Non-alcoholic steatohepatitis (NASH) can lead to cirrhosis and hepatocellular carcinoma. Currently, lifestyle modification is the only effective treatment. We have shown that human amnion epithelial cells (hAECs) reduce inflammation and fibrosis in toxin-induced liver injury models. We examined the effect of these cells and the soluble factors released by the cells into culture medium (hAEC conditioned medium [hAEC-CM]) in a diet-induced murine NASH model. METHODS: C57BL/6J male mice received a Western "fast food diet" for 42 weeks. Group 1 received an intraperitoneal injection of 2 × 106 hAECs at week 34, group 2 received an additional hAEC dose at week 38, and group 3 received thrice weekly hAEC-CM injections intraperitoneal for 8 weeks from week 34. Liver fibrosis area, inflammation, and fibrosis regulators were measured by immunohistochemistry, qPCR, and gelatin zymography. Metabolic parameters were also assessed. RESULTS: Fast food diet-fed mice demonstrated peri-cellular hepatic fibrosis, inflammation, and steatosis typical of NASH. Liver fibrosis area was reduced by 40% in hAEC-treated and hAEC-CM-treated mice. hAEC treatment significantly reduced pSMAD 2/3 signaling and the number of activated hepatic stellate cells and liver macrophages. Matrix metalloproteinase 2 and 9 gene and protein expression were variably affected. hAEC treatment did not alter the NASH activity score or metabolic parameters such as bodyweight, total cholesterol, or glucose tolerance. CONCLUSION: Human amnion epithelial cell and hAEC-CM significantly reduced hepatic inflammation and fibrosis in a diet-induced non-alcoholic fatty liver disease model. Although hAEC and hAEC-CM did not affect the metabolic components of NASH, their therapeutic potential is promising and warrants further investigation.

The Human Amnion Epithelial Cell Secretome Decreases Hepatic Fibrosis in Mice with Chronic Liver Fibrosis.

Background: Hepatic stellate cells (HSCs) are the primary collagen-secreting cells in the liver. While HSCs are the major cell type involved in the pathogenesis of liver fibrosis, hepatic macrophages also play an important role in mediating fibrogenesis and fibrosis resolution. Previously, we observed a reduction in HSC activation, proliferation, and collagen synthesis following exposure to human amnion epithelial cells (hAEC) and hAEC-conditioned media (hAEC-CM). This suggested that specific factors secreted by hAEC might be effective in ameliorating liver fibrosis. hAEC-derived extracellular vesicles (hAEC-EVs), which are nanosized (40-100 nm) membrane bound vesicles, may act as novel cell-cell communicators. Accordingly, we evaluated the efficacy of hAEC-EV in modulating liver fibrosis in a mouse model of chronic liver fibrosis and in human HSC. Methods: The hAEC-EVs were isolated and characterized. C57BL/6 mice with CCl4-induced liver fibrosis were administered hAEC-EV, hAEC-CM, or hAEC-EV depleted medium (hAEC-EVDM). LX2 cells, a human HSC line, and bone marrow-derived mouse macrophages were exposed to hAEC-EV, hAEC-CM, and hAEC-EVDM. Mass spectrometry was used to examine the proteome profile of each preparation. Results: The extent of liver fibrosis and number of activated HSCs were reduced significantly in CCl4-treated mice given hAEC-EVs, hAEC-CM, and hAEC EVDM compared to untreated controls. Hepatic macrophages were significantly decreased in all treatment groups, where a predominant M2 phenotype was observed. Human HSCs cultured with hAEC-EV and hAEC-CM displayed a significant reduction in collagen synthesis and hAEC-EV, hAEC-CM, and hAEC-EVDM altered macrophage polarization in bone marrow-derived mouse macrophages. Proteome analysis showed that 164 proteins were unique to hAEC-EV in comparison to hAEC-CM and hAEC-EVDM, and 51 proteins were co-identified components with the hAEC-EV fraction. Conclusion: This study provides novel data showing that hAEC-derived EVs significantly reduced liver fibrosis and macrophage infiltration to an extent similar to hAEC-EVDM and hAEC-CM. hAEC-EV-based therapy may be a potential therapeutic option for liver fibrosis.

A Pilot Study Evaluating the Safety of Intravenously Administered Human Amnion Epithelial Cells for the Treatment of Hepatic Fibrosis.

