The human placenta is a transient yet crucial organ that plays a key role in sustaining the relationship between the maternal and fetal organisms. Despite its historical classification as "biowaste," placental tissues have garnered increasing attention since the early 1900s for their significant medical potential, particularly in wound repair and surgical application. As ethical considerations regarding human placental derivatives have largely been assuaged in many countries, they have gained significant attention due to their versatile applications in various biomedical fields, such as biomedical engineering, regenerative medicine, and pharmacology. Moreover, there is a substantial trend toward various animal product substitutions in laboratory research with human placental derivatives, reflecting a broader commitment to advancing ethical and sustainable research methodologies. This review provides a comprehensive examination of the current applications of human placental derivatives, explores the mechanisms behind their therapeutic effects, and outlines the future potential and directions of this rapidly advancing field.

Several immunosuppressive therapies have been proposed as key treatment options for critically ill patients since the first appearance of severe acute respiratory syndrome coronavirus 2. Mesenchymal stem cells (MSCs) from different sources have been considered for their potential to attenuate the cytokine storm associated to COVID-19 and the consequent multi-organ failure, providing evidence for safe and efficacious treatments. Among them, administration of umbilical cord-derived MSCs (UC-MSCs) has demonstrated a significant increase in survival rates, largely due to their potent immunosuppressive properties.

We applied next-generation sequencing (NGS) analysis to compare the transcriptomic profiles of MSCs isolated from two gestational sources: amniotic fluid (AF) obtained during prenatal diagnosis and their clinically relevant umbilical cord counterparts, for which datasets were publicly available. A full meta-analysis was performed to identify suitable GEO and NGS datasets for comparison between AF- and UC-MSC samples.

Transcriptome analysis revelaed significant differences between groups, despite both cell lines being strongly involved in the tissue development, crucial to achieve the complex task of wound healing. Significantly enriched hallmark genes suggest AF-MSC superior immunomodulatory features against signaling pathways actively involved in the cytokine storm (i.e., IL-2/STAT, TNF-a/NFkB, IL-2/STAT5, PI3K/AKT/mTOR).

The data presented here suggest that AF-MSCs hold significant promise for treating not only COVID-19-associated cytokine storms but also a variety of other inflammatory syndromes (i.e., those induced by bacterial infections, autoimmune disorders, and therapeutic interventions). Realizing the full potential of AF-MSCs as a comprehensive therapeutic approach in inflammatory disease management will require more extensive clinical trials and in-depth mechanistic studies.

Background: Ovarian cancer accounts for more deaths than any other cancer of the female reproductive system. Despite standard care, recurrence due to tumor spread and chemoresistance is common, highlighting the need for novel therapies. Mesenchymal stromal cells from the human amniotic membrane (hAMSC) and the intact amniotic membrane (hAM) are promising due to their secretion of tumor-modulating bioactive factors, accessibility from biological waste, and ethical favorability. Furthermore, unlike isolated cells, hAM provides an easier, clinically translatable product. We previously demonstrated that hAMSC can inhibit tumor cell proliferation, both in contact and transwell settings, suggesting that hAMSC secrete bioactive factors able to target tumor cells. This study evaluates the anti-tumor effects of bioactive factors from hAMSC and hAM conditioned medium (CM) on ovarian cancer cells in 2D and 3D models, alone or with paclitaxel. Methods: The impact of CM, alone or with paclitaxel, was tested on ovarian cancer cell proliferation, migration, invasion, and on angiogenesis. Results: hAMSC-CM and hAM-CM inhibited the proliferation and migration in 2D cultures and reduced spheroid growth and invasion in 3D models. Combining CM with paclitaxel enhanced anti-tumor effects in both settings. Conclusions: hAMSC-CM and hAM-CM show therapeutic potential against ovarian cancer, with synergistic benefits when combined with paclitaxel.

Molecular biomarker profiling is an emerging field in maternal-fetal health with the potential to transform early detection and prediction of placental dysfunction. By analysing a range of biomarkers in maternal blood, researchers and clinicians can gain crucial insights into placental health, enabling timely interventions to enhance fetal and maternal outcomes. Placental structural function is vital for fetal growth and development, and disruptions can lead to serious pregnancy complications like preeclampsia. While conventional methods such as ultrasound and Doppler velocimetry offer valuable information on fetal growth and blood flow, they have limitations in predicting placental dysfunction before clinical signs manifest. In contrast, molecular biomarker profiling can provide a more comprehensive assessment by measuring proteins, metabolites, and microRNAs (miRNAs) in maternal blood, reflecting the placenta's endocrine and metabolic functions. This approach offers a deeper understanding of placental health and function, aiding in early detection and prediction of complications. Challenges in developing molecular biomarker profiling include pinpointing specific molecular changes in the placenta linked to pathologies, timing predictions of conditions before clinical onset, and understanding how placental dysfunction affects maternal metabolism. Validating specific biomarkers and integrating them effectively into clinical practice requires further research. This review underscores the significance of molecular biomarker profiling as a powerful tool for early detection and prediction of placental dysfunction in maternal-fetal health. Through an exploration of biomarker analysis, we delve into how a deeper understanding of placental health can potentially improve outcomes for both mother and baby. Furthermore, we address the critical need to validate the utility of biomarkers and effectively integrate them into clinical practice.

