From fetal development and beyond: human term placenta as a source of stem cells for regenerative medicine

Over the past decade, human term placenta has become more than a vital organ during pregnancy, but also a precious reservoir of cells. An increasing number of studies have shown that these cells harbor beneficial properties. In fact, we and others have reported the therapeutic effects of placental cells in preclinical models of lung and liver fibrosis, sepsis, inflammatory bowel disease, autoimmune encephalomyelitis, cardiac ischemia and hind limb ischemia. Remarkably, the diseases which were most attenuated by placental cell treatment were those with underlying altered immune reactions. We have significantly contributed to the understanding of the immune-modulatory properties of cells from the amniotic membrane in vitro, showing that they can reduce the proliferation of T cell subsets, down regulate Th1 and Th17 subsets, and increase T lymphocytes with regulatory functions (Treg). Contributing to their regenerative potential, placenta-derived cells have been reported to secrete a variety of growth factors that could act on progenitor and/or resident cells to favor tissue regeneration. For example, placental cells can release pro-angiogenic factors, such as hepatocyte growth factor, and mediators in extracellular matrix degradation, such as matrix metalloproteinases (MMPs). Moreover, the release of growth factors could stimulate resident stem cells to proliferate, altogether contributing to tissue regeneration. Interestingly, to further substantiate the observation that secreted factors are the main players in the therapeutic properties of placental cells, an increasing number of studies have shown that these beneficial effects are evident when conditioned medium obtained from cell culture is used or when cells are cultured in transwell systems.In conclusion, the placenta is a rich resource of therapeutic derivatives, such as cells, their secreted factors, and also others such as amniotic membrane patches, the latter of which have been successfully used in medicine for over a century. More recently, placental cells and their derivatives are being testing in clinical trials in patients with immune-dysregulated diseases.This work was supported by Fondazione Poliambulanza, Fondazione Cariplo, Ministero dell’Istruzione, dell’Università e della Ricerca, and Ministero della Salute

AEC and AFMSC Transplantation Preserves Fertility of Experimentally Induced Rat Varicocele by Expressing Differential Regenerative Mechanisms

Amniotic membrane and amniotic fluid derived cells are regarded as a promising stem cell source for developing regenerative medicine techniques, although they have never been tested on male infertility diseases such as varicocele (VAR). The current study aimed to examine the effects of two distinct cell sources, human Amniotic Fluid Mesenchymal Stromal Cells (hAFMSCs) and amniotic epithelial cells (hAECs), on male fertility outcomes in a rat induced VAR model. To explain cell-dependent enhancement of reproductive outcomes in rats transplanted with hAECs and hAFMSCs, insights on testis morphology, endocannabinoid system (ECS) expression and inflammatory tissue response have been carried out alongside cell homing assessment. Both cell types survived 120 days post-transplantation by modulating the ECS main components, promoting proregenerative M2 macrophages (Mφ) recruitment and a favorable anti-inflammatory IL10 expression pattern. Of note, hAECs resulted to be more effective in restoring rat fertility rate by enhancing both structural and immunoresponse mechanisms. Moreover, immunofluorescence analysis revealed that hAECs contributed to CYP11A1 expression after transplantation, whereas hAFMSCs moved towards the expression of Sertoli cell marker, SOX9, confirming a different contribution into the mechanisms leading to testis homeostasis. These findings highlight, for the first time, a distinct role of amniotic membrane and amniotic fluid derived cells in male reproduction, thus proposing innovative targeted stem-based regenerative medicine protocols for remedying high-prevalence male infertility conditions such as VAR

Editorial: Perinatal derivatives and the road to clinical translation, Volume II

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Shaping the Future of Perinatal Cells: Lessons From the Past and Interpretations of the Present