Liver cirrhosis is the 6th leading cause of death in adults aged 15-59 years in high-income countries. For many who progress to cirrhosis, the only prospect for survival is liver transplantation. While there is some indication that mesenchymal stem cells may be useful in reversing established liver fibrosis, there are limitations to their widespread use - namely their rarity, the need for extensive serial passaging and the associated potential for genomic instability and cellular senescence. To this end, we propose the use of allogeneic amnion epithelial cells. This clinical trial will assess the safety of intravenously delivered allogeneic human amnion epithelial cells (hAECs) in patients with compensated liver cirrhosis. This will also provide clinical data that will inform phases 2 and 3 clinical trials with the ultimate goal of developing hAECs as a therapeutic option for patients with cirrhosis who are at significant risk of disease progression. We will recruit 12 patients with compensated cirrhosis, based on their hepatic venous pressure gradient, for a dose escalation study. Patients will be closely monitored in the first 24 h post-infusion, then via daily telephone interviews until clinical assessment on day 5. Long term follow up will include standard liver tests, transient elastography and hepatic ultrasound. Ethics approval was obtained from Monash Health for this trial 16052A, "A Pilot Study Evaluating the Safety of Intravenously Administered Human Amnion Epithelial Cells for the Treatment of Liver Fibrosis, A First in Adult Human Study." The trial will be conducted in accordance to Monash Health Human Ethics guidelines. Outcomes from this study will be disseminated in the form of conference presentations and submission to a peer reviewed journal. This trial has been registered on the Australian and New Zealand Clinical Trials Registry ACTRN12616000437460.

Coffee Intake Is Associated with a Lower Liver Stiffness in Patients with Non-Alcoholic Fatty Liver Disease, Hepatitis C, and Hepatitis B.

There is emerging evidence for the positive effects or benefits of coffee in patients with liver disease. We conducted a retrospective cross-sectional study on patients with non-alcoholic fatty liver disease (NAFLD), hepatitis C virus (HCV), and hepatitis B virus (HBV) infection to determine the effects of coffee intake on a non-invasive marker of liver fibrosis: liver stiffness assessed by transient elastography (TE). We assessed coffee and tea intake and measured TE in 1018 patients with NAFLD, HCV, and HBV (155 with NAFLD, 378 with HCV and 485 with HBV). Univariate and multivariate regression models were performed taking into account potential confounders. Liver stiffness was higher in males compared to females (p < 0.05). Patients with HBV had lower liver stiffness than those with HCV and NAFLD. After adjustment for age, gender, smoking, alcohol consumption, M or XL probe, and disease state (NAFLD, HCV, and HBV status), those who drank 2 or more cups of coffee per day had a lower liver stiffness (p = 0.044). Tea consumption had no effect (p = 0.9). Coffee consumption decreases liver stiffness, which may indicate less fibrosis and inflammation, independent of disease state. This study adds further evidence to the notion of coffee maybe beneficial in patients with liver disease.

Direct hydrogel encapsulation of pluripotent stem cells enables ontomimetic differentiation and growth of engineered human heart tissues.

Human engineered heart tissues have potential to revolutionize cardiac development research, drug-testing, and treatment of heart disease; however, implementation is limited by the need to use pre-differentiated cardiomyocytes (CMs). Here we show that by providing a 3D poly(ethylene glycol)-fibrinogen hydrogel microenvironment, we can directly differentiate human pluripotent stem cells (hPSCs) into contracting heart tissues. Our straight-forward, ontomimetic approach, imitating the process of development, requires only a single cell-handling step, provides reproducible results for a range of tested geometries and size scales, and overcomes inherent limitations in cell maintenance and maturation, while achieving high yields of CMs with developmentally appropriate temporal changes in gene expression. We demonstrate that hPSCs encapsulated within this biomimetic 3D hydrogel microenvironment develop into functional cardiac tissues composed of self-aligned CMs with evidence of ultrastructural maturation, mimicking heart development, and enabling investigation of disease mechanisms and screening of compounds on developing human heart tissue.

Enhanced stem cell-derived cardiomyocyte differentiation in suspension culture by delivery of nitric oxide using S-nitrosocysteine.

