CM from intact hAM: an easily obtained product with relevant implications for translation in regenerative medicine.

BACKGROUND: It is now well established that factors (free or in extracellular vesicles) secreted by mesenchymal stromal cells (MSC) are important mediators of MSC regenerative actions. Herein we produced the secretome (conditioned medium, CM) from MSC isolated from the amniotic membrane (hAMSC) and CM from the intact amniotic membrane (hAM, no manipulation or enzymatic digestion) in order to potentially identify an effective, easy and less expensive secretome to produce for potential applications in regenerative medicine. Given that immunomodulation is a key mechanism of action through which hAMSC contributes to tissue regeneration, we used a comprehensive panel of in vitro immunomodulatory tests to compare the CMs. METHODS: Amniotic membranes were either cut into fragments or used for hAMSC isolation. CMs from hAMSC at passages 0 and 2 were collected after a standard 5-day culture while CM from hAM was collected after a 2- and 5-day culture. Immunomodulation was assessed in terms of PBMC and T-cell proliferation, T-cell subset polarization, T-regulatory cell induction, cell cytotoxicity and monocyte differentiation toward antigen-presenting cells. Furthermore, we performed a comparison between CM obtained from single donors and pooled CM. We also assessed the impact of lyophilization on the immunomodulatory properties of CM. RESULTS: We demonstrate that CM from hAM has comparable immunomodulatory properties to CM from hAMSC at passages 0 and 2. Furthermore, we demonstrate that pooled CMs have similar effects when compared to CM from single donors used separately. Finally, we demonstrate that lyophilization does not alter the in vitro immunomodulatory properties of CM from hAM and hAMSC. CONCLUSIONS: The results presented herein support the possibility to produce secretome from intact hAM and open the prospect to highly improve the scalability of the GMP production process while reducing the costs and time related to the process of cell isolation and expansion. Moreover, the possibility of having a lyophilized secretome that maintains its original properties would allow for a ready-to-use product with easier handling, shipping and storage. The use of a lyophilized product will also facilitate clinicians by permitting customized reconstitution volumes and methods according to the most suitable formula required by the clinical application.

Human amniotic mesenchymal stromal cells support the ex vivo expansion of cord blood hematopoietic stem cells.

Currently, more than 30 000 allogeneic hematopoietic stem cell (HSC) transplantations have been performed for the treatment of hematological and nonhematological diseases using HSC from umbilical cord blood (CB). However, the wide utilization of CB as a source of HSC is limited by the low number of cells recovered. One strategy to expand ex vivo CB-HSC is represented by the use of bone marrow mesenchymal stromal cells (BM-MSCs) as a feeder to enhance HSC proliferation while maintaining HSC stemness. Indeed, BM-MSCs have been recognized as one of the most relevant players in the HSC niche. Thus, it has been hypothesized that they can support the ex vivo expansion of HSC by mimicking the physiological microenvironment present in the hematopoietic niche. Due to the role of placenta in supporting fetal hematopoiesis, MSC derived from the amniotic membrane (hAMSC) of human term placenta could represent an interesting alternative to BM-MSC as a feeder layer to enhance the proliferation and maintain HSC stemness. Therefore, in this study we investigated if hAMSC could support the ex vivo expansion of HSC and progenitor cells. The capacity of hAMSCs to support the ex vivo expansion of CB-HSC was evaluated in comparison to the control condition represented by the CB-CD34+ cells without a feeder layer. The coculture was performed at two different CD34+ :MSC ratios (1:2 and 1:8) in both cell-to-cell contact and transwell setting. After 7 days, the cells were collected and analyzed for phenotype and functionality. Our results suggest that hAMSCs represent a valuable alternative to BM-MSC to support: (a) the ex vivo expansion of CB-HSC in both contact and transwell systems, (b) the colony forming unit ability, and (c) long-term culture initiating cells ability. Overall, these findings may contribute to address the unmet need of high HSC content in CB units available for transplantation.

The Cells and Extracellular Matrix of Human Amniotic Membrane Hinder the Growth and Invasive Potential of Bladder Urothelial Cancer Cells.

