Human amnion epithelial cell therapy reduces hypertension-induced vascular stiffening and cognitive impairment.

Vascular inflammation and fibrosis are hallmarks of hypertension and contribute to the development of cardiovascular disease and cognitive impairment. However, current anti-hypertensive drugs do not treat the underlying tissue damage, such as inflammation-associated fibrosis. Human amnion epithelial cells have several properties amenable for treating vascular pathology. This study tested the effect of amnion epithelial cells on vascular pathology and cognitive impairment during hypertension. Male C57Bl6 mice (8-12 weeks) were administered vehicle (saline; n = 58) or angiotensin II (0.7 mg/kg/d, n = 56) subcutaneously for 14 d. After surgery, a subset of mice were injected with 106 amnion epithelial cells intravenously. Angiotensin II infusion increased systolic blood pressure, aortic pulse wave velocity, accumulation of aortic leukocytes, and aortic mRNA expression of collagen subtypes compared to vehicle-infused mice (n = 9-11, P < 0.05). Administration of amnion epithelial cells attenuated these effects of angiotensin II (P < 0.05). Angiotensin II-induced cognitive impairment was prevented by amnion epithelial cell therapy (n = 7-9, P < 0.05). In the brain, amnion epithelial cells modulated some of the inflammatory genes that angiotensin II promoted differential expression of (n = 6, p-adjusted < 0.05). These findings suggest that amnion epithelial cells could be explored as a potential therapy to inhibit vascular pathology and cognitive impairment during hypertension.

Protection against brain injury after ischemic stroke by intravenous human amnion epithelial cells in combination with tissue plasminogen activator.

Background: Thrombolytic agents such as tissue plasminogen activator (tPA) are the only drug class approved to treat ischemic stroke and are usually administered within 4.5 h. However, only ~20% of ischemic stroke patients are eligible to receive the therapy. We previously demonstrated that early intravenous administration of human amnion epithelial cells (hAECs) can limit brain inflammation and infarct growth in experimental stroke. Here, we have tested whether hAECs exert cerebroprotective effects in combination with tPA in mice. Methods: Male C57Bl/6 mice were subjected to middle cerebral artery occlusion for 60 min followed by reperfusion. Immediately following reperfusion, vehicle (saline, n = 31) or tPA (10 mg/kg; n = 73) was administered intravenously. After 30 min of reperfusion, tPA-treated mice were injected intravenously with either hAECs (1×106; n = 32) or vehicle (2% human serum albumin; n = 41). A further 15 sham-operated mice were treated with vehicle (n = 7) or tPA + vehicle (n = 8). Mice were designated to be euthanised at 3, 6 or 24 h post-stroke (n = 21, 31, and 52, respectively), and brains were collected to assess infarct volume, blood-brain barrier (BBB) disruption, intracerebral bleeding and inflammatory cell content. Results: There was no mortality within 6 h of stroke onset, but a high mortality occurred in tPA + saline-treated mice between 6 h and 24 h post-stroke in comparison to mice treated with tPA + hAECs (61% vs. 27%, p = 0.04). No mortality occurred within 24 h of sham surgery in mice treated with tPA + vehicle. We focused on early infarct expansion within 6 h of stroke and found that infarction was ~50% larger in tPA + saline- than in vehicle-treated mice (23 ± 3 mm3 vs. 15 ± 2 mm3, p = 0.02) but not in mice receiving tPA + hAECs (13 ± 2 mm3, p < 0.01 vs. tPA + saline) in which intracerebral hAECs were detected. Similar to the profiles of infarct expansion, BBB disruption and intracerebral bleeding in tPA + saline-treated mice at 6 h was 50-60% greater than in vehicle-treated controls (2.6 ± 0.5 vs. 1.6 ± 0.2, p = 0.05) but not after tPA + hAECs treatment (1.7 ± 0.2, p = 0.10 vs. tPA + saline). No differences in inflammatory cell content were detected between treatment groups. Conclusion: When administered following tPA in acute stroke, hAECs improve safety and attenuate infarct growth in association with less BBB disruption and lower 24 h mortality.

Improving cell viability using counterflow centrifugal elutriation.