The development of cell-based treatments for heart disease relies on the creation of functionally mature stem cell-derived cardiomyocytes employing in vitro culture suspension systems, a process which remains a formidable and expensive endeavor. The use of nitric oxide as a signaling molecule during differentiation has demonstrated the potential for creating increased numbers of spontaneously contracting embryoid bodies in culture; however, the effects of nitric oxide signaling on the function and maturation of stem cell-derived cardiomyocytes is not well understood. In this study, the effects of nitric oxide on mouse embryonic stem cell-derived cardiomyocyte contractile activity, protein, and gene expression, and calcium handling were quantified. Embryoid bodies (EBs) formed using the hanging drop method, were treated with the soluble nitric oxide donor S-nitrosocysteine (CysNO) over a period of 18 days in suspension culture and spontaneous contractile activity was assessed. On day 8, selected EBs were dissociated to form monolayers for electrophysiological characterization using calcium transient mapping. Nitric oxide treatment led to increased numbers of stem cell-derived cardiomyocytes (SC-CMs) relative to non-treated EBs after 8 days in suspension culture. Increased incidence of spontaneous contraction and frequency of contraction were observed from days 8-14 in EBs receiving nitric oxide treatment in comparison to control. Expression of cardiac markers and functional proteins was visualized using immunocytochemistry and gene expression was assessed using qPCR. Cardiac-specific proteins were present in both CysNO-treated and control SC-CMs; however, CysNO treatment during differentiation significantly increased ßMHC gene expression in SC-CMs relative to control SC-CMs. Furthermore, increased calcium transient velocity and decreased calcium transient duration was observed for CysNO-treated SC-CMs in comparison to control SC-CMs. Soluble nitric oxide donors, including S-nitrosocysteine, have advantages over other bioactive molecules for use in scalable culture systems in driving cardiac differentiation, since they are inexpensive and the diffusivity of nitric oxide is relatively high. By enabling maintenance of spontaneous contraction in suspension culture and progressing electrophysiological function of resulting SC-CMs toward a more mature phenotype, long-term application of S-nitrosocysteine was shown to be beneficial during cardiac differentiation. Taken together, these results demonstrate the efficiency of nitric oxide as a signaling compound, with implications in the improvement of pluripotent stem cell-derived cardiomyocyte maturation in large-scale culture systems.

Conductive interpenetrating networks of polypyrrole and polycaprolactone encourage electrophysiological development of cardiac cells.

Conductive and electroactive polymers have the potential to enhance engineered cardiac tissue function. In this study, an interpenetrating network of the electrically-conductive polymer polypyrrole (PPy) was grown within a matrix of flexible polycaprolactone (PCL) and evaluated as a platform for directing the formation of functional cardiac cell sheets. PCL films were either treated with sodium hydroxide to render them more hydrophilic and enhance cell adhesion or rendered electroactive with PPy grown via chemical polymerization yielding PPy-PCL that had a resistivity of 1.0 ± 0.4 kΩ cm, which is similar to native cardiac tissue. Both PCL and PPy-PCL films supported cardiomyocyte attachment; increasing the duration of PCL pre-treatment with NaOH resulted in higher numbers of adherent cardiomyocytes per unit area, generating cell densities which were more similar to those on PPy-PCL films (1568 ± 126 cells mm(-2), 2880 ± 439 cells mm(-2), 3623 ± 456 cells mm(-2) for PCL with 0, 24, 48 h of NaOH pretreatment, respectively; 2434 ± 166 cells mm(-2) for PPy-PCL). When cardiomyocytes were cultured on the electrically-conductive PPy-PCL, more cells were observed to have peripheral localization of the gap junction protein connexin-43 (Cx43) as compared to cells on NaOH-treated PCL (60.3 ± 4.3% vs. 46.6 ± 5.7%). Cx43 gene expression remained unchanged between materials. Importantly, the velocity of calcium wave propagation was faster and calcium transient duration was shorter for cardiomyocyte monolayers on PPy-PCL (1612 ± 143 µm/s, 910 ± 63 ms) relative to cells on PCL (1129 ± 247 µm/s, 1130 ± 20 ms). In summary, PPy-PCL has demonstrated suitability as an electrically-conductive substrate for culture of cardiomyocytes, yielding enhanced functional properties; results encourage further development of conductive substrates for use in differentiation of stem cell-derived cardiomyocytes and cardiac tissue engineering applications. STATEMENT OF SIGNIFICANCE: Current conductive materials for use in cardiac regeneration are limited by cytotoxicity or cost in implementation. In this manuscript, we demonstrate for the first time the application of a biocompatible, conductive polypyrrole-polycaprolactone film as a platform for culturing cardiomyocytes for cardiac regeneration. This study shows that the novel conductive film is capable of enhancing cell-cell communication through the formation of connexin-43, leading to higher velocities for calcium wave propagation and reduced calcium transient durations among cultured cardiomyocyte monolayers. Furthermore, it was demonstrated that chemical modification of polycaprolactone through alkaline-mediated hydrolysis increased overall cardiomyocyte adhesion. The results of this study provide insight into how cardiomyocytes interact with conductive substrates and will inform future research efforts to enhance the functional properties of cardiomyocytes, which is critical for their use in pharmaceutical testing and cell therapy.

Soluble factors derived from human amniotic epithelial cells suppress collagen production in human hepatic stellate cells.