Bladder cancer is one of the most common cancers among men in industrialized countries and on the global level incidence and mortality rates are increasing. In spite of progress in surgical treatment and chemotherapy, the prognosis remains poor for patients with muscle-invasive bladder cancer. Therefore, there is a great need for the development of novel therapeutic approaches. The human amniotic membrane (hAM) is a multi-layered membrane that comprises the innermost part of the placenta. It has unique properties that make it suitable for clinical use, such as the ability to promote wound healing and decrease scarring, low immunogenicity, and immunomodulatory, antimicrobial and anticancer properties. This study aimed to investigate the effect of (i) hAM-derived cells and (ii) hAM scaffolds on the growth dynamics, proliferation rate, and invasive potential of muscle-invasive bladder cancer T24 cells. Our results show that 24 and 48 h of co-culturing T24 cells with hAM-derived cells (at 1:1 and 1:4 ratios) diminished the proliferation rate of T24 cells. Furthermore, when seeded on hAM scaffolds, namely (1) epithelium of hAM (e-hAM), (2) basal lamina of hAM (denuded; d-hAM), and (3) stroma of hAM (s-hAM), the growth dynamic of T24 cells was altered and proliferation was reduced, even more so by the e-hAM scaffolds. Importantly, despite their muscle-invasive potential, the T24 cells did not disrupt the basal lamina of hAM scaffolds. Furthermore, we observed a decrease in the expression of epithelial-mesenchymal transition (EMT) markers N-cadherin, Snail and Slug in T24 cells grown on hAM scaffolds and individual T24 cells even expressed epithelial markers E-cadherin and occludin. Our study brings new knowledge on basic mechanisms of hAM affecting bladder carcinogenesis and the results serve as a good foundation for further research into the potential of hAM-derived cells and the hAM extracellular matrix to serve as a novel bladder cancer treatment.

B Lymphocytes as Targets of the Immunomodulatory Properties of Human Amniotic Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSC) from the amniotic membrane of human term placenta (hAMSC), and the conditioned medium generated from their culture (CM-hAMSC) offer significant tools for their use in regenerative medicine mainly due to their immunomodulatory properties. Interestingly, hAMSC and their CM have been successfully exploited in preclinical disease models of inflammatory and autoimmune diseases where depletion or modulation of B cells have been indicated as an effective treatment, such as inflammatory bowel disease, lung fibrosis, would healing, collagen-induced arthritis, and multiple sclerosis. While the interactions between hAMSC or CM-hAMSC and T lymphocytes, monocytes, dendritic cells, and macrophages has been extensively explored, how they affect B lymphocytes remains unclear. Considering that B cells are key players in the adaptive immune response and are a central component of different diseases, in this study we investigated the in vitro properties of hAMSC and CM-hAMSC on B cells. We provide evidence that both hAMSC and CM-hAMSC strongly suppressed CpG-activated B-cell proliferation. Moreover, CM-hAMSC blocked B-cell differentiation, with an increase of the proportion of mature B cells, and a reduction of antibody secreting cell formation. We observed the strong inhibition of B cell terminal differentiation into CD138+ plasma cells, as further shown by a significant decrease of the expression of interferon regulatory factor 4 (IRF-4), PR/SET domain 1(PRDM1), and X-box binding protein 1 (XBP-1) genes. Our results point out that the mechanism by which CM-hAMSC impacts B cell proliferation and differentiation is mediated by secreted factors, and prostanoids are partially involved in these actions. Factors contained in the CM-hAMSC decreased the CpG-uptake sensors (CD205, CD14, and TLR9), suggesting that B cell stimulation was affected early on. CM-hAMSC also decreased the expression of interleukin-1 receptor-associated kinase (IRAK)-4, consequently inhibiting the entire CpG-induced downstream signaling pathway. Overall, these findings add insight into the mechanism of action of hAMSC and CM-hAMSC and are useful to better design their potential therapeutic application in B-cell mediated diseases.

Amniotic MSCs reduce pulmonary fibrosis by hampering lung B-cell recruitment, retention, and maturation.