Since their discovery and characterization, mesenchymal stromal cells (MSC) have been a topic of great interest in regenerative medicine. Over the last 10 years, detailed studies investigated the properties of MSC from perinatal tissues and have indicated that these cells may represent important tools for restoring tissue damage or promoting regeneration and repair of the tissue microenvironment. At first, perinatal tissue-derived MSC drew attention due to their potential differentiation capacities suggested by their early embryological origin. It is nowadays accepted that perinatal tissue-derived MSC are promising for a wide range of regenerative medicine applications because of their unique immune modulatory properties, rather than their differentiation ability. As a matter of fact, the activation and function of various cells of the innate and adaptive immune systems are suppressed and modulated by MSC from different perinatal tissues, such as human term placenta. However, the mechanisms by which they act on immune cells to facilitate tissue repair during pathological processes remain to be thoroughly elucidated to develop safe and efficient therapeutic approaches. In addition to immune modulatory ability, several other peculiar characteristics of placenta MSC, less explored and/or more debated, are being investigated. These include an understanding of the anti-microbial properties and the role of placental MSC in tumor progression. Moreover, a thorough investigation on preparation methods, bioactive factors, mechanisms of action of the cell secretome, and the development of potency assays to predict clinical efficacy of placenta MSC and their products, are necessary to provide a solid basis for their clinical application

Mesenchymal Stromal Cells: From Therapeutic Option to Therapeutic Target

As our understanding of mesenchymal stromal cells (MSC) has evolved, they have come to be recognized as an integral part of the tumor tissue, and the exploitability of their intrinsic features in the field of oncology has reached a standstill. Currently, there are 1621 registered clinical trials (clinicaltrials.gov) on “mesenchymal cells”, and yet none of them are exploring or explored unmodified MSC as a therapeutic option for cancer. Indeed, the therapeutic potential of these cells in oncology relies on their ability to migrate towards sites of injury and inflammation and function as delivery systems for the local release of therapeutics [1], recently reported as also occurring via exosomes [2]. Since the first characterization of these cells in healthy bone marrow (BM) by Friedenstein, MSC have been described in various tissues of healthy and diseased bodies, performing a plethora of functions mostly aimed towards the governance of tissue homeostasis. When considering pathological conditions and cancer, in particular, evidence suggests that the ability of local or recruited MSC to maintain a steady state is compromised, and the cells become integrated into the newly formed organ at the expense of its healthy counterpart. Cancer-associated MSC (CA-MSC) have been shown to promote multi-organ metastasis and to govern tumor immune surveillance [3]. Furthermore, BM-derived cancer-associated fibroblasts (CAFs) promote angiogenesis in breast cancer [4] and exhibit a unique inflammatory profile depending on the location. Indeed, several studies have highlighted that the microenvironment can reprogram stromal cells and inflammatory cytokines released in the tumor microenvironment (TME), ultimately boosting the immune-suppressive properties of MSC and favoring tumor growth [5,6]. This Special Issue, entitled “Role of Mesenchymal Stromal Cells (MSC) in Cancer Progression and Cancer Therapy”, aims to further explore the fate of MSC within malignancies, encompassing the crosstalk between the stroma and the tumor as well as that between the different stromal components. A crucial phase of cancer progression is the ability of the tumor to communicate with its surroundings. To this end, Aasebø and colleagues dissected the bidirectional communication between MSC and acute myeloid leukemia (AML) cells [7]. The authors performed a proteomic analysis of AML cells isolated from 40 patients and evaluated how healthy MSC influence the proteomic pattern of cancer cells. The results showed that, overall, patient heterogeneity was maintained upon challenge with MSC; nevertheless, the authors observed a reduction in patient heterogeneity for a minority of proteins, including extracellular matrix molecules, proteases (i.e., protein modifiers) and soluble adhesion molecules, thus highlighting possible targetable pathways. In line with this study, Fallati and colleagues reviewed the ability of MSC to orchestrate a leukemia-supportive microenvironment [8]. The authors detailed the capacity of the cells to contribute to the different stages of cancer and provided an overview of the potential mechanisms of action. They described the possible contribution of MSC to leukemogenesis and the cells’ ability to generate a leukemia-permissive environment by acting directly on tumor cells and indirectly via the microenvironment. Lastly, the authors reviewed the mechanisms of chemoprotection that involve MSC and are based upon a bi-directional exchange of soluble factors (metabolites, amino acids, etc.), extracellular vesicles (EVs) and nanotube-based connections. The emergence of chemoresistance is, indeed, a major clinical problem for tumors in cases where chemotherapy remains the frontline treatment. Železnik Ramuta and colleagues reviewed 42 studies published between 2001 and 2022 evaluating the role of MSC in chemoresistance [9]. The results highlighted the existence of various mechanisms involved in this specific function of MSC, most of them affecting signaling pathways related to apoptosis and proliferation. Sentek and colleagues illustrated the relevance of the niche to interactions between endogenous lung-resident mesenchymal stem cells (LRMSC) and tumor cells [10]. They described peculiar features of “cancer-educated” LR-MSCs and discussed their potential to differentiate into CAFs and pericytes and, ultimately, favor tumor progression. Papait and colleagues acknowledged the role of CAFs in cancer development, addressing the main open questions regarding these controversial cells [11]. They clarified different aspects related to phenotype identification and subtype specification and discussed some of the most advanced technologies involved in the process. Finally, they reviewed pre-clinical and clinical attempts to target CAFs in various types of cancer. As cancer progression is also defined by stroma-to-stroma interactions, Çakır and colleagues investigated the capacity of melanoma-associated fibroblasts (MAFs) to modulate macrophage functions [12]. The authors observed that the MAFs were able to shape the functional phenotype of macrophages and elicit IL-10 secretory production in these cells via both the cyclo-oxygenase pathway and IDO, thus regulating tumor immunity. With their innate features having been unraveled, more questions will be raised concerning the safety and translational relevance of MSC in cancer. This Special Issue highlights the need for a deeper understanding of the fundamental processes that regulate MSC biology in health and disease to develop a clinically relevant therapeutic strategy that takes into consideration the recipient’s environment as a bi-directional type of communication