BACKGROUND: Cell viability is an important release criterion in the manufacturing of cell therapy products. Low cell viability can have significant impact on product quality and manufacturing efficiency. Counterflow centrifugation technology has been applied to the manufacturing of cell therapy products, to enable cell separation based on size and density. This study evaluated the utility of counterflow centrifugation technology for dead cell removal to improve cell viability of the final product. METHODS: Jurkat cell cultures with low and high dead cell burden were subjected to counterflow centrifugal elutriation to determine the correlation between process parameters (e.g., flow rate, centrifugal force) and processing outcomes (i.e., cell recovery and viability). Subsequently, the optimized parameters were applied to dead cell elutriation using expanded T cells and freshly isolated human amniotic epithelial cells (hAECs). The efficiency of dead cell removal, cell function and post-thaw viability were compared. RESULTS: Elutriation using a low flow rate allowed better control of viable cell recovery from both low and high dead cell burden cultures of Jurkat cells. The viability of T cells and hAECs was improved by counterflow centrifugal processing, from 80.67% ± 2.33 to 94.73% ± 1.19 and 79.19% ± 5.35 to 90.34% ± 3.59, respectively. Processing increased the proliferation rate of T cells, while the metabolic activity of hAECs was unchanged. CONCLUSION: Counterflow centrifugal elutriation can be added as an integrated step to the automated wash-and-concentrate protocol for cell manufacturing to remove dead cells and improve cell viability of the final product.

Impact of chemically defined culture media formulations on extracellular vesicle production by amniotic epithelial cells.

The therapeutic properties of cell derived extracellular vesicles (EVs) make them promising cell-free alternative to regenerative medicine. However, clinical translation of this technology relies on the ability to manufacture EVs in a scalable, reproducible, and cGMP-compliant manner. To generate EVs in sufficient quantity, a critical step is the selection and development of culture media, where differences in formulation may influence the EV manufacturing process. In this study, we used human amniotic epithelial cells (hAECs) as a model system to explore the effect of different formulations of chemically defined, commercially sourced media on EV production. Here, we determined that cell viability and proliferation rate are not reliable quality indicators for EV manufacturing. The levels of tetraspanins and epitope makers of EVs were significantly impacted by culture media formulations. Mass spectrometry-based proteomic profiling revealed proteome composition of hAEC-EVs and the influence of media formulations on composition of EV proteome. This study has revealed critical aspects including cell viability and proliferation rate, EV yield, and tetraspanins, surface epitopes and proteome composition of EVs influenced by media formulations, and further insight into standardised EV production culture media that should be considered in clinical-grade scalable EV manufacture for generation of therapeutic EVs.

Prematurity negatively affects regenerative properties of human amniotic epithelial cells in the context of lung repair.

There is a growing appreciation of the role of lung stem/progenitor cells in the development and perpetuation of chronic lung disease including idiopathic pulmonary fibrosis. Human amniotic epithelial cells (hAECs) were previously shown to improve lung architecture in bleomycin-induced lung injury, with the further suggestion that hAECs obtained from term pregnancies possessed superior anti-fibrotic properties compared with their preterm counterparts. In the present study, we aimed to elucidate the differential effects of hAECs from term and preterm pregnancies on lung stem/progenitor cells involved in the repair. Here we showed that term hAECs were better able to activate bronchioalveolar stem cells (BASCs) and type 2 alveolar epithelial cells (AT2s) compared with preterm hAECs following bleomycin challenge. Further, we observed that term hAECs restored TGIF1 and TGFß2 expression levels, while increasing c-MYC expression despite an absence of significant changes to Wnt/ß-catenin signaling. In vitro, term hAECs increased the average size and numbers of BASC and AT2 colonies. The gene expression levels of Wnt ligands were higher in term hAECs, and the expression levels of BMP4, CCND1 and CDC42 were only increased in the BASC and AT2 organoids co-cultured with hAECs from term pregnancies but not preterm pregnancies. In conclusion, term hAECs were more efficient at activating the BASC niche compared with preterm hAECs. The impact of gestational age and/or complications leading to preterm delivery should be considered when applying hAECs and other gestational tissue-derived stem and stem-like cells therapeutically.

Automated Counterflow Centrifugal System for Small-Scale Cell Processing.

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and product concentration are essential components for most manufacturing processes. However, at the early stages of product development, these steps are often performed manually. Manual dead-end centrifugation for buffer exchange is labor-intensive, costly, and not scalable. A closed automated system can effectively eliminate this laborious step, but implementation can be challenging. Here, we describe a newly developed cell processing device that is suitable for small- to medium-scale cell processing and aims to bridge the gap between manual processing and large-scale automation. This protocol can be easily applied to various cell types and processes by modifying the flow rate and centrifugation speed. Our protocol demonstrated high cell recovery with shorter processing times in comparison to the manual process. Cells recovered from the automated process also maintained their proliferation rates. The device can be applied as a modular component in a closed manufacturing process to accommodate steps such as buffer exchange, cell formulation, and cryopreservation.