Growing evidence suggests a mechanistic link between inflammation and the development and progression of fibrotic processes. Mesenchymal stromal cells derived from the human amniotic membrane (hAMSCs), which display marked immunomodulatory properties, have been shown to reduce bleomycin-induced lung fibrosis in mice, possibly by creating a microenvironment able to limit the evolution of chronic inflammation to fibrosis. However, the ability of hAMSCs to modulate immune cells involved in bleomycin-induced pulmonary inflammation has yet to be elucidated. Herein, we conducted a longitudinal study of the effects of hAMSCs on alveolar and lung immune cell populations upon bleomycin challenge. Immune cells collected through bronchoalveolar lavage were examined by flow cytometry, and lung tissues were used to study gene expression of markers associated with different immune cell types. We observed that hAMSCs increased lung expression of T regulatory cell marker Foxp3, increased macrophage polarization toward an anti-inflammatory phenotype (M2), and reduced the antigen-presentation potential of macrophages and dendritic cells. For the first time, we demonstrate that hAMSCs markedly reduce pulmonary B-cell recruitment, retention, and maturation, and counteract the formation and expansion of intrapulmonary lymphoid aggregates. Thus, hAMSCs may hamper the self-maintaining inflammatory condition promoted by B cells that continuously act as antigen presenting cells for proximal T lymphocytes in injured lungs. By modulating B-cell response, hAMSCs may contribute to blunting of the chronicization of lung inflammatory processes with a consequent reduction of the progression of the fibrotic lesion.

Effect of human amniotic epithelial cells on pro-fibrogenic resident hepatic cells in a rat model of liver fibrosis.

Myofibroblasts are key fibrogenic cells responsible for excessive extracellular matrix synthesis characterizing the fibrotic lesion. In liver fibrosis, myofibroblasts derive either from activation of hepatic stellate cells (HSC) and portal fibroblasts (PF), or from the activation of fibroblasts that originate from ductular epithelial cells undergoing epithelial-mesenchymal transition. Ductular cells can also indirectly promote myofibroblast generation by activating TGF-ß, the main fibrogenic growth factor, through αvß6 integrin. In addition, after liver injury, liver sinusoidal cells can lose their ability to maintain HSC quiescence, thus favouring HSC differentiation towards myofibroblasts. The amniotic membrane and epithelial cells (hAEC) derived thereof have been shown to decrease hepatic myofibroblast levels in rodents with liver fibrosis. In this study, in a rat model of liver fibrosis, we investigated the effects of hAEC on resident hepatic cells contributing to myofibroblast generation. Our data show that hAEC reduce myofibroblast numbers with a consequent reduction in fibronectin and collagen deposition. Interestingly, we show that hAEC strongly act on specific myofibroblast precursors. Specifically, hAEC reduce the activation of PF rather than HSC. In addition, hAEC target reactive ductular cells by inhibiting their proliferation and αvß6 integrin expression, with a consequent decrease in TGF-ß activation. Moreover, hAEC counteract the transition of ductular cells towards fibroblasts, while it does not affect injury-induced and fibrosis-promoting sinusoidal alterations. In conclusion, among the emerging therapeutic applications of hAEC in liver diseases, their specific action on PF and ductular cells strongly suggests their application in liver injuries involving the expansion and activation of the portal compartment.

Internalization of nanopolymeric tracers does not alter characteristics of placental cells.

