Editorial: Tissue Repair and Regenerative Mechanisms by Stem/Progenitor Cells and Their Secretome

Tissue Repair and Regenerative Mechanisms by Stem/Progenitor Cells and Their Secretome Regenerative medicine is a branch of translational research which is energizing and empowering clinical practice. A multitude of novel approaches proposed during the recent years is slowly but dramatically transforming the health care system, harnessing the power of repairing, replacing, restoring, and regenerating human organs and tissues affected by various degenerative disorders and diseases. During the twentieth century, hundreds of thousands of patients with end-stage diseases have been rescued by solid organ transplants as ultimate treatment option. In the past 3 decades, cell-based therapies have been gaining importance since they can contribute to regeneration of failing organs or damaged tissues by direct replacement of the lost cells or by facilitating the body’s natural regenerative processes by removing roadblocks. A growing armamentarium of therapeutic options, spanning from bioartificial organs and tissues, stem and progenitor cells, biomaterials, cell secretome, and extracellular vesicles have become available as medical treatments substituting the standard pharmaceutics. Examples of “classical” cellular therapies are peripheral blood stem cell or stromal cell transplantations, and more recently, allogenic hepatocyte or pancreatic islet transplants. While pancreas transplantation remains the gold standard in diabetes patients where the insulin injection fails to control symptoms, transplantation of islets of Langerhans has been recognized as a successful cell-based treatment in type 1 diabetes. The mechano-enzymatic separation of endocrine tissue from the exocrine pancreatic parenchyma required several decades to become a standardized clinical approach approved by Medical Product Agencies worldwide

Immunomodulatory properties of extracellular vesicles isolated from bone marrow of patients with neuroblastoma: role of PD-L1 and HLA-G

IntroductionExtracellular vesicles (EVs) can be released by any cell and are crucial for cell-to-cell communications. EVs have been characterized in patients with solid and hematological tumors, where they play an important role in tumor progression and metastasis. EVs may express different surface proteins derived from the parental cells, including immunomodulatory molecules, such as HLA-G and PDL1.MethodsWe isolated EV from bone marrow (BM) samples of patients with Neuroblastoma (NB) and healthy controls and we analyzed the expression of CD56, GD2 and immune checkpoints on EV by flow cytometry. Next, we analyzed the function of T cells in vitro in the presence or absence of NB patients' BM-derived EV, in terms of proliferation and cytokine production. Finally, we analyzed the correlation between the expression of immune checkpoints on EV and the clinical outcome of patients.ResultsWe found a higher expression of CD56 on EVs derived from BM of patients with NB than in those from healthy donors (HD). However, CD56 expression was not dependent on BM infiltration of NB cells. Moreover, the analysis of GD2 expression revealed that only a small fraction of EVs was released by infiltrating NB cells, whereas the majority may derive from BM-resident cells. BM-derived EVs from NB patients display a higher expression of HLA-G and PD-L1 than those derived from HD. Nonetheless, such EVs are able to modulate T cell immune responses. We measured a robust response, in vitro, towards a common bacterial antigen, including the release of GM-CSF and proinflammatory cytokines, like IFN-a and IL-6, from mononuclear cells. Some of these immunomodulatory features are dependent on the expression of HLA-G and PD-L1, whereas others may rely on other mechanism(s). Finally, a high expression of CD56, HLA-G and PD-L1 on BM-derived EVs may represent a good prognostic factor.ConclusionsWe described the presence of HLA-G and PDL1-bearing EVs in the BM of NB patients, which may represent a mechanism performed by resident BM cells to counteract the inflammation occurring in the BM microenvironment of NB patients

Standard Protocols for Characterising Primary and In Vitro-Generated Human Hepatocytes

Hepatocyte-like cells (HLCs) derived from pluripotent stem cells (PSCs) or direct reprogramming are an unlimited source of human hepatocytes for biomedical applications. HLCs are used to model human diseases, develop precise drugs and establish groundbreaking regenerative cell-based therapies. Primary human hepatocytes are the gold standard for studying human liver biology and pathology. However, their widespread use is limited by their rapid dedifferentiation in vitro, reliance on transplant-rejected donor organs, poor scalability and significant batch-to-batch variations. Therefore, high-quality 'off-the-shelf' HLCs are needed to overcome those limitations. Basic stepwise differentiation protocols have been developed to generate HLCs from PSCs. To evaluate the quality of the in vitro generated products, HLCs have been phenotyped using various methods. This review discusses various biological assays and methods available for the robust evaluation of HLC quality, emphasising the importance of using 24-h cultured primary human hepatocytes (PHHs) as a reference standard for comparison.</p

Application of Perinatal Derivatives on Oncological Preclinical Models: A Review of Animal Studies.

