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).

Corrigendum: Phase I trial outcome of amnion cell therapy in patients with ischemic stroke (I-ACT).

[This corrects the article DOI: 10.3389/fnins.2023.1153231.].

Phase I trial outcome of amnion cell therapy in patients with ischemic stroke (I-ACT).

Background: We proposed a Phase I dose escalation trial to assess the safety of allogeneic human amniotic epithelial cells (hAECs) in stroke patients with a view to informing the design for a Phase II trial. Methods: The design is based on 3 + 3 dose escalation design with additional components for measuring MR signal of efficacy as well as the effect of hAECs (2-8 × 106/kg, i.v.) on preventing immunosuppression after stroke. Results: Eight patients (six males) were recruited within 24 h of ischemic stroke onset and were infused with hAECs. We were able to increase the dose of hAECs to 8 × 106 cells/kg (2 × 106/kg, n = 3; 4 × 106/kg, n = 3; 8 × 106/kg, n = 2). The mean age is 68.0 ± 10.9 (mean ± SD). The frequencies of hypertension and hyperlipidemia were 87.5%, diabetes was 37.5%, atrial fibrillation was 50%, ischemic heart disease was 37.5% and ever-smoker was 25%. Overall, baseline NIHSS was 7.5 ± 3.1, 7.8 ± 7.2 at 24 h, and 4.9 ± 5.4 at 1 week (n = 8). The modified Rankin scale at 90 days was 2.1 ± 1.2. Supplemental oxygen was given in five patients during hAEC infusion. Using pre-defined criteria, two serious adverse events occurred. One patient developed recurrent stroke and another developed pulmonary embolism whilst in rehabilitation. For the last four patients, infusion of hAECs was split across separate infusions on subsequent days to reduce the risk for fluid overload. Conclusion: Our Phase I trial demonstrates that a maximal dose of 2 × 106/kg hAECs given intravenously each day over 2 days (a total of 4 × 106/kg) is safe and optimal for use in a Phase II trial. Clinical trial registration: ClinicalTrials.gov, identifier ACTRN12618000076279P.

Assessing the impact of gestational age of donors on the efficacy of amniotic epithelial cell-derived extracellular vesicles in experimental bronchopulmonary dysplasia.

BACKGROUND AND RATIONALE: Extracellular vesicles (EVs) are a potential cell-free regenerative medicine. Human amniotic epithelial cells (hAECs) are a viable source of cell therapy for diseases like bronchopulmonary dysplasia (BPD). However, little is known about the impact of gestational age of the donor on the quality of hAEC-derived EVs. AIMS: To determine the impact of gestational age on hAEC-derived EVs in experimental BPD. RESULTS: Term hAEC-derived EVs displayed a significantly higher density of surface epitopes (CD142 and CD133) and induced greater macrophage phagocytosis compared to preterm hAEC-EVs. However, T cell proliferation was more significantly suppressed by preterm hAEC-EVs. Using a model of experimental BPD, we observed that term but not preterm hAEC-EVs improved tissue-to-airspace ratio and septal crest density. While both term and preterm hAEC-EVs reduced the levels of inflammatory cytokines on postnatal day 7, the improvement in lung injury was associated with increased type II alveolar cells which was only observed in term hAEC-EV treatment group. Furthermore, only neonatal term hAEC-EVs reduced airway hyper-responsiveness, mitigated pulmonary hypertension and protected against right ventricular hypertrophy at 6 weeks of age. CONCLUSION: Term hAEC-EVs, but not preterm hAEC-EVs, have therapeutic efficacy in a mouse model of BPD-like lung injury. Therefore, the impact of donor criteria should be considered when applying perinatal cells-derived EV therapy for clinical use.

Sulforaphane improves endothelial function and reduces placental oxidative stress in vitro.