First-In-Human Administration of Allogeneic Amnion Cells in Premature Infants With Bronchopulmonary Dysplasia: A Safety Study.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease that mainly affects premature babies who require ventilator support. The pathogenesis of BPD is complex but includes vascular maldevelopment, alveolarization arrest, and lung inflammation. There is no cure for BPD. Clinical care is limited to supportive respiratory measures. A population of stem-like cells derived from placental membranes, human amnion epithelial cells (hAECs), has shown therapeutic promise in preclinical models of BPD. With a view to future efficacy trials, we undertook a first-in-human clinical trial of hAECs in babies with BPD to assess the safety of these cells. In a single-center, open-label phase I trial, we administered allogeneic hAECs (1 × 106 per kilogram bodyweight) by intravenous infusion to six premature babies with BPD. The primary outcomes of the study were focused on safety, including local site reaction, anaphylaxis, infection, features of rejection, or tumor formation. Outcomes to discharge from neonatal unit were studied. The hAECs were well tolerated. In the first baby, there was transient cardiorespiratory compromise during cell administration consistent with a pulmonary embolic event. Following changes to cell administration methods, including introduction of an inline filter, and reducing the cell concentration and the rate of cell infusion, no such events were observed in the subsequent five babies. We did not see evidence of any other adverse events related to cell administration. Allogeneic hAECs can be safely infused into babies with established BPD. Future randomized clinical trials to assess efficacy in this patient population are justified. Stem Cells Translational Medicine 2018;7:628-635.

Role of activin A in the pathogenesis of endothelial cell dysfunction in preeclampsia.

Circulating markers for endothelial activation such as endothelin-1 (ET-1), ICAM-1 and VCAM-1 are elevated in women with preeclampsia. Using human umbilical vein endothelial cells (HUVECs) as an in vitro model of the maternal vasculature, we show that activin A and preeclamptic serum upregulate ET-1, ICAM-1, and VCAM-1 in HUVECs. Further, we show that follistatin, a specific binding protein for activin, mitigates the upregulation of ET-1, ICAM-1 and VCAM-1 in HUVECs exposed to either activin A or preeclamptic serum. These data are consistent with activin A contributing to the pathophysiology of preeclampsia and suggest that therapies targeting activin signalling are worth exploring.

Evaluating the Impact of Human Amnion Epithelial Cells on Angiogenesis.

The effects of human amnion epithelial cells (hAECs) on angiogenesis remain controversial. It is yet unknown if the presence of inflammation and/or gestational age of hAEC donors have an impact on angiogenesis. In this study, we examined the differences between term and preterm hAECs on angiogenesis in vitro and in vivo. Conditioned media from term hAECs induced the formation of longer huVEC tubules on Matrigel. Both term and preterm hAECs expressed VEGFA, PDGFB, ANGPT1, and FOXC1, which significantly increased after TNFα and IFNγ stimulation. In the presence of TNFα and IFNγ, coculture with term hAECs reduced gene transcription of Tie-2 and Foxc1 in huVECs, while coculture with preterm hAECs increased gene transcription of PDGFRα and PDGFRß and reduced gene transcription of FOXC1 in huVECs. In vivo assessment of angiogenesis using vWF immunostaining revealed that hAEC treatment decreased angiogenesis in a bleomycin model of lung fibrosis but increased angiogenesis in a neonatal model of hyperoxia-induced lung injury. In summary, our findings suggested that the impact of hAECs on angiogenesis may be influenced by the presence of inflammation and underlying pathology.

Activin A contributes to the development of hyperoxia-induced lung injury in neonatal mice.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is one of the leading causes of morbidity and mortality in babies born prematurely, yet there is no curative treatment. In recent years, a number of inhibitors against TGFß signaling have been tested for their potential to prevent neonatal injury associated with hyperoxia, which is a contributing factor of BPD. In this study, we assessed the contribution of activin A-a member of the TGFß superfamily-to the development of hyperoxia-induced lung injury in neonatal mice. METHODS: We placed newborn C57Bl6 mouse pups in continuous hyperoxia (85% O2) to mimic many aspects of BPD including alveolar simplification and pulmonary inflammation. The pups were administered activin A receptor type IIB-Fc antagonist (ActRIIB-Fc) at 5 mg/kg or follistatin at 0.1 mg/kg on postnatal days 4, 7, 10, and 13. RESULTS: Treatment with ActRIIB-Fc and follistatin protected against hyperoxia-induced growth retardation. ActRIIB-Fc also reduced pulmonary leukocyte infiltration, normalized tissue: airspace ratio and increased septal crest density. These findings were associated with reduced phosphorylation of Smad3 and decreased matrix metalloproteinase (MMP)-9 activity. CONCLUSION: This study suggests that activin A signaling may contribute to the pathology of bronchopulmonary dysplasia.

Measuring respiratory function in mice using unrestrained whole-body plethysmography.

Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.

Human amnion epithelial cells mediate lung repair by directly modulating macrophage recruitment and polarization.