Effects of conditioned medium from human amniotic mesenchymal tissue cell cultures on prostate cancer cells

It has been recently demonstrated that human amniotic mesenchymal tissue cells (hAMTC) derived from term placenta inhibit lymphocyte proliferation and significantly reduce the growth of haemopoietic and non haemopoietic cancer cell lines (HeLa and Saos cells) in vitro (1). The aim of our study was to evaluate the effects of hAMTC-conditioned medium (CM) on two human prostate cancer cells lines: LNCaP, androgen responsive and well differentiated, and PC-3, androgen unresponsive and less differentiated. Cells were grown in their standard culture conditions in the absence or in the presence of various concentrations (0.001–50%) of hAMTC-CM or their own exhausted medium. Cell numbers were determined by using a haemocytometer, after three days. Moreover, E- and N-cadherin expression was evaluated in PC-3 cells cultured in medium with 0.01, 1 or 25% hAMTC-CM by Immunocytochemistry and Western blot analysis. Our findings indicate that hAMTC-CM reduces the growth of both PC-3 and LNCaP cells. The effect is more pronounced in PC-3 cells in which inhibition is about 25% vs control (p<0.001) at a very low concentration (0.001%) and reaches the maximum (about 55% vs control, p<0.001) with the highest concentration used (50%). In LNCaP cells only the highest concentration of hAMTC-CM (50%) inhibits cell proliferation (about 40% vs control, p<0.001). Interestingly, growth of LNCaP cells is reduced by their own exhausted medium, while proliferation of PC-3 cells is not affected by their spent medium. Both E- and N-cadherin expression have been detected at the membrane level in untreated PC-3 cells and the localization does not change in hAMTC-CM-treated cells. Preliminary data obtained by Western blot analysis seem to indicate an increase in both E- and N-cadherin levels. Our findings show that hAMTC-CM reduces prostate cancer cell proliferation in relationship to their androgen sensitivity and modifies the expression levels of adhesion molecules. Experiments are in progress to determine the mechanisms which underlie the observed effects and assess if hAMTC-CM can determine any variation in the differentiation status of prostate cancer cells

Immunological and Differentiation Properties of Amniotic Cells Are Retained After Immobilization in Pectin Gel