Thanks to its unique biological properties, the human amniotic membrane (AM) has shown promising results for guided bone regeneration (GBR), but displays some limitations such as poor space-maintaining ability. This study thus aimed to develop a new amnion/chorion membrane (ACM), with better mechanical properties as well as comparable or improved biological properties for GBR. We first developed a new decellularization method of ACM (DL-ACM) which was validated by DNA staining and quantification, and its cytocompatibility was established in vitro. The thickness of DL-ACM was significantly increased over thirty-fivefold, and its tearing strength and compression strength significantly increased more than tenfold compared to the decellularized AM (DL-AM). In vivo, DL-ACM demonstrated its biocompatibility subcutaneously, and its osteogenic properties were compared to DL-AM and a gold standard membrane in a GBR defect model in rats. Micro-CT and histomorphometric analysis showed that DL-ACM significantly promoted early bone regeneration after 1 week and significantly increased bone regeneration compared to the empty defect and the gold standard membrane over time. In this study, we developed a simple and reproducible method to produce an acellular, non-cytotoxic, and biocompatible DL-ACM. This new membrane is as effective as AM to promote early bone regeneration while demonstrating better biomechanical properties.

Exosomes have emerged as promising therapeutic agents in regenerative medicine. This review introduces a novel cell type-oriented perspective to systematically analyze exosomal properties in regenerative therapies. To our knowledge, this review is the first to comprehensively compare exosomes based on cellular source type, offering unprecedented insights into selecting optimal exosome producers for targeted regenerative applications. Factors beyond cellular origin influencing exosomal therapeutic efficacy, such as donor sites and collection methods, are also explored here. By synthesizing key advances, we propose promising research directions in the end. We aim to accelerate the development of more effective exosome-based regenerative therapies and highlight underexplored directions in this rapidly evolving field.

Somatic Cell Nuclear Transfer (SCNT) has transformed animal genetic improvement, gene-editing in model production, xenotransplantation, and conservation efforts for endangered species. However, SCNT-derived embryos occasionally display developmental abnormalities, and following embryo transfer, the miscarriage rate is high. Gene-edited fetuses may experience birth defects, resulting in decreased survival rates. Correct selection of nuclear donor cells is essential for the success of somatic cell cloning. Fibroblasts are the most commonly used cells, but their rapid proliferation increases the risk of genetic mutation, impairing embryo development and production. Conversely, amniotic cells have slower proliferation rates, decreasing the mutation risk during cultivation. Amniotic cells are thus better SCNT candidates than fibroblasts because they offer genomic stability, low tumorigenic and teratogenic risks, reduced immunogenicity, high differentiation potential, ease of accessibility, and fewer ethical concerns. Cells derived from first-generation gene-edited animals exhibit stable genetic structures, reduced susceptibility to genetic alterations and artificial modifications, closely resembling natural cells, and enhanced compatibility with SCNT procedures. Amniotic cells derived from gene-edited sheep fetuses used as nuclear donor cells for SCNT successfully recloned three healthy second-generation gene-edited sheep. Using amniotic cells as nuclear donor cells for SCNT did not significantly alter embryo cleavage rates, blastocyst formation, or fetal birth compared to edited fibroblasts (p > 0.05). However, fetal survival rates were significantly higher than edited fibroblasts (p < 0.05). The results support the potential of amniotic cells as SCNT alternatives, suggesting a promising strategy to improve gene-edited fetus survival rates using first-generation gene-edited sheep-derived amniotic cells.

Increasing evidence shows extracellular vesicles (EVs) are primarily responsible for the beneficial effects of cell-based therapies. EVs derived from mesenchymal stromal cells (MSCs) show promise as a source of EVs for cell-free therapies. The human placental fetal-maternal interface is a rich and abundant source of MSCs from which EVs can be isolated. This study focusses on chorionic MSCs (CMSC) located on the fetal aspect of the interface and decidual MSCs (DMSC) on the maternal aspect. This study used Ligand-based Exosome Affinity Purification (LEAP) chromatography to isolate EVs from well-characterized placental hTERT-transduced CMSC29 and DMSC23 cell lines, which retain many important stem cell-like properties of primary CMSC and DMSC, respectively. After initial biophysical characterization of the EVs isolated from each cell line, the biological activities and the protein, lipid and small RNA contents of CMSC29-EVs and DMSC23-EVs were compared and assessed. LEAP-purified EVs from both sources were validated at the biophysical level by Spectradyne, Cryo-Transmission Electron Microscopy (Cryo-TEM), and Western blot analysis. EVs from each type were labelled with the live cell stain PKH26 and their in vitro uptake and internalization by human dermal fibroblast cells was assessed, as well as their phosphorylation of the protein kinase B/AKT (AKT) pathway. The protein and lipid contents were analyzed by mass spectrometry and the nucleic acid content by RNA sequencing (RNA-seq). Lastly, the biological activities of the EVs were evaluated in a BioMAP® Diversity PLUS® screen system across a panel of 12 human primary cell-based systems and in vitro cell proliferation. EVs isolated from both DMSC23 and CMSC29 significantly increased proliferation of fibroblasts and showed phosphorylation of the AKT pathway. Protein mass spectrometry analysis identified a large number of proteins including cell surface receptors, cytokines, chemokines, matrix molecules and enzymes in both EV types. Lipidomic analysis identified species including phosphatidylcholine, triacylglycerides and diacylglycerides in both DMSC23 and CMSC29-derived EVs. There were some significant differences in identified microRNAs (miRNAs) between the two EV types. The top differentially expressed miRNAs between the two EV types show pathways association with matrix interaction, transcriptional regulation, proliferation, cellular protein modification processes, and vasculogenesis. Differences were also detected between DMSC23- and CMSC29-EVs in the biological activity they displayed in the BioMAP® Diversity PLUS® screen.

Chronic wounds are a common clinical problem that necessitate the exploration of novel regenerative therapies. We report a method to investigate the in vitro wound healing capacity of an innovative biomaterial, which is based on amniotic membrane-derived stem cells (AMSCs) embedded in an alginate hydrogel matrix. The aim of this study was to prepare an sodium alginate-based hydrogel, cross-linked calcium chloride (CaCl2) with the active ingredient AMSC (AMSC/Alg-H) and to evaluate its in vitro effectiveness for wound closure.