BACKGROUND: Intravenous infusion of human amniotic epithelial cells (hAECs) has been shown to ameliorate hepatic fibrosis in murine models. Hepatic stellate cells (HSCs) are the principal collagen-secreting cells in the liver. The aim of this study was to investigate whether factors secreted by hAECs and present in hAEC-conditioned medium (CM) have anti-fibrotic effects on activated human HSCs. METHODS: Human AECs were isolated from the placenta and cultured. Human hepatic stellate cells were exposed to hAEC CM to determine potential anti-fibrotic effects. RESULTS: HSCs treated for 48 h with hAEC CM displayed a significant reduction in the expression of the myofibroblast markers α-smooth muscle actin and platelet-derived growth factor. Expression of the pro-fibrotic cytokine transforming growth factor-ß1 (TGF-ß1) and intracellular collagen were reduced by 45% and 46%, respectively. Human AEC CM induced HSC apoptosis in 11.8% of treated cells and reduced HSC proliferation. Soluble human leukocyte antigen-G1, a hAEC-derived factor, significantly decreased TGF-ß1 and collagen production in activated HSCs, although the effect on collagen production was less than that of hAEC CM. The reduction in collagen and TGF-B1 could not be attributed to PGE2, relaxin, IL-10, TGF-B3, FasL or TRAIL. CONCLUSIONS: Human AEC CM treatment suppresses markers of activation, proliferation and fibrosis in human HSCs as well as inducing apoptosis and reducing proliferation. Human AEC CM treatment may be effective in ameliorating liver fibrosis and warrants further study.

Biomimetic materials design for cardiac tissue regeneration.

Cardiovascular disease is the leading cause of death worldwide. In the absence of sufficient numbers of organs for heart transplant, alternate approaches for healing or replacing diseased heart tissue are under investigation. Designing biomimetic materials to support these approaches will be essential to their overall success. Strategies for cardiac tissue engineering include injection of cells, implantation of three-dimensional tissue constructs or patches, injection of acellular materials, and replacement of valves. To replicate physiological function and facilitate engraftment into native tissue, materials used in these approaches should have properties that mimic those of the natural cardiac environment. Multiple aspects of the cardiac microenvironment have been emulated using biomimetic materials including delivery of bioactive factors, presentation of cell-specific adhesion sites, design of surface topography to guide tissue alignment and dictate cell shape, modulation of mechanical stiffness and electrical conductivity, and fabrication of three-dimensional structures to guide tissue formation and function. Biomaterials can be engineered to assist in stem cell expansion and differentiation, to protect cells during injection and facilitate their retention and survival in vivo, and to provide mechanical support and guidance for engineered tissue formation. Numerous studies have investigated the use of biomimetic materials for cardiac regeneration. Biomimetic material design will continue to exploit advances in nanotechnology to better recreate the cellular environment and advance cardiac regeneration. Overall, biomimetic materials are moving the field of cardiac regenerative medicine forward and promise to deliver new therapies in combating heart disease.

Human amniotic epithelial cell transplantation induces markers of alternative macrophage activation and reduces established hepatic fibrosis.

Chronic hepatic inflammation from multiple etiologies leads to a fibrogenic response that can progress to cirrhosis and liver failure. Transplantation of human amniotic epithelial cells (hAEC) from term delivered placenta has been shown to decrease mild to moderate hepatic fibrosis in a murine model. To model advanced human liver disease and assess the efficacy of hAEC therapy, we transplanted hAEC in mice with advanced hepatic fibrosis. Immunocompetent C57BL/6 mice were administered carbon tetrachloride (CCl(4)) twice weekly resulting in bridging fibrosis by 12 weeks. hAEC (2 × 10(6)) were infused via the tail vein at week 8 or weeks 8 and 10 (single and double dose, respectively). Human cells were detected in mouse liver four weeks after transplantation showing hAEC engraftment. CCl(4) treated mice receiving single or double hAEC doses showed a significant but similar decrease in liver fibrosis area associated with decreased activation of collagen-producing hepatic stellate cells and decreased hepatic protein levels of the pro-fibrogenic cytokine, transforming growth factor-beta1. CCl(4) administration caused hepatic T cell infiltration that decreased significantly following hAEC transplantation. Hepatic macrophages play a crucial role in both fibrogenesis and fibrosis resolution. Mice exposed to CCl(4) demonstrated increased numbers of hepatic macrophages compared to normal mice; the number of macrophages decreased significantly in CCl(4) treated mice given hAEC. These mice had significantly lower hepatic protein levels of the chemokine monocyte chemoattractant protein-1 than mice given CCl(4) alone. Alternatively activated M2 macrophages are associated with fibrosis resolution. CCl(4) treated mice given hAEC showed increased expression of genes associated with M2 macrophages including YM-1, IL-10 and CD206. We provide novel data showing that hAEC transplantation induces a wound healing M2 macrophage phenotype associated with reduction of established hepatic fibrosis that justifies further investigation of this potential cell-based therapy for advanced hepatic fibrosis.