Mesenchymal stromal cells from the human amniotic membrane (i.e., human amniotic mesenchymal stromal cells [hAMSCs]) of term placenta are increasingly attracting attention for their applications in regenerative medicine. Osteochondral defects represent a major clinical problem with lifelong chronic pain and compromised quality of life. Great promise for osteochondral regeneration is held in hydrogel-based constructs that have a flexible composition and mimic the physiological structure of cartilage. Cell loading within a hydrogel represents an advantage for regenerative purposes, but the encapsulation steps can modify cell properties. As pectin gels have also been explored as cell vehicles on 3D scaffolds, the aim of this study was to explore the possibility to include hAMSCs in pectin gel. Immobilization of hAMSCs into pectin gels could expand their application in cell-based bioengineering strategies. hAMSCs were analyzed for their viability and recovery from the pectin gel and for their ability to differentiate toward the osteogenic lineage and to maintain their immunological characteristics. When treated with a purposely designed pectin/hydroxyapatite gel biocomposite, hAMSCs retained their ability to differentiate toward the osteogenic lineage, did not induce an immune response, and retained their ability to reduce T cell proliferation. Taken together, these results suggest that hAMSCs could be used in combination to pectin gels for the study of novel osteochondral regeneration strategies

Human acellular amniotic membrane implantation for lower third nasal reconstruction: a promising therapy to promote wound healing

BACKGROUND: The lower third of the nose is one of the most important cosmetic units of the face, and its reconstructive techniques remain a big challenge. As an alternative approach to repair or regenerate the nasal tissue, the biomaterial-based strategy has been extensively investigated. The aim of this study is to determine the safety and efficacy of human acellular amniotic membrane (HAAM) to repair the full-thickness defects in the lower third of the nose in humans. METHODS: In this study, 180 patients who underwent excision of skin lesions of the lower third of the nose from 2012 to 2016 were included; of the patients, 92 received HAAM and Vaseline gauze treatments, and the other 88 patients received Vaseline gauze treatment only. The haemostasis time and the duration of operation were recorded during surgery; after surgery, the time to pain disappearance, scab formation and wound healing, and the wound healing rate were measured. RESULTS: Immediately after the HAAM implantation, a reduction of the haemostasis time and an accelerated disappearance of pain were observed. Compared with the control group, the formation and detachment of scab in patients who received the HAAM implantation were notably accelerated, postoperatively. When the diameter of the lesion exceeded 5\u2009mm, the HAAM implantation was found to enhance the wound healing, although this enhancement was not seen when the diameter was less than 5\u2009mm. Additionally, the HAAM implantation significantly reduced bleeding, wound infection and scar formation, postoperatively. CONCLUSIONS: HAAM-assisted healing is a promising therapy for lower third nasal reconstruction leading to rapid wound healing and fewer complications and thus has considerable potential for extensive clinical application in repairing skin wounds. TRIAL REGISTRATION: ChiCTR1800017618, retrospectively registered on July 08, 2018

HIPGEN: a randomized, multicentre phase III study using intramuscular PLacenta-eXpanded stromal cells therapy for recovery following hip fracture arthroplasty : a study design

Aims The aim of the HIPGEN consortium is to develop the first cell therapy product for hip fracture patients using PLacental-eXpanded (PLX-PAD) stromal cells. Methods HIPGEN is a multicentre, multinational, randomized, double-blind, placebo-controlled trial. A total of 240 patients aged 60 to 90 years with low-energy femoral neck fractures (FNF) will be allocated to two arms and receive an intramuscular injection of either 150 × 106 PLX-PAD cells or placebo into the medial gluteal muscle after direct lateral implantation of total or hemi hip arthroplasty. Patients will be followed for two years. The primary endpoint is the Short Physical Performance Battery (SPPB) at week 26. Secondary and exploratory endpoints include morphological parameters (lean body mass), functional parameters (abduction and handgrip strength, symmetry in gait, weightbearing), all-cause mortality rate and patient-reported outcome measures (Lower Limb Measure, EuroQol five-dimension questionnaire). Immunological biomarker and in vitro studies will be performed to analyze the PLX-PAD mechanism of action. A sample size of 240 subjects was calculated providing 88% power for the detection of a 1 SPPB point treatment effect for a two-sided test with an α level of 5%. Conclusion The HIPGEN study assesses the efficacy, safety, and tolerability of intramuscular PLX-PAD administration for the treatment of muscle injury following arthroplasty for hip fracture. It is the first phase III study to investigate the effect of an allogeneic cell therapy on improved mobilization after hip fracture, an aspect which is in sore need of addressing for the improvement in standard of care treatment for patients with FNF