This hydrogel preparation involved combining sterile solutions of AMSC, sodium alginate, and CaCl2, followed by rinsing with serum-free media. The cells were cultured in different 6-well plates, namely sodium alginate, calcium chloride, AMSC, Alg-H, and AMSC/Alg-H, in complete medium with 10% FBS. The hydrogel was successfully formulated, as confirmed by characterization techniques including Scanning Electron Microscopy (SEM), Fourier Transform Infrared (FTIR) spectroscopy, Differential Scanning Calorimetry (DSC), Cytotoxicity Studies, TGF-β1 Level Measurement by ELISA, and Cell Scratch Wound Assay.

Cryo-EM characterization of the Alg-H preparation successfully demonstrated the encapsulation of MSCs. FTIR and DSC analyses indicate that crosslinking transpires in Alg-H encapsulating AMSC. The AMSC/Alg-H preparation showed no significant difference in toxicity compared to HaCaT cells (p < 0.05), indicating it was not toxic to HaCaT cells. Furthermore, in the scratch wound assay test at 24 hours, the AMSC/Alg-H preparation achieved 100% wound closure, outperforming both AMSC and Alg-H alone. In vitro assessment revealed that AMSC/Alg-H significantly enhanced key wound healing processes, including cell proliferation and migration, compared to Alg-H.

Our study demonstrated the promising potential of AMSC/Alg-H as an enhanced regenerative therapy for in vitro wound healing. AMSC/Alg-H was able to maintain the viability of AMSCs and facilitate the formation of tissue-like structures.

The immunomodulatory, anti-inflammatory and regenerative properties of the human amniotic mesenchymal stromal cells (hAMSCs) secretome are acknowledged but the understanding of the specific bioactive components remains incomplete. To address these limitations, the present investigation aimed to profile the proteins and peptides content of the hAMSC secretome through sample pretreatment and fractionation on 10 kDa molecular cut-off FASP (Filter Aided Sample Preparation) device and LC-MS analysis. The filter retained protein fraction underwent trypsin digestion, while the unretained was collected unchanged for intact small proteins and peptides analysis. This combined approach (C-FASP) collects in a single step two complementary fractions, advantageously saving sample volume and time of analysis. The bottom-up analysis of the C-FASP proteins fraction >10 kDa confirmed our previous findings, establishing a set of proteins consistently characterizing the hAMSC secretome. The analysis of the fraction <10 kDa, never been investigated to our knowledge, identified peptide fragments of thymosin beta 4 and beta 10, collagen alpha 1 chains I and III, alpha-enolase, and glyceraldehyde-3-phosphate dehydrogenase, involved in wound healing, anti-inflammatory response, tissue repair and regeneration, key biological activities of the secretome. C-FASP provided a comprehensive molecular profile of the hAMSC secretome offering new insights for enhanced therapeutic applications in regenerative medicine. SIGNIFICANCE: In this investigation we originally present the comprehensive proteomic investigation of the human amniotic mesenchymal stromal cell secretome by combining the analysis of the proteome and of the peptidome following sample pretreatment and fractionation by Filter Aided Sample Preparation (FASP) with 10 kDa molecular cut-off in coupling with LC-MS analysis. The proteome fraction retained by FASP filter was analyzed after enzymatic digestion, while the unretained fraction, below 10 kDa molecular mass, was analyzed unchanged in its intact form. This dual approach provides novel insights, previously unexplored, into the molecular components potentially responsible for the immunomodulatory and anti-inflammatory properties of the hAMSC secretome. These findings could significantly enhance the therapeutic potential of hAMSCs in regenerative medicine.

Perinatal stem cells have prominent applications in cell therapy and regenerative medicine. Among them, human Wharton's jelly mesenchymal stem/stromal cells (hWJMSCs) and human amniotic epithelial stem cells (hAESCs) have been widely used. However, the distinction in the therapeutic potential of hWJMSCs and hAESCs is poorly understood. In this study, we reported the phenotypic differences between these two distinct cell types and provided the first systematic comparison of their therapeutic potential in terms of immunomodulation, extracellular matrix (ECM) remodelling, angiogenesis and antioxidative stress using proteomics. The results revealed that the two cell types presented different protein expression profiles and were both promising candidates for cell therapy. Both types of cells demonstrated angiogenic and antifibrotic potential, whereas hAESCs presented superior immunological tolerance and antioxidant properties, which were supported by a series of relevant in vitro assays. Our study provides clues for the selection of appropriate cell types for diverse indications in cell therapy, which contributes to the advancement of their clinical translation and application.

Advanced bioinformatics analysis, such as systems biology (SysBio) and artificial intelligence (AI) approaches, including machine learning (ML) and deep learning (DL), is increasingly present in stem cell (SC) research. An approximate timeline on these developments and their global impact is still lacking. We conducted a scoping review on the contribution of SysBio and AI analysis to SC research and therapy development based on literature published in PubMed between 2000 and 2024. We identified an 8 to 10-fold increase in research output related to all 3 search terms between 2000 and 2021, with a 10-fold increase in AI-related production since 2010. Use of SysBio and AI still predominates in preclinical basic research with increasing use in clinically oriented translational medicine since 2010. SysBio- and AI-related research was found all over the globe, with SysBio output led by the (US, n = 1487), (UK, n = 1094), Germany (n = 355), The Netherlands (n = 339), Russia (n = 215), and France (n = 149), while for AI-related research the US (n = 853) and UK (n = 258) take a strong lead, followed by Switzerland (n = 69), The Netherlands (n = 37), and Germany (n = 19). The US and UK are most active in SCs publications related to AI/ML and AI/DL. The prominent use of SysBio in ESC research was recently overtaken by prominent use of AI in iPSC and MSC research. This study reveals the global evolution and growing intersection among AI, SysBio, and SC research over the past 2 decades, with substantial growth in all 3 fields and exponential increases in AI-related research in the past decade.

