Jean Tan

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 do not abrogate pulmonary fibrosis in mice with impaired macrophage function.

Since current treatments for both acute and chronic lung diseases are less than ideal, there has been recent interest in the use of cell-based therapies for inflammatory lung disease. Specifically, human amnion epithelial cells (hAECs) have been shown to reduce bleomycin-induced lung injury and prevent subsequent loss of respiratory function, primarily through modulation of the host immune response. The precise mechanisms of this effect remain unclear. We aimed to investigate the potential of hAECs to mitigate bleomycin-induced lung injury in surfactant protein C deficient (Sftpc-/-) mice, which are highly susceptible to pulmonary injury as a result of impairment of macrophage function. Primary hAECs were administered to wild-type (Sftpc+/+) and Sftpc-/- mice 24 h after exposure to bleomycin. Compared to Sftpc+/+ mice receiving bleomycin alone, Sftpc+/+ mice administered hAECs 24 h after bleomycin exposure had decreased expression of proinflammatory genes, decreased macrophage and neutrophil infiltration, fibrosis, collagen content, and α-smooth muscle actin as well as a significant improvement in lung function. Compared to Sftpc-/- mice given bleomycin alone, Sftpc-/- mice administered hAECs 24 h after bleomycin did not have a decrease in inflammatory gene expression or a reduction in macrophage pulmonary infiltration. Subsequently, Sftpc-/- mice did not show any decrease in pulmonary fibrosis or improvement of lung function after hAEC administration. The ability of hAECs to mitigate bleomycin-induced lung injury is abolished in Sftpc-/- mice, suggesting that hAECs require normal host macrophage function to exert their reparative effects.

Preterm human amnion epithelial cells have limited reparative potential

The collection and use of stem cells from the fetal membranes as cell therapy for a variety of lung diseases, including preterm lung disease, have been previously proposed. To date, only cells from term amnion have been assessed. In the setting of a future therapy for the preterm neonate, it would be ideal if autologous cells could be given. However, the reparative and anti-inflammatory actions of stem cells isolated from preterm amnions have not been evaluated. In this study, with a view to developing an autologous cell therapy for preterm lung injury, we compared the differentiation potential and efficacy of term versus preterm human amnion epithelial cells (hAECs) to protect against inflammation and fibrosis in a bleomycin mouse model of lung injury. We found that, unlike term hAECs, preterm hAECs did not differentiate into a lung lineage following culture in small airway growth media. Preterm hAECs also exerted significantly less protective effects than term hAEC following acute lung injury. Specifically, preterm hAEC did not improve Ashcroft scoring or collagen deposition in the lung despite a reduction in activated myofibroblasts. Term hAECs expressed double the levels of HLA-G compared to preterm hAECs. These findings indicate that while hAECs can be isolated from term and preterm amnions in similar numbers, they bear distinctive characteristics, which may impact upon their clinical use.

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, TGFβ, 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

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

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.

Regulatory T-Cells: Potential Regulator of Tissue Repair and Regeneration

The identification of stem cells and growth factors as well as the development of biomaterials hold great promise for regenerative medicine applications. However, the therapeutic efficacy of regenerative therapies can be greatly influenced by the host immune system, which plays a pivotal role during tissue repair and regeneration. Therefore, understanding how the immune system modulates tissue healing is critical to design efficient regenerative strategies. While the innate immune system is well known to be involved in the tissue healing process, the adaptive immune system has recently emerged as a key player. T-cells, in particular, regulatory T-cells (Treg), have been shown to promote repair and regeneration of various organ systems. In this review, we discuss the mechanisms by which Treg participate in the repair and regeneration of skeletal and heart muscle, skin, lung, bone, and the central nervous system.

To Protect and to Preserve: Novel Preservation Strategies for Extracellular Vesicles

Extracellular vesicles (EVs)-based therapeutics are based on the premise that EVs shed by stem cells exert similar therapeutic effects and these have been proposed as an alternative to cell therapies. EV-mediated delivery is an effective and efficient system of cell-to-cell communication which can confer therapeutic benefits to their target cells. EVs have been shown to promote tissue repair and regeneration in various animal models such as, wound healing, cardiac ischemia, diabetes, lung fibrosis, kidney injury, and many others. Given the unique attributes of EVs, considerable thought must be given to the preservation, formulation and cold chain strategies in order to effectively translate exciting preclinical observations to clinical and commercial success. This review summarizes current understanding around EV preservation, challenges in maintaining EV quality, and also bioengineering advances aimed at enhancing the long-term stability of EVs.

Amniotic Membrane Stem Cell Populations

Amnion membrane has long been used to facilitate wound healing and repair. However, the recent recognition that the fetal membranes, both amnion and chorion, contain a variety of cell populations with stem cell or stem cell-like properties has led to renewed interest in the regenerative medicine potential of these tissues that are otherwise regarded as medical waste. There are two main populations of stem cell-like cells in the amnion—amnion mesenchymal stem cells and amnion epithelial cells. While they possess similar properties there are also some important differences that may be important for future clinical applications. Studies using these cells to date have been mainly limited to experimental animal work but have addressed diverse applications such as lung disease, diabetes, neurological disorders, liver disease, ischaemic disorders. It would appear that for most of these applications the cells are not implanting and differentiating into niche lineages to effect organ repair but rather are targeting host immune responses to injury to drive these towards reparative pathways. Specifically, the cells appear to critically modulate host macrophage and T cell responses. Most recently, it has been shown that the cells themselves may not be required to effect repair but that cell-conditioned media may be sufficient. Exploring what cell secreted products effect the reparative actions is now an urgent focus of attention. These insights will likely better direct translation of the experimental research into clinical trials, many of which are poised to begin.

Amnion Epithelial Cells for Lung Diseases

Amnion epithelial cells, derived from the amnion fetal membranes, are pluripotent and immunomodulatory. They have been shown to both prevent and repair acute lung injury in a number of diverse experimental models. They are readily isolated from term placentae in sufficient numbers suitable for clinical application. They also have a number of features that make them particularly attractive as a future cell therapy including being immune-privileged and nontumorigenic. This chapter reviews the use of amnion epithelial cells as a cell therapy for lung injury, addressing likely mechanisms of action and future clinical trials.