In the cell therapy scenario, efficient tracing of transplanted cells is essential for investigating cell migration and interactions with host tissues. This is fundamental to provide mechanistic insights which altogether allow for the understanding of the translational potential of placental cell therapy in the clinical setting. Mesenchymal stem/stromal cells (MSC) from human placenta are increasingly being investigated for their potential in treating patients with a variety of diseases. In this study, we investigated the feasibility of using poly (methyl methacrylate) nanoparticles (PMMA-NPs) to trace placental MSC, namely those from the amniotic membrane (hAMSC) and early chorionic villi (hCV-MSC). We report that PMMP-NPs are efficiently internalized and retained in both populations, and do not alter cell morphofunctional parameters. We observed that PMMP-NP incorporation does not alter in vitro immune modulatory capability of placental MSC, a characteristic central to their reparative/therapeutic effects in vitro. We also show that in vitro, PMMP-NP uptake is not affected by hypoxia. Interestingly, after in vivo brain ischaemia and reperfusion injury achieved by transient middle cerebral artery occlusion (tMCAo) in mice, iv hAMSC treatment resulted in significant improvement in cognitive function compared to PBS-treated tMCAo mice. Our study provides evidence that tracing placental MSC with PMMP-NPs does not alter their in vitro and in vivo functions. These observations are grounds for the use of PMMP-NPs as tools to investigate the therapeutic mechanisms of hAMSC and hCV-MSC in preclinical models of inflammatory-driven diseases.

Amniotic mesenchymal cells from pre-eclamptic placentae maintain immunomodulatory features as healthy controls.

Pre-eclampsia (PE) is one of the most severe syndromes in human pregnancy, and the underlying mechanisms of PE have yet to be determined. Pre-eclampsia is characterized by the alteration of the immune system's activation status, an increase in inflammatory Th1/Th17/APC cells, and a decrease in Th2/Treg subsets/cytokines. Moreover, inflammatory infiltrates have been detected in the amniotic membranes of pre-eclamptic placentae, and to this date limited data are available regarding the role of amniotic membrane cells in PE. Interestingly, we and others have previously shown that human amniotic mesenchymal stromal cells (hAMSC) possess anti-inflammatory properties towards almost all immune cells described to be altered in PE. In this study we investigated whether the immunomodulatory properties of hAMSC were altered in PE. We performed a comprehensive study of cell phenotype and investigated the in vitro immunomodulatory properties of hAMSC isolated from pre-eclamptic pregnancies (PE-hAMSC), comparing them to hAMSC from normal pregnancies (N-hAMSC). We demonstrate that PE-hAMSC inhibit CD4/CD8 T-cell proliferation, suppress Th1/Th2/Th17 polarization, induce Treg and block dendritic cells and M1 differentiation switching them to M2 cells. Notably, PE-hAMSC generated a more prominent induction of Treg and higher suppression of interferon-γ when compared to N-hAMSC, and this was associated with higher transforming growth factor-ß1 secretion and PD-L2/PD-L1 expression in PE-hAMSC. In conclusion, for the first time we demonstrate that there is no intrinsic impairment of the immunomodulatory features of PE-hAMSC. Our results suggest that amniotic mesenchymal stromal cells do not contribute to the disease, but conversely, could participate in offsetting the inflammatory environment which characterizes PE.

Amniotic membrane mesenchymal cells-derived factors skew T cell polarization toward Treg and downregulate Th1 and Th17 cells subsets.

We previously demonstrated that cells derived from the mesenchymal layer of the human amniotic membrane (hAMSC) and their conditioned medium (CM-hAMSC) modulate lymphocyte proliferation in a dose-dependent manner. In order to understand the mechanisms involved in immune regulation exerted by hAMSC, we analyzed the effects of CM-hAMSC on T-cell polarization towards Th1, Th2, Th17, and T-regulatory (Treg) subsets. We show that CM-hAMSC equally suppresses the proliferation of both CD4(+) T-helper (Th) and CD8(+) cytotoxic T-lymphocytes. Moreover, we prove that the CM-hAMSC inhibitory ability affects both central (CD45RO(+)CD62L(+)) and effector memory (CD45RO(+)CD62L(-)) subsets. We evaluated the phenotype of CD4(+) cells in the MLR setting and showed that CM-hAMSC significantly reduced the expression of markers associated to the Th1 (T-bet(+)CD119(+)) and Th17 (RORγt(+)CD161(+)) populations, while having no effect on the Th2 population (GATA3(+)CD193(+)/GATA3(+)CD294(+)cells). T-cell subset modulation was substantiated through the analysis of cytokine release for 6 days during co-culture with alloreactive T-cells, whereby we observed a decrease in specific subset-related cytokines, such as a decrease in pro-inflammatory, Th1-related (TNFα, IFNγ, IL-1ß), Th2 (IL-5, IL-6), Th9 (IL-9), and Th17 (IL-17A, IL-22). Furthermore, CM-hAMSC significantly induced the Treg compartment, as shown by an induction of proliferating CD4(+)FoxP3(+) cells, and an increase of CD25(+)FoxP3(+) and CD39(+)FoxP3(+) Treg in the CD4(+) population. Induction of Treg cells was corroborated by the increased secretion of TGF-ß. Taken together, these data strengthen the findings regarding the immunomodulatory properties of CM-hAMSC derived from human amniotic membrane MSC, and in particular provide insights into their effect on regulation of T cell polarization.