Wound healing is a complicated process, especially when surgical, traumatic, burn, or pathological injury occurs, which requires different kinds of dressing covers including hydrogels, hydrocolloids, alginates foams and films for treatment. The human amniotic membrane (hAM) is a biodegradable extracellular matrix with unique and tailorable physicochemical and biological properties, generated by the membrane itself or other cells that are located on the membrane surface. It is noted as a promising aid for wound healing and tissue regeneration due to the release of growth factors and cytokines, and its antibacterial and immunosuppressive properties. Moreover, hAM has optimal physical, biological, and mechanical properties, which makes it a much better option as a regenerative skin treatment than existing alternative materials. In addition, this layer has a structure with different layers and cells with different functions, which act as a regenerative geometry and reservoir of bioactive substances and cells for wound healing. In the present work, the structural and biological features of hAM are introduced as well as the application of this layer in different forms of composites to enhance wound healing. Future studies are recommended to detect possible further functionalization to enhance the hAM effectiveness on wound healing.

The rotator cuff tendon-bone interface tissue exhibits high heterogeneity in its composition and structure, with collagen being its primary component. Here, we prepared tissue-engineered decellularized live hyaline cartilage grafts (dLHCG), this dLHCG scaffold's bioactive ECM mainly consists of collagen II, proteoglycans, and fibronectin, presenting a cartilage-like lacuna microstructure. The dLHCG scaffold loaded human amniotic mesenchymal stem cells (hAMSCs) and adipose stem cells (ADSCs) were implanted into the interface. The dLHCG scaffold could maintain the pluripotency of stem cells, supporting the proliferation, osteogenic differentiation, and tenogenic differentiation of the MSCs. The collagen II, through the integrin α2β1-FAK-JNK signaling axis, promotes Runx-2 activation, playing a better regulatory role in the early osteogenic differentiation of MSCs, enhancing bone defect repair through an endochondral ossification process. The in vivo rat model demonstrated that 12 weeks post-operation, the MSC-loaded dLHCG scaffold group exhibited continuous aligned collagen fibers at the tendon-bone interface, with significantly enhanced biomechanical function compared to the control group. The dLHCG scaffold create an efficient interface, which promoting the restoration of the soft-hard gradient structure tissue at the junction between the scaffold and the host tissue, thereby providing a rational and promising strategy for the rapid healing of the rotator cuff injury.

Chronic wounds are defined as those with disturbances in normal healing. They involve symptoms like exudate, odor, pain or impaired mobility, severely impacting life quality. In the case of patients with additional comorbidities, these are known to aggravate the healing impairment. Amniotic membrane (AM) is gaining attention for its regenerative potential, as it has shown promise in treating hard-to-heal wounds, such as diabetic foot ulcers. This work examines a series of five patients who, while suffering an array of other chronic conditions, were treated with AM for the management of non-healing chronic ulcers. Inclusion criteria involved patients with lesions that have been active at least for six weeks and resistant to multiple treatments, accompanied by complex underlying pathologies affecting cardiovascular, immune or renal functions. Exclusion criteria included untreated active infections and patients undergoing other experimental treatments. The mean age of the patients was 68.4 ± 5.2 years. Wounds were treated once a week with AM, following standardized procedures. The variables measured included pain levels, microorganism presence, wound reduction and the number of AM applications to recovery. The median pain VAS score decreased significantly from seven at the start to two at the end of procedures. Four out of five patients achieved complete epithelialization, while the remaining patient showed significant reductions of 40% in wound size after 14 months. Our results confirm how the application of AM is a safe and effective resource for the management of chronic wounds in patients with serious comorbidities, enhancing patients' quality of life, firstly by reducing pain, later by allowing recovery. Future research, including molecular analyses of wound exudates before and after AM treatment, can contribute to better understanding and fine tuning of this therapeutic resource.

Burn wounds require optimal medical management due to associated psycho-emotional and socioeconomic impacts and severe pain. The use of synthetic and biological dressings improves healing and reduces burn wound complications. The present study aimed to compare the outcomes of using human amniotic membrane (hAM) dressings and conventional silver sulfadiazine (SSDZ) ointment dressings in the management of second-degree burn wounds.

Fifty patients who participated in this clinical trial were divided into two groups via simple randomization. All the enrolled patients, who had burnt in the last 24 h, had thermal damage mechanisms and were suffering from less than 20% second-degree heat-burn wounds on the skin surface. The target group (n = 25) was treated with hAM, and the control group (n = 25) was treated with SSDZ ointment. The researcher-designed checklist was used to determine the clinical performance in the follow-up assessments on days 7, 14, and 30.

No significant differences were detected in terms of sex, age, or percentage of burn wounds (p > 0.05). Wound epithelialization at days 7, 14, and 30, scar formation, wound pigmentation, pain severity, analgesia requirements, and hospital stay length (on day 30) were significantly lower in the target group (treated with hAM) than in the control group (treated with SSDZ ointment) (p < 0.05). However, treatment costs in the target group ($170) were significantly higher than those in the control group ($71) (p < 0.001).

Despite its higher cost, hAM, as a technology-based therapy dressing, demonstrates superiority over SSDZ ointment in terms of wound healing and pain management.