Human amnion epithelial cells (hAECs) have been shown to modulate inflammation and restore normal lung structure and respiratory function following bleomycin challenge in immune-competent mice. These effects are exerted despite a lack of significant engraftment of hAECs, suggesting that immunomodulatory effect mechanisms are at play. In this study, using the bleomycin model of injury, we explored the interactions between hAECs and macrophages. We administered 4 million hAECs intraperitoneally to C57Bl6 mice 24 h following a bleomycin challenge. Using FACS analysis and qPCR, we showed that hAEC administration significantly reduced macrophage infiltration into the lungs and that the majority of the pulmonary macrophages were of the M2 phenotype. Using bone marrow-derived macrophages, we then showed that hAEC-conditioned media could alter macrophage polarization, migration, and phagocytosis, without affecting macrophage survival or proliferation in vitro. This study provides the first evidence that hAECs directly influence macrophage behavior in a proreparative manner and suggests that hAECs are able to mediate these effects independently of other immune cell types.

Human amnion epithelial cells modulate hyperoxia-induced neonatal lung injury in mice.

BACKGROUND AIMS: Human amnion epithelial cells (hAECs) prevent pulmonary inflammation and injury in fetal sheep exposed to intrauterine lipopolysaccharide. We hypothesized that hAECs would similarly mitigate hyperoxia-induced neonatal lung injury. METHODS: Newborn mouse pups were randomized to either normoxia (inspired O2 content (FiO2) = 0.21, n = 60) or hyperoxia (FiO2 = 0.85, n = 57). On postnatal days (PND) 5, 6 and 7, hAECs or sterile saline (control) was administered intraperitoneally. All animals were assessed at PND 14. RESULTS: Hyperoxia was associated with lung inflammation, alveolar simplification and reduced postnatal growth. Administration of hAECs to hyperoxia-exposed mice normalized body weight and significantly attenuated some aspects of hyperoxia-induced lung injury (mean linear intercept and septal crest density) and inflammation (interleukin-1α, interleukin-6, transforming growth factor-ß and platelet-derived growth factor-ß). However, hAECs did not significantly alter changes to alveolar airspace volume, septal tissue volume, tissue-to-airspace ratio, collagen content or leukocyte infiltration induced by hyperoxia. CONCLUSIONS: Intraperitoneal administration of hAECs to neonatal mice partially reduced hyperoxia-induced lung inflammation and structural lung damage. These observations suggest that hAECs may be a potential therapy for neonatal lung disease.

Human amnion epithelial cells repair established lung injury.

With a view to developing a cell therapy for chronic lung disease, human amnion epithelial cells (hAECs) have been shown to prevent acute lung injury. Whether they can repair established lung disease is unknown. We aimed to assess whether hAECs can repair existing lung damage induced in mice by bleomycin and whether the timing of cell administration influences reparative efficacy. In addition, we aimed to characterize the effect of hAECs on fibroblast proliferation and activation, investigating possible mechanisms of reparative action. hAECs were administered intraperitoneally (IP) either 7 or 14 days after bleomycin exposure. Lungs were assessed 7 days after hAEC administration. Bleomycin significantly reduced body weight and induced pulmonary inflammation and fibrosis at 14 and 21 days. Delivery of hAECs 7 days after bleomycin had no effect on lung injury, whereas delivery of hAECs 14 days after bleomycin normalized lung tissue density, collagen content, and α-SMA production, in association with a reduction in pulmonary leucocytes and lung expression of TGF-ß, PDGF-α, and PDGF-ß. In vitro, hAECs reduced proliferation and activation of primary mouse lung fibroblasts. Our findings suggest that the timing of hAEC administration in the course of lung disease may impact on the ability of hAECs to repair lung injury.

Human mesenchymal stem cells reduce lung injury in immunocompromised mice but not in immunocompetent mice.

BACKGROUND: The immunomodulatory and immunosuppressive capacity of human mesenchymal stem cells (hMSC) is well recognized, but efficacies of hMSC in immunocompetent and immunocompromised animals have never been directly compared. OBJECTIVES: We aimed to compare the efficacy of hMSC in preventing bleomycin-induced lung injury in immunocompromised SCID and immunocompetent C57Bl/6 mice. METHODS: SCID and C57Bl/6 mice were subjected to a single bolus intranasal instillation of bleomycin to induce lung injury. One million hMSC were administered intravenously 24 h following the induction of bleomycin lung injury. RESULTS: hMSC xenotransplantation into SCID mice resulted in transient improvements in lung weight and tidal volume and to persistent improvement in inspiratory duty cycle, inspiratory flow rate and inspiration/expiration ratio. We did not observed protective effects in C57Bl/6 mice. This correlated with histological changes, where hMSC administration reduced Ashcroft scores, collagen deposition and inflammatory influx in the lungs of SCID mice, but not in those of C57Bl/6 mice. CONCLUSION: The application of hMSC for the treatment of acute and chronic lung injury is significantly affected by the immune status of the recipient. Lack of hMSC-mediated repair observed in C57Bl/6 mice was likely to be due to limitations of their immune privilege and differential priming of hMSC in immunocompetent versus immunocompromised hosts.