The increasing cancer incidence has certified oncological management as one of the most critical challenges for the coming decades. New anticancer strategies are still needed, despite the significant advances brought to the forefront in the last decades. The most recent, promising therapeutic approaches have benefitted from the application of human perinatal derivatives (PnD), biological mediators with proven benefits in several fields beyond oncology. To elucidate preclinical results and clinic outcomes achieved in the oncological field, we present a narrative review of the studies resorting to animal models to assess specific outcomes of PnD products. Recent preclinical evidence points to promising anticancer effects offered by PnD mediators isolated from the placenta, amniotic membrane, amniotic fluid, and umbilical cord. Described effects include tumorigenesis prevention, uncontrolled growth or regrowth inhibition, tumor homing ability, and adequate cell-based delivery capacity. Furthermore, PnD treatments have been described as supportive of chemotherapy and radiological therapies, particularly when resistance has been reported. However, opposite effects of PnD products have also been observed, offering support and trophic effect to malignant cells. Such paradoxical and dichotomous roles need to be intensively investigated. Current hypotheses identify as explanatory some critical factors, such as the type of the PnD biological products used or the manufacturing procedure to prepare the tissue/cellular treatment, the experimental design (including human-relevant animal models), and intrinsic pathophysiological characteristics. The effective and safe translation of PnD treatments to clinical practice relies on the collaborative efforts of all researchers working with human-relevant oncological preclinical models. However, it requires proper guidelines and consensus compiled by experts and health workers who accurately describe the methodology of tissue collection, PnD isolation, manufacturing, preservation, and delivery to the final user

Bone marrow-derived extracellular vesicles from multiple myeloma patients promote adaptive immune dysfunction via HLA-G, PD-1, and PD-L1

IntroductionExtracellular vesicles (EVs) are critical mediators of intercellular communication and contribute to cancer progression and immune regulation.MethodsWe characterized EVs isolated from bone marrow (BM) plasma harvested from healthy donors and patients affected by Multiple Myeloma (MM) by Nano Tracking Analysis and by flow cytometry.ResultsEVs from MM patients were significantly more abundant and enriched in CD138, supporting their partial origin from malignant plasma cells, with additional input from BM resident cells, including monocytes and NK cells. Phenotypic profiling revealed increased expression of immune checkpoint molecules HLA-G, PD-1, and PD-L1 on MM-derived EVs compared to healthy controls. Functionally, MM-EVs suppressed Staphylococcal enterotoxin B (SEB)-induced T cell activation, as evidenced by reduced IFN-γ production and CD4+ T cell proliferation. Such effects were partially reversed by HLA-G blockade. Moreover, MM-derived EVs modulated cytokine secretion profiles suppressing IL-2, IFN-α, TNF-α, and IL-6, and enhancing GM-CSF, with some changes attributed to HLA-G and PD-L1 activity. Transcriptomic analysis showed higher HLA-G expression in patients with gain of chromosome 1q, suggesting a link between high-risk cytogenetics and EV-driven immune suppression. While clinical correlations were not observed, likely due to limited sample size, these findings underscore the immunosuppressive role of MM-derived EVs.DiscussionHLA-G+, PD-1+, and PD-L1+ EVs contribute to immune dysfunction in MM and represent promising targets to restore anti-tumor immunity

General consensus on multimodal functions and validation analysis of perinatal derivatives for regenerative medicine applications.

Perinatal tissues, such as placenta and umbilical cord contain a variety of somatic stem cell types, spanning from the largely used hematopoietic stem and progenitor cells to the most recently described broadly multipotent epithelial and stromal cells. As perinatal derivatives (PnD), several of these cell types and related products provide an interesting regenerative potential for a variety of diseases. Within COST SPRINT Action, we continue our review series, revising and summarizing the modalities of action and proposed medical approaches using PnD products: cells, secretome, extracellular vesicles, and decellularized tissues. Focusing on the brain, bone, skeletal muscle, heart, intestinal, liver, and lung pathologies, we discuss the importance of potency testing in validating PnD therapeutics, and critically evaluate the concept of PnD application in the field of tissue regeneration. Hereby we aim to shed light on the actual therapeutic properties of PnD, with an open eye for future clinical application. This review is part of a quadrinomial series on functional/potency assays for validation of PnD, spanning biological functions, such as immunomodulation, anti-microbial/anti-cancer, anti-inflammation, wound healing, angiogenesis, and regeneration