Localized prostate cancer (PCa) is one of the most common malignancies in the United States. Despite continued refinement of robot assisted radical prostatectomy (RARP) surgical methods, post-surgical erectile dysfunction and urinary incontinence remain significant challenges due to iatrogenic injury of local nervous tissue. Thus, the development of therapeutic strategies, including the use of biologic adjuncts to protect and/or enhance recovery and function of nerves following RARP is of growing interest. Perinatal tissue allografts have been investigated as one such biologic adjunct to nerve sparing RARP. However, knowledge regarding their clinical efficacy in hastening return of potency and continence as well as the potential underpinning biological mechanisms involved remains understudied. Thus, the objective of this literature review was to summarize published basic science and clinical studies supporting and evaluating the use of perinatal allografts for nerve repair and their clinical efficacy as adjuncts to RARP, respectively.

The literature as of May 2024 was reviewed non-systematically using PubMed, EMBASE, Scopus, and Web of Science databases. The search terms utilized were "robotic prostatectomy", "prostate cancer", "nerve sparing", "perinatal tissue", "allograft", "potency", and "continence" alone or in combination. All articles were reviewed and judged for scientific merit by authors RP and JM, only peer-reviewed studies were considered.

Eight studies of perinatal tissue allograph use in RARP were deemed worthy of inclusion in this nonsystematic review.

Incontinence and impotence remain significant comorbidities despite continued advancement in surgical technique. However, basic science research has demonstrated potential neurotrophic, anti-fibrotic, and anti-inflammatory properties of perinatal tissue allografts, and clinical studies have shown that patients who receive an intra-operative prostatic perinatal membrane wrap have faster return to potency and continence.

Human amniotic membrane (hAM), the innermost placental layer, has unique properties that allow for a multitude of clinical applications. It is a common misconception that birth-derived tissue products, such as dual-layered dehydrated amnion-amnion graft (dHAAM), are similar regardless of the manufacturing steps. A commercial dHAAM product, Axolotl Biologix DualGraft™, was assessed for biological and mechanical characteristics. Testing of dHAAM included antimicrobial, cellular biocompatibility, proteomics analysis, suture strength, and tensile, shear, and compressive modulus testing. Results demonstrated that the membrane can be a scaffold for fibroblast growth (cellular biocompatibility), containing an average total of 7678 unique proteins, 82,296 peptides, and 96,808 peptide ion variants that may be antimicrobial. Suture strength results showed an average pull force of 0.2 N per dHAAM sample (equating to a pull strength of 8.5 MPa). Tensile modulus data revealed variation, with wet samples showing 5× lower stiffness than dry samples. The compressive modulus and shear modulus displayed differences between donors (lots). This study emphasizes the need for standardized processing protocols to ensure consistency across dHAAM products and future research to explore comparative analysis with other amniotic membrane products. These findings provide baseline data supporting the potential of amniotic membranes in clinical applications.

Human amniotic mesenchymal stromal cells (hAMSCs) have unique immunomodulatory properties making them attractive candidates for regenerative applications in inflammatory diseases. Most of their beneficial properties are mediated through their secretome. The bioactive factors concurring to its therapeutic activity are still unknown. Evidence suggests synergy between the two main components of the secretome, soluble factors and vesicular fractions, pivotal in shifting inflammation and promoting self-healing. Biological variability and the absence of quality control (QC) protocols hinder secretome-based therapy translation to clinical applications. Moreover, vesicular secretome contains a multitude of particles with varying size, cargos and functions whose complexity hinders full characterization and comprehension. This study achieved a significant advancement in secretome characterization by utilizing native, FFF-based separation and characterizing extracellular vesicles derived from hAMSCs. This was accomplished by obtaining dimensionally homogeneous fractions then characterized based on their protein content, potentially enabling the identification of subpopulations with diverse functionalities. This method proved to be successful as an independent technique for secretome profiling, with the potential to contribute to the standardization of a qualitative method. Additionally, it served as a preparative separation tool, streamlining populations before ELISA and LC-MS characterization. This approach facilitated the categorization of distinctive and recurring proteins, along with the identification of clusters associated with vesicle activity and functions. However, the presence of proteins unique to each fraction obtained through the FFF separation tool presents a challenge for further analysis of the protein content within these cargoes.

The intricate interplay between the developing placenta and fetal-maternal interactions is critical for pregnancy outcomes. Despite advancements, gaps persist in understanding biomechanics, transport processes, and blood circulation parameters, all of which are crucial for safe pregnancies. Moreover, the complexity of fetal-maternal interactions led to conflicting data and methodological variations. This review presents a comprehensive overview of current knowledge on fetal-maternal interface structures, with a particular focus on the first trimester. More in detail, the embryological development, structural characteristics, and physiological functions of placental chorionic plate and villi, fetal membranes and umbilical cord are discussed. Furthermore, a description of the main structures and features of maternal and fetal fluid dynamic exchanges is provided. However, ethical constraints and technological limitations pose still challenges to studying early placental development directly, which calls for sophisticated in vitro, microfluidic organotypic models for advancing our understanding. For this, knowledge about key in vivo parameters are necessary for their design. In this scenario, the integration of data from later gestational stages and mathematical/computational simulations have proven to be useful tools. Notwithstanding, further research into cellular and molecular mechanisms at the fetal-maternal interface is essential for enhancing prenatal care and improving maternal and fetal health outcomes.

Vascular tissue engineering is a promising approach for regenerating damaged blood vessels and developing new therapeutic approaches for heart disease treatment. To date, different sources of cells have been recognized that offer assistance within the recovery of heart supply routes and veins with distinctive capacities and are compelling for heart regeneration. However, some challenges still remain that need to be overcome to establish the full potential application of these cells. In this paper, we review the different cell sources used for vascular tissue engineering, focusing on extraembryonic tissue-derived cells (ESCs), and elucidate their roles in cardiovascular disease. In addition, we highlight the intricate interplay between mechanical and biochemical factors in regulating mesenchymal stem cell (MSC) differentiation, offering insights into optimizing their application in vascular tissues.