INTRODUCTION: The maternal endothelial dysfunction characteristic of preeclampsia arises, in part, from excessive placental production of anti-angiogenic factors, including soluble Flt-1, soluble endoglin and activin A, inducing oxidative stress. We assessed whether the antioxidant and NRF2-activator sulforaphane could mitigate endothelial and trophoblast dysfunction in vitro. METHODS: We induced dysfunction in human umbilical vein endothelial cells (HUVECs) with TNF-α, assessing endothelial activation and dysfunction (endothelin-1, vascular cell adhesion molecule; VCAM1, intracellular adhesion molecule; ICAM1, e-selectin and endothelial permeability) in the presence or absence of sulforaphane. We also assessed the effects of sulforaphane in mitigating hypoxic and hyperoxic injury in term placental explants by measuring secretion of anti-angiogenic factors. To assess the role of NRF2 we silenced NRF2 in HUVECs and primary trophoblast cells. RESULTS: Sulforaphane reduced TNF-α mediated HUVEC secretion of endothelin-1, VCAM1, ICAM1 and E-selectin, and prevented increased endothelial permeability. In placental explants, sulforaphane reduced the secretion of soluble Flt-1, soluble endoglin and activin A. Sulforaphane induced activation and nuclear translocation of NRF2 in HUVECs, inducing heme oxygenase 1. NRF2 silencing blocked some but not all of sulforaphane's effects in HUVECs. NRF2 silencing did not prevent sulforaphane's inhibition of trophobast secretion of soluble Flt-1 or activin A. CONCLUSION: In reducing placental and endothelial oxidative stress, sulforaphane may offer a new adjuvant therapeutic approach for the treatment of preeclampsia.

Amnion Epithelial Cell-Derived Exosomes Restrict Lung Injury and Enhance Endogenous Lung Repair.

Idiopathic pulmonary fibrosis (IPF) is characterized by chronic inflammation, severe scarring, and stem cell senescence. Stem cell-based therapies modulate inflammatory and fibrogenic pathways by release of soluble factors. Stem cell-derived extracellular vesicles should be explored as a potential therapy for IPF. Human amnion epithelial cell-derived exosomes (hAEC Exo) were isolated and compared against human lung fibroblasts exosomes. hAEC Exo were assessed as a potential therapy for lung fibrosis. Exosomes were isolated and evaluated for their protein and miRNA cargo. Direct effects of hAEC Exo on immune cell function, including macrophage polarization, phagocytosis, neutrophil myeloperoxidase activity and T cell proliferation and uptake, were measured. Their impact on immune response, histological outcomes, and bronchioalveolar stem cell (BASC) response was assessed in vivo following bleomycin challenge in young and aged mice. hAEC Exo carry protein cargo enriched for MAPK signaling pathways, apoptotic and developmental biology pathways and miRNA enriched for PI3K-Akt, Ras, Hippo, TGFß, and focal adhesion pathways. hAEC Exo polarized and increased macrophage phagocytosis, reduced neutrophil myeloperoxidases, and suppressed T cell proliferation directly. Intranasal instillation of 10 µg hAEC Exo 1 day following bleomycin challenge reduced lung inflammation, while treatment at day 7 improved tissue-to-airspace ratio and reduced fibrosis. Administration of hAEC Exo coincided with the proliferation of BASC. These effects were reproducible in bleomycin-challenged aged mice. The paracrine effects of hAECs can be largely attributed to their exosomes and exploitation of hAEC Exo as a therapy for IPF should be explored further. Stem Cells Translational Medicine 2018;7:180-196.

Resveratrol mitigates trophoblast and endothelial dysfunction partly via activation of nuclear factor erythroid 2-related factor-2.