Role of Chromatin Structural Changes in Regulating Human CYP3A Ontogeny

Non-standard abbreviations: bp(s), base pair(s); C/EBP, CCAAT/enhancer binding protein; ChIP, chromatin immunoprecipitation; CLEM4, constitutive liver enhancer module 4; Cq, quantification cycle; DME, drug metabolizing enzyme; HNF4α, hepatocyte nuclear factor 4 alpha; PXR, pregnane X receptor; qPCR, quantitative polymerase chain reaction; TBP, TATAbox binding protein; TFIID, transcription factor II D; TSS, transcription start site; USF1, upstream stimulatory factor 1; XREM, xenobiotic-response enhancer module DMD #69344 3 Abstract Variability in drug metabolizing enzyme developmental trajectories contributes to interindividual differences in susceptibility to chemical toxicity and adverse drug reactions, particularly in the first years of life. Factors linked to these interindividual differences are largely unknown, but molecular mechanisms regulating ontogeny are likely involved. To evaluate chromatin structure dynamics as a likely contributing mechanism, age-dependent changes in modified and variant histone occupancy were evaluated within known CYP3A4 and 3A7 regulatory domains. Chromatin immunoprecipitation using fetal or postnatal human hepatocyte chromatin pools followed by quantitative polymerase chain reaction DNA amplification was used to determine relative chromatin occupancy by modified and variant histones. Chromatin structure representing a poised transcriptional state (bivalent chromatin), indicated by the occupancy by modified histones associated with both active and repressed transcription, was observed for CYP3A4 and most 3A7 regulatory regions in both postnatal and fetal livers. However, the CYP3A4 regulatory regions had significantly greater occupancy by modified histones associated with repressed transcription in the fetal liver. Conversely, some modified histones associated with active transcription exhibited greater occupancy in the postnatal liver. CYP3A7 regulatory regions also had significantly greater occupancy by modified histones associated with repressed transcription in the fetus. The occupancy by modified histones observed is consistent with chromatin structural dynamics contributing to CYP3A4 ontogeny, although the data is less conclusive regarding CYP3A7. Interpretation of the latter data may be confounded by cell-type heterogeneity in the fetal liver. DMD #69344

Expert Consideration on Regulatory Aspects for Perinatal Derivatives in Clinical Settings.

Perinatal derivatives (PnD) are drawing growing interest among the scientific community as an unrestricted source of multipotent stem cells, secretome, and biological matrices. They are useful for the treatment of diseases that currently have limited or no effective therapeutic options, but they require the development of regenerative approaches. With this development, the question of regulation of donation, processing, and distribution has therefore become more important. Within the European Cooperation in Science and Technology (COST) community, we compiled a group of international experts on PnD technologies, who revised and compared existing EU national regulations. Notably, despite clear European directives, each EU Country has developed their own implementation and standard levels for cell- and tissue-based therapies. To enable extended applications of PnD treatments within the EU community and worldwide, harmonization is highly recommended. This paper aims to provide an overview of the various options available to introduce PnD into clinical practice. For this purpose, the different aspects resulting from (1) the type of PnD, (2) the amount of available data, (3) the degree of manipulation, and (4) the intended application and the process toward a possible commercialization will be presented. In the future, it will be important to find a balance between regulatory requirements and the best medical quality of the PnD product

Correction of a urea cycle defect after ex vivo gene editing of human hepatocytes

Ornithine transcarbamylase deficiency (OTCD) is a monogenic disease of ammonia metabolism in hepatocytes. Severe disease is frequently treated by orthotopic liver transplantation. An attractive approach is the correction of a patient's own cells to regenerate the liver with gene-repaired hepatocytes. This study investigates the efficacy and safety of ex vivo correction of primary human hepatocytes. Hepatocytes isolated from an OTCD patient were genetically corrected ex vivo, through the deletion of a mutant intronic splicing site achieving editing efficiencies >60% and the restoration of the urea cycle in vitro. The corrected hepatocytes were transplanted into the liver of FRGN mice and repopulated to high levels (>80%). Animals transplanted and liver repopulated with genetically edited patient hepatocytes displayed normal ammonia, enhanced clearance of an ammonia challenge and OTC enzyme activity, as well as lower urinary orotic acid when compared to mice repopulated with unedited patient hepatocytes. Gene expression was shown to be similar between mice transplanted with unedited or edited patient hepatocytes. Finally, a genome-wide screening by performing CIRCLE-seq and deep sequencing of >70 potential off-targets revealed no unspecific editing. Overall analysis of disease phenotype, gene expression, and possible off-target editing indicated that the gene editing of a severe genetic liver disease was safe and effective. Keywords: CRISPR; FRGN; ex vivo; genome editing; hepatocyte transplantation; liver-humanized mouse; primary hepatocytes; urea cycle disorder