Mesenchymal stromal cell (MSC)-based advanced therapy medicinal products (ATMPs) are being tried in a vast range of clinical applications. These cells can be isolated from different donor tissues by using several methods, or they can even be derived from induced pluripotent stem cells or embryonic stem cells. However, ATMP heterogeneity may impact product identity and potency, and, consequently, clinical trial outcomes. In this review, we discuss these topics and the need to establish minimal criteria regarding the manufacturing of MSCs so that these innovative therapeutics may be better positioned to contribute to the advancement of regenerative medicine.

Mesenchymal stromal/stem cells (MSCs) are a heterogeneous population of multipotent cells that can be obtained from various tissues, such as dental pulp, adipose tissue, bone marrow and placenta. MSCs have gained importance in the field of regenerative medicine because of their promising role in cell therapy and their regulatory abilities in tissue repair and regeneration. However, a better characterization of these cells and their products is necessary to further potentiate their clinical application. In this study, we used unbiased high-resolution mass spectrometry-based proteomic analysis to investigate the impact of distinct priming strategies, such as hypoxia and IFN-γ treatment, on the composition and therapeutic functionality of the secretome produced by MSCs derived from the amniotic membrane of the human placenta (hAMSCs). Our investigation revealed that both types of priming improved the therapeutic efficacy of hAMSCs, and these improvements were related to the secretion of functional factors present in the conditioned medium (CM) and exosomes (EXOs), which play crucial roles in mediating the paracrine effects of MSCs. In particular, hypoxia was able to induce a pro-angiogenic, innate immune response-activating, and tissue-regenerative hAMSC phenotype, as highlighted by the elevated production of regulatory factors such as VEGFA, PDGFRB, ANGPTL4, ENG, GRO-γ, IL8, and GRO-α. IFN-γ priming, instead, led to an immunosuppressive profile in hAMSCs, as indicated by increased levels of TGFB1, ANXA1, THBS1, HOMER2, GRN, TOLLIP and MCP-1. Functional assays validated the increased angiogenic properties of hypoxic hAMSCs and the enhanced immunosuppressive activity of IFN-γ-treated hAMSCs. This study extends beyond the direct priming effects on hAMSCs, demonstrating that hypoxia and IFN-γ can influence the functional characteristics of hAMSC-derived secretomes, which, in turn, orchestrate the production of functional factors by peripheral blood cells. This research provides valuable insights into the optimization of MSC-based therapies by systematically assessing and comparing the priming type-specific functional features of hAMSCs. These findings highlight new strategies for enhancing the therapeutic efficacy of MSCs, particularly in the context of multifactorial diseases, paving the way for the use of hAMSC-derived products in clinical practice.

Biological aging is defined as a progressive decline in tissue function that eventually results in cell death. Accelerated biologic aging results when the telomere length is shortened prematurely secondary to damage from biological or environmental stressors, leading to a defective reparative mechanism. Stem cells therapy may have a potential role in influencing (counteract/ameliorate) biological aging and maintaining the function of the organism. Mesenchymal stem cells, also called mesenchymal stromal cells (MSCs) are multipotent stem cells of mesodermal origin that can differentiate into other types of cells, such as adipocytes, chondrocytes, and osteocytes. MSCs influence resident cells through the secretion of paracrine bioactive components such as cytokines and extracellular vesicles (EVs). This review examines the changes in telomere length, cellular senescence, and normal biological age, as well as the factors contributing to telomere shortening and accelerated biological aging. The role of MSCs-especially those derived from gestational tissues-in prevention of telomere shortening (TS) and accelerated biological aging is explored. In addition, the strategies to prevent MSC senescence and improve the antiaging therapeutic application of MSCs and MSC-derived EVs in influencing telomere length and cellular senescence are reviewed.

Regenerative medicine harnesses stem cells' capacity to restore damaged tissues and organs. In vitro methods employing specific bioactive molecules, such as growth factors, bio-inductive scaffolds, 3D cultures, co-cultures, and mechanical stimuli, steer stem cells toward the desired differentiation pathways, mimicking their natural development. Chondrogenesis presents a challenge for regenerative medicine. This intricate process involves precise modulation of chondro-related transcription factors and pathways, critical for generating cartilage. Cartilage damage disrupts this process, impeding proper tissue healing due to its unique mechanical and anatomical characteristics. Consequently, the resultant tissue often forms fibrocartilage, which lacks adequate mechanical properties, posing a significant hurdle for effective regeneration. This review comprehensively explores studies showcasing the potential of amniotic mesenchymal stem cells (AMSCs) and amniotic epithelial cells (AECs) in chondrogenic differentiation. These cells exhibit innate characteristics that position them as promising candidates for regenerative medicine. Their capacity to differentiate toward chondrocytes offers a pathway for developing effective regenerative protocols. Understanding and leveraging the innate properties of AMSCs and AECs hold promise in addressing the challenges associated with cartilage repair, potentially offering superior outcomes in tissue regeneration.

Endothelial barrier disruption of human pulmonary vascular endothelial cells (HPVECs) is an important pathogenic factor for acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Mesenchymal stem cells-exosome (MSCs-Exo) represents an ideal carrier for cell-free therapy. The therapeutic implication and underlying mechanism of human placental MSCs-Exo (HPMSCs-Exo) in ALI/ARDS need to be further explored.

HPMSCs-Exo was extracted from HPMSCs and characterized. Then, the therapeutic effects of exosomes were evaluated in ALI mice and HPVECs. RNA-sequencing was applied to reveal the miRNA profile of HPMSCs-Exo and differentially expressed genes (DEGs) in HPMSCs-Exo-pretreated HPVECs. The targets of miRNAs were predicted by bioinformatics methods and correlated to DEGs. Finally, the role of hsa-miR-148a-3p/ROCK1 pathway in HPVECs has been further discussed.