Gestational stage affects amniotic epithelial cells phenotype, methylation status, immunomodulatory and stemness properties.

Stem cells isolated from amniotic epithelium (AECs) have shown great potential in cell-based regenerative therapies. Because of their fetal origin, these cells exhibit elevated proliferation rates and plasticity, as well as, immune tolerance and anti-inflammatory properties. These inherent attitudes make AECs well-suited for both allogenic and xenogenic cellular transplants in animal models. Since in human only at term amnion is easily obtainable after childbirth, limited information are so far available concerning the phenotypic and functional difference between AECs isolated from early and late amnia. To this regard, the sheep animal model offers an undoubted advantage in allowing the easy collection of both types of AECs in large quantity. The aim of this study was to determine the effect of gestational age on ovine AECs (oAECs) phenotype, immunomodulatory properties, global DNA methylation status and pluripotent differentiation ability towards mesodermic and ectodermic lineages. The immunomodulatory property of oAECs in inhibiting lymphocyte proliferation was mainly unaffected by gestational age. Conversely, gestation considerably affected the expression of surface markers, as well the expression and localization of pluripotency markers. In detail, with progression of gestation the mRNA expression of NANOG and SOX2 markers was reduced, while the ones of TERT and OCT4A was unaltered; but at the end of gestation NANOG, SOX2 and TERT proteins mainly localized outside the nuclear compartment. Regarding the differentiation ability, LPL (adipogenic-specific gene) mRNA content significantly increased in oAECs isolated from early amnia, while OCN (osteogenic-specific gene) and NEFM (neurogenic-specific gene) mRNA content significantly increased in oAECs isolated from late amnia, suggesting that gestational stage affected cell plasticity. Finally, the degree of global DNA methylation increased with gestational age. All these results indicate that gestational age is a key factor capable of influencing morphological and functional properties of oAECs, and thus probably affecting the outcome of cell transplantation therapies.

Feasibility and potential of in utero foetal membrane-derived cell transplantation.

Cells isolated from foetal membranes of human term placenta display multiple properties, including some features of stem/progenitor cells, together with immunomodulatory actions and the ability to secrete bioactive soluble factors. Whilst such properties support the potential applicability of these cells in transplantation settings aimed at regenerating/repairing tissues in adults, theoretically, using these cells in prenatal treatment strategies may also be achievable. To assess the feasibility of a foetal membrane-derived cell-based therapeutic treatment during foetal development, we firstly addressed the question of whether in utero transplantation using these cells was possible. To this end, we assessed postnatal microchimerism after transplantation of amniotic membrane-derived cells (a mixture of both mesenchymal stromal/stem cells and epithelial cells) in foetal sheep. Transplantation was performed with or without human umbilical cord blood mononuclear cells and chorionic membrane-derived mesenchymal stromal/stem cells, and was followed by a postnatal booster cell injection. Lambs were euthanized 2-4 months postnatally and their organs/tissues were analysed for microchimerism through detection of human DNA. Human DNA was found in almost all tissues of all of the lambs, with the seemingly random appearance of human cells in some of the analysed tissues suggesting long-term human microchimerism and donor cell migration after in utero/postnatal booster xenotransplation. Differences in microchimerism tissue distribution between animals transplanted with different cell types are discussed. This pilot study adds to ongoing efforts by different investigators to explore the potential of in utero cellular transplantation, and warrants further investigation of using foetal membrane-derived cells for prenatal cell therapies.