INTRODUCTION: Maternal endothelial dysfunction underlying preeclampsia arises from excessive placental release of anti-angiogenic factors, such as soluble fms-like tyrosine kinase-1 (sFlt1), soluble endoglin (sEng) and activin A. Resveratrol, an activator of the nuclear factor erythroid 2-related factor-2 (Nrf2) transcription factor, mediates the gene expression of antioxidant and vasoprotective factors that may counter the endothelial damage imposed by these anti-angiogenic factors. The objective of this study was to assess whether resveratrol could reduce placental oxidative stress and production of anti-angiogenic factors in vitro and/or improve in vitro markers of endothelial dysfunction via Nrf2 activation. METHOD: We used in vitro term placental explants to assess the effects of resveratrol on placental oxidative stress and production of sFlt1, sEng and activin A. Using human umbilical vein endothelial cells we investigated the effects of resveratrol on markers of in vitro endothelial dysfunction, including the expression of intercellular adhesion molecule 1 (ICAM1), vascular cell adhesion molecule 1 (VCAM1), E-selectin and endothelin-1, and endothelial permeability. To confirm that resveratrol mediated its effects via Nrf2, we examined the impact of resveratrol on the same in vitro markers of endothelial and placental dysfunction following Nrf2 knockdown. RESULTS: Resveratrol significantly decreased placental oxidative stress and the production of sFlt1 and activin A. Resveratrol significantly mitigated tumor necrosis factor-α stimulated endothelial expression of ICAM1, VCAM1, E-selectin and endothelin-1 and prevented an increase in endothelial monolayer permeability. Nrf2 knockdown abolished some of the protective effects of resveratrol on endothelial cells, but not in primary trophoblast cells. CONCLUSION: Features of placental and endothelial dysfunction characteristic of preeclampsia are improved by resveratrol in vitro, partially via the modulation of Nrf2.

Human amnion cells reverse acute and chronic pulmonary damage in experimental neonatal lung injury.

BACKGROUND: Despite advances in neonatal care, bronchopulmonary dysplasia (BPD) remains a significant contributor to infant mortality and morbidity. While human amnion epithelial cells (hAECs) have shown promise in small and large animal models of BPD, there is scarce information on long-term benefit and clinically relevant questions surrounding administration strategy remain unanswered. In assessing the therapeutic potential of hAECs, we investigated the impact of cell dosage, administration routes and timing of treatment in a pre-clinical model of BPD. METHODS: Lipopolysaccharide was introduced intra-amniotically at day 16 of pregnancy prior to exposure to 65% oxygen (hyperoxia) at birth. hAECs were administered either 12 hours (early) or 4 days (late) after hyperoxia commenced. Collective lung tissues were subjected to histological analysis, multikine ELISA for inflammatory cytokines, FACS for immune cell populations and 3D lung stem cell culture at neonatal stage (postnatal day 7 and 14). Invasive lung function test and echocardiography were applied at 6 and 10 weeks of age. RESULTS: hAECs improved the tissue-to-airspace ratio and septal crest density in a dose-dependent manner, regardless of administration route. Early administration of hAECs, coinciding with the commencement of postnatal hyperoxia, was associated with reduced macrophages, dendritic cells and natural killer cells. This was not the case if hAECs were administered when lung injury was established. Fittingly, early hAEC treatment was more efficacious in reducing interleukin-1ß, tumour necrosis factor alpha and monocyte chemoattractant protein-1 levels. Early hAEC treatment was also associated with reduced airway hyper-responsiveness and normalisation of pressure-volume loops. Pulmonary hypertension and right ventricle hypertrophy were also prevented in the early hAEC treatment group, and this persisted until 10 weeks of age. CONCLUSIONS: Early hAEC treatment appears to be advantageous over late treatment. There was no difference in efficacy between intravenous and intratracheal administration. The benefits of hAEC administration resulted in long-term improvements in cardiorespiratory function.

Amnion Epithelial Cells Promote Lung Repair via Lipoxin A4.