The results showed that HPMSCs-Exo could downregulate Rho-associated coiled-coil-containing protein kinase 1 (ROCK1), upregulate the expression of zonula occludens-1 (ZO-1) and F-actin, promote HPVECs migration and tube formation, reduce cytoskeletal disorders and cell permeability, and thus improve ALI/ARDS. RNA-sequencing revealed the DEGs were mainly enriched in cell junction, angiogenesis, inflammation, and energy metabolism. HPMSCs-Exo contains multiple miRNAs which are associated with cytoskeletal function; the expression abundance of hsa-miR-148a-3p is the highest. Bioinformatic analysis identified ROCK1 as a target of hsa-miR-148a-3p. The overexpression of hsa-miR-148a-3p in HPMSCs-Exo promoted the migration and tube formation of HPVECs and reduced ROCK1 expression. However, the overexpression of ROCK1 on HPVECs reduced the therapeutic effect of HPMSCs-Exo.

HPMSCs-Exo represents a protective regimen against endothelial barrier disruption of HPVECs in ALI/ARDS, and the hsa-miR-148a-3p/ROCK1 pathway plays an important role in this therapeutics implication.

Myocardial infarction (MI) is a leading cause of death worldwide, resulting in extensive loss of cardiomyocytes and subsequent heart failure. Inducing cardiac differentiation of stem cells is a potential approach for myocardial regeneration therapy to improve post-MI prognosis. Mesenchymal stem cells (MSCs) have several advantages, including immune privilege and multipotent differentiation potential. This study aimed to explore the feasibility of chemically inducing human amniotic membrane MSCs (hAMSCs) to differentiate into cardiomyocytes in vitro. Human amniotic membrane (AM) samples were obtained from routine cesarean sections at Far Eastern Memorial Hospital. The isolated cells exhibited spindle-shaped morphology and expressed surface antigens CD73, CD90, CD105, and CD44, while lacking expression of CD19, CD11b, CD19, CD45, and HLA-DR. The SSEA-1, SSEA-3, and SSEA-4 markers were also positive, and the cells displayed the ability for tri-lineage differentiation into adipocytes, chondrocytes, and osteoblasts. The expression levels of MLC2v, Nkx2.5, and MyoD were analyzed using qPCR after applying various protocols for chemical induction, including BMP4, ActivinA, 5-azacytidine, CHIR99021, and IWP2 on hAMSCs. The group treated with 5 ng/ml BMP4, 10 ng/ml Activin A, 10 μM 5-azacytidine, 7.5 μM CHIR99021, and 5 μM IWP 2 expressed the highest levels of these genes. Furthermore, immunofluorescence staining demonstrated the expression of α-actinin and Troponin T in this group. In conclusion, this study demonstrated that hAMSCs can be chemically induced to differentiate into cardiomyocyte-like cells in vitro. However, to improve the functionality of the differentiated cells, further investigation of inductive protocols and regimens is needed.

Among neurological diseases, multiple sclerosis (MS) affects mostly young adults and can cause long-term disability. While most medications with approval from regulatory agencies are very effective in treating MS disease, they are unable to repair the tissue damage found in the central nervous system (CNS). Consequently, Cell-based therapy particularly using mesenchymal stem/stromal cells (MSCs), holds promise for neuroprotection and tissue repair in MS treatment. Furthermore, placenta-derived MSCs (PLMSCs) have shown the potential to treat MS due to their abundance, noninvasive isolation from discarded tissues, no ethical problems, anti-inflammatory, and reparative properties. Accordingly, good manufacturing practices (GMPs) plays a crucial part in clinical SCs manufacturing. The purpose of our article is to discuss GMP-grade PLMSC protocols for treating MS as well as other clinical applications.

Placental tissue obtained of a healthy donor during the caesarean delivery and PLMSCs isolated by GMP standards. Flow cytometry was used to assess the expression of the CD markers CD34, CD105, CD90, and CD73 in the MSCs and the mesodermal differentiation ability was evaluated. Furthermore, Genetic evaluation of PLMSCs was done by G-banded karyotyping and revealed no chromosomal instability. In spite of the anatomical origin of the starting material, PLMSCs using this method of culture were maternal in origin.

We hope that our protocol for clinical manufacturing of PLMSCs according to GMP standards will assist researchers in isolating MSCs from placental tissue for clinical and pre-clinical applications.

Amniotic membrane (AM), the innermost layer of the placenta, is an exceptionally effective biomaterial with divers applications in clinical medicine. It possesses various biological functions, including scar reduction, anti-inflammatory properties, support for epithelialization, as well as anti-microbial, anti-fibrotic and angio-modulatory effects. Furthermore, its abundant availability, cost-effectiveness, and ethical acceptability make it a compelling biomaterial in the field of medicine. Given the potential unavailability of fresh tissue when needed, the preservation of AM is crucial to ensure a readily accessible and continuous supply for clinical use. However, preserving the properties of AM presents a significant challenge. Therefore, the establishment of standardized protocols for the collection and preservation of AM is vital to ensure optimal tissue quality and enhance patient safety. Various preservation methods, such as cryopreservation, lyophilization, and air-drying, have been employed over the years. However, identifying a preservation method that effectively safeguards AM properties remains an ongoing endeavor. This article aims to review and discuss different sterilization and preservation procedures for AM, as well as their impacts on its histological, physical, and biochemical characteristics.

Isolation of tissue-specific fetal stem cells and derivation of primary organoids is limited to samples obtained from termination of pregnancies, hampering prenatal investigation of fetal development and congenital diseases. Therefore, new patient-specific in vitro models are needed. To this aim, isolation and expansion of fetal stem cells during pregnancy, without the need for tissue samples or reprogramming, would be advantageous. Amniotic fluid (AF) is a source of cells from multiple developing organs. Using single-cell analysis, we characterized the cellular identities present in human AF. We identified and isolated viable epithelial stem/progenitor cells of fetal gastrointestinal, renal and pulmonary origin. Upon culture, these cells formed clonal epithelial organoids, manifesting small intestine, kidney tubule and lung identity. AF organoids exhibit transcriptomic, protein expression and functional features of their tissue of origin. With relevance for prenatal disease modeling, we derived lung organoids from AF and tracheal fluid cells of congenital diaphragmatic hernia fetuses, recapitulating some features of the disease. AF organoids are derived in a timeline compatible with prenatal intervention, potentially allowing investigation of therapeutic tools and regenerative medicine strategies personalized to the fetus at clinically relevant developmental stages.

Early gene therapy studies held great promise for the cure of heritable diseases, but the occurrence of various genotoxic events led to a pause in clinical trials and a more guarded approach to progress. Recent advances in genetic engineering technologies have reignited interest, leading to the approval of the first gene therapy product targeting genetic mutations in 2017. Gene therapy (GT) can be delivered either in vivo or ex vivo. An ex vivo approach to gene therapy is advantageous, as it allows for the characterization of the gene-modified cells and the selection of desired properties before patient administration. Autologous cells can also be used during this process which eliminates the possibility of immune rejection. This review highlights the various stages of ex vivo gene therapy, current research developments that have increased the efficiency and safety of this process, and a comprehensive summary of Human Immunodeficiency Virus (HIV) gene therapy studies, the majority of which have employed the ex vivo approach.

In recent years, the amnion (AM) has emerged as a versatile tool for stimulating tissue regeneration and has been of immense interest for clinical applications. AM is an abundant and cost-effective tissue source that does not face strict ethical issues for biomedical applications. The outstanding biological attributes of AM, including side-dependent angiogenesis, low immunogenicity, anti-inflammatory, anti-fibrotic, and antibacterial properties facilitate its usage for tissue engineering and regenerative medicine. However, the clinical usage of thin AM sheets is accompanied by some limitations, such as handling without folding or tearing and the necessity for sutures to keep the material over the wound, which requires additional considerations. Therefore, processing the decellularized AM (dAM) tissue into a temperature-sensitive hydrogel has expanded its processability and applicability as an injectable hydrogel for minimally invasive therapies and a source of bioink for the fabrication of biomimetic tissue constructs by recapitulating desired biochemical cues or pre-defined architectural design. This article reviews the multi-functionality of dAM hydrogels for various biomedical applications, including skin repair, heart treatment, cartilage regeneration, endometrium regeneration, vascular graft, dental pulp regeneration, and cell culture/carrier platform. Not only recent and cutting-edge research is reviewed but also available commercial products are introduced and their main features and shortcomings are elaborated. Besides the great potential of AM-derived hydrogels for regenerative therapy, intensive interdisciplinary studies are still required to modify their mechanical and biological properties in order to broaden their therapeutic benefits and biomedical applications. Employing additive manufacturing techniques (e.g., bioprinting), nanotechnology approaches (e.g., inclusion of various bioactive nanoparticles), and biochemical alterations (e.g., modification of dAM matrix with photo-sensitive molecules) are of particular interest. This review article aims to discuss the current function of dAM hydrogels for the repair of target tissues and identifies innovative methods for broadening their potential applications for nanomedicine and healthcare.

The therapeutic effect of mesenchymal stromal cells (MSCs) has been described for a variety of disorders, including those affecting musculoskeletal tissues. In this context, the literature reports several data about the regenerative effectiveness of MSCs derived from bone marrow, adipose tissue, and an amniotic membrane (BMSCs, ASCs, and hAMSCs, respectively), either when expanded or when acting as clinical-grade biologic pillars of products used at the point of care. To date, there is no evidence about the superiority of one source over the others from a clinical perspective. Therefore, a reliable characterization of the tissue-specific MSC types is mandatory to identify the most effective treatment, especially when tailored to the target disease. Because molecular characterization is a crucial parameter for cell definition, the need for reliable normalizers as housekeeping genes (HKGs) is essential. In this report, the stability levels of five commonly used HKGs (ACTB, EF1A, GAPDH, RPLP0, and TBP) were sifted into BMSCs, ASCs, and hAMSCs. Adult and fetal/neonatal MSCs showed opposite HKG stability rankings. Moreover, by analyzing MSC types side-by-side, comparison-specific HKGs emerged. The effect of less performant HKG normalization was also demonstrated in genes coding for factors potentially involved in and predicting MSC therapeutic activity for osteoarthritis as a model musculoskeletal disorder, where the choice of the most appropriate normalizer had a higher impact on the donors rather than cell populations when compared side-by-side. In conclusion, this work confirms HKG source-specificity for MSCs and suggests the need for cell-type specific normalizers for cell source or condition-tailored gene expression studies.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related morbidity and mortality worldwide. Current therapeutic approaches suffer significant side effects and lack of clear understanding of their molecular targets. Recent studies reported the anticancer effects, immunomodulatory properties, and antiangiogenic effects of the human amniotic membrane (hAM). hAM is a transparent protective membrane that surrounds the fetus. Preclinical studies showed pro-apoptotic and antiproliferative properties of hAM treatment on cancer cells. Herein, we present the latest findings of the application of the hAM in combating HCC tumorigenesis and the underlying molecular pathogenies and the role of transforming growth factor-beta (TGFβ), P53, WNT/beta-catenin, and PI3K/AKT pathways. The emerging clinical applications of hAM in cancer therapy provide evidence for its diverse and unique features and suitability for the management of a wide range of pathological conditions.