Human amnion epithelial cells (hAECs) have been shown to possess potent immunomodulatory properties across a number of disease models. Recently, we reported that hAECs influence macrophage polarization and activity, and that this step was dependent on regulatory T cells. In this study, we aimed to assess the effects of hAEC-derived proresolution lipoxin-A4 (LXA4) on T-cell, macrophage, and neutrophil phenotype and function during the acute phase of bleomycin-induced lung injury. Using C57Bl6 mice, we administered 4 million hAECs intraperitoneally 24 hours after bleomycin challenge. Outcomes were measured at days 3, 5, and 7. hAEC administration resulted in significant changes to T-cell, macrophage, dendritic cell, and monocyte/macrophage infiltration and phenotypes. Endogenous levels of lipoxygenases, LXA4, and the lipoxin receptor FPR2 were elevated in hAEC-treated animals. Furthermore, we showed that the effects of hAECs on macrophage phagocytic activity and T-cell suppression are LXA4 dependent, whereas the inhibition of neutrophil-derived myleoperoxidase by hAECs is independent of LXA4. This study provides the first evidence that lipid-based mediators contribute to the immunomodulatory effects of hAECs and further supports the growing body of evidence that LXA4 is proresolutionary in lung injury. This discovery of LXA4-dependent communication between hAECs, macrophages, T cells, and neutrophils is important to the understanding of hAEC biodynamics and would be expected to inform future clinical applications. Stem Cells Translational Medicine 2017;6:1085-1095.

Amnion cell-mediated immune modulation following bleomycin challenge: controlling the regulatory T cell response.

INTRODUCTION: The immunomodulatory properties of human amnion epithelial cells (hAECs) have been previously described in several disease models. We previously reported on the ability of hAECs to influence macrophage phenotype and chemotaxis. In this study, we aim to elucidate the contribution of regulatory T cells (Tregs) to macrophage polarisation and downstream effects on inflammation and fibrosis in a bleomycin model of lung injury. METHODS: Either CD45(+)/FoxP3(+) Tregs or CD45(+)/FoxP3 (-) non-Tregs were adoptively transferred into Rag1 (-/-) mice immediately prior to bleomycin challenge. Four million hAECs were administered 24 hours later. Outcomes were measured 7 or 14 days later. RESULTS: Mitigation of lung inflammation and fibrosis was observed only in animals that received both hAECs and Tregs. hAEC treatment also induced the maturation of non-Tregs into FoxP3-expressing Tregs. This event was found to be transforming growth factor-beta (TGFß)-dependent. Furthermore, polarisation of macrophages from M1 to M2 occurred only in animals that received hAECs and Tregs. CONCLUSIONS: This study provides the first evidence that Tregs are required for hAEC-mediated macrophage polarisation and consequential mitigation of bleomycin-induced lung injury. Uncovering the interactions between hAECs, macrophages, and T-cell subsets is central to understanding the mechanisms by which hAECs elicit lung repair.

Transplantation of retrovirally transduced bone marrow prevents autoimmune disease in aged mice by peripheral tolerance mechanisms.

Transplantation of bone marrow (BM) engineered to express self-antigen has been shown to protect 100% of young mice from myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), with thymic clonal deletion as a tolerance mechanism. Here, we asked whether aged mice can also be tolerised following transplantation with self-antigen-engineered BM and whether castration-induced thymus regrowth can enhance this outcomes. Then, 50% of aged mice were protected from EAE regardless of castration-induced thymus regrowth. EAE-free and diseased mice demonstrated MOG-specific lymphocyte proliferation and antibody production regardless of castration-induced thymus regrowth, consistent with lack of intrathymic deletion of self-antigen-reactive T cells. Although low chimerism levels ( < 4%) were observed, EAE-free mice showed significantly higher chimerism levels in lymphocytes in peripheral lymphoid organs compared with thymus. CD4(+)CD25(+) regulatory T cells were elevated in lymph nodes of EAE-free mice. We conclude that transplantation of self-antigen expressing BM protects 50% of aged mice and castration-induced thymic regrowth had no effect on outcomes. Peripheral tolerance mechanisms are implicated since protection is associated with higher chimerism levels in peripheral T and B lymphocytes and with elevated regulatory T cells.