Pregnancy requires major immunomodulatory changes, both systemically and locally, as the maternal immune system needs to be modulated to tolerate the semi-allogeneic foetus. Decidual macrophages and stromal cells, but also foetal tissues are involved in this immune tolerance, for example by inducing M2 macrophages and regulatory T cells. However, it is so far unknown whether foetal membrane cells such as amnion epithelial cells (AECs) can influence human macrophage polarisation. In this study, a human in vitro macrophage assay was employed to demonstrate that conditioned medium (CM) from AECs derived from term placentas induces M2 macrophage polarisation, and to compare AEC culture conditions aiming for efficient M2 polarisation. Macrophage colony-stimulating factor (M-CSF), a well-known M2-inducing cytokine, was found to be secreted by AECs, and M-CSF was partly responsible for the observed M2-polarising effect of AECs. In addition, the M2-polarising effect remained after removal of extracellular vesicles (EVs) from AEC-CM, suggesting the involvement of soluble but not of EV-associated mediators. Taken together, this study shows that AECs may contribute to the induction of the vital immunotolerant environment at the foetal-maternal interface. Based on their immunomodulatory effects observed here and in in vivo studies, AECs could be harnessed as cytotherapeutics for inflammatory disorders.

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.

Despite a growing interest in Amniotic Epithelial Cell (AEC)-based therapies, the immune responses triggered by AEC transplantation in vivo remain poorly characterized. In particular, how direct exposure to AECs within the central nervous system (CNS) shapes the local immune environment is currently unknown. Herein we describe a novel CNS- specific immunoregulatory pathway induced by intracisternal delivery of human AECs. Local immune responses induced by AECs in the brain led to recruitment of immunosuppressive Arginase 1+ (ARG1+) macrophages and a novel population of myeloid-derived suppressor cells with eosinophilic characteristics, which we term Eo- MDSCs. We further demonstrate that Eo-MDSCs produce Maresin 2 (MaR2), a specialized pro-resolving mediator (SPM) involved in the resolution of inflammation. In a mouse model of Multiple Sclerosis (MS), treatment of established disease with AECs induced immunological responses that resulted in reduced numbers of pathogenic macrophages and T helper (TH)17 cells, increased anti-inflammatory T cell subsets, and enhanced myelin phagocytosis, all of which led to functional recovery. These findings suggest that AEC therapy has the potential to target CNS-intrinsic inflammatory processes in MS, providing a strong rationale for translation into the clinic. ### Competing Interest Statement The authors have declared no competing interest. Alltid Litt Sterkere Swedish Research Council, https://ror.org/03zttf063 Karolinska Institutet KID funding NEURO Stockholm Ollie and Elof Ericssons foundation Swedish Neurofonden Loo and Hans Osterman Foundation for Medical Research Eva and Oscar Ahréns foundation Karolinska Fonder Gunvor och Josef Anérs Stiftelse MS Forskningsfonden

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.

Congenital and chronic liver diseases have a substantial health burden worldwide. The most effective treatment available for these patients is whole organ transplantation; however, due to the severely limited supply of donor livers and the side effects associated with the immunosuppressive regimen required to accept allograft, the mortality rate in patients with end-stage liver disease is annually rising. Stem cell-based therapy aims to provide alternative treatments by either cell transplantation or bioengineered construct transplantation. Human amnion epithelial cells (AEC) are a widely available, ethically neutral source of cells with the plasticity and potential of multipotent stem cells and immunomodulatory properties of perinatal cells. AEC have been proven to be able to achieve functional improvement towards hepatocyte-like cells, capable of rescuing animals with metabolic disorders; however, they showed limited metabolic activities in vitro. Decellularised extracellular matrix (ECM) scaffolds have gained recognition as adjunct biological support. Decellularised scaffolds maintain native ECM components and the 3D architecture instrumental of the organ, necessary to support cells’ maturation and function. We combined ECM-scaffold technology with primary human AEC, which we demonstrated being equipped with essential ECM-adhesion proteins, and evaluated the effects on AEC differentiation into functional hepatocyte-like cells (HLC). This novel approach included the use of a custom 4D bioreactor to provide constant oxygenation and media perfusion to cells in 3D cultures over time. We successfully generated HLC positive for hepatic markers such as ALB, CYP3A4 and CK18. AEC-derived HLC displayed early signs of hepatocyte phenotype, secreted albumin and urea, and expressed Phase-1 and -2 enzymes. The combination of liver-specific ECM and bioreactor provides a system able to aid differentiation into HLC, indicating that the innovative perfusion ECM-scaffold technology may support the functional improvement of multipotent and pluripotent stem cells, with important repercussions in the bioengineering of constructs for transplantation.

Perinatal derivatives have been proposed as adjunct therapeutic strategies or innovative treatments. Undoubtedly, perinatal derivatives can offer the opportunity and source material to isolate multipotent stem cells, but both maternal- and fetal-derived tissues can be processed and transformed into engineered tissues or advanced biomedical devices, whose potential remains to be fully elucidated. Promising preclinical and clinical results collected so far clearly foresee an escalation of such novel treatments. Market forecasts predict exponential growth in such advanced medicinal products during the next decade, with a pragmatic innovation for medicine into a more advanced biomedical version, enlarging the portfolio for treating a wide range of congenital and acute conditions. However, all these promising and fascinating therapeutic possibilities cannot gain a solid and recognized role in established medical practice without rigid and harmonized manufacturing strategies. The implementation of strategies according to guidelines and directives compiled by Regulatory Agencies, in conformity to (European) Pharmacopoeia and for Good Manufacturing Practice -conforming production of such products, represent critical steps required to translate perinatal technologies into effective therapeutic approaches. During the past 5 years, a panel of European experts and developers, gathered under the umbrella of the COST Sprint Action, supported by the European Cooperation in Science and Technology action, had the opportunity to revise and summarize experience and recommendations for a fruitful and proficient generation of perinatal biomedical products. In order to facilitate the creation and potential commercialization of perinatal bioengineered and advanced pharmaceutical products and technologies, such a collection of data and recommendations is described and discussed here.

Background & AimPlacenta is a non-controversial and readily available source of cells for regenerative medicine. Human amnion epithelial cells (hAEC) from term placenta, once transplanted in immune-competent recipients, have been reported to engraft and survive, boosting the innate capacity of regeneration or correcting congenital disorders. Recently, it has been proposed that hAEC therapeutic potential is not mediated by intact cells only, but efficiently supported by hAEC secretome. Extracellular vesicles are secreted by all cells, but their protein and nucleic acid cargo varies significantly according to cell of origin.We coupled complete profiling of surface molecules and enzymes on intact hAEC and hAEC-derived EVs (hAEV), with cargo analysisMethods, Results & ConclusionhAEC from 20 full-term placentae were isolated according to cGMP procedures, and flow cytometric evaluation validated both hAEC and hAEV identity and surface enzymes. We purified hAEV and sequenced non-coding and microRNA cargo (Illumina MiSeq technology). Finally, we quantified and screened soluble factors (by Luminex and OLINK technologies)We identified several mediators and enzymes on the surface of intact hAEC, transferred to hAEVs. Amnion cells characteristically lack HLA class 2 expression and express both class 1a and non-polymorphic class 1b. We measured the constitutive presence of membrane-bound HLA-G on hAEC and hAEV, in addition to soluble isoforms. Both hAEC and hAEV modulate immune cells, in a dose-dependent matter. hAEC/hAEV purinergic mediators modulated immune effector cells (T-, B- and NK-cells), while soluble mediators induced the macrophage switch from M1 to M2. Finally, we qualified and sequenced biomolecules contained in small and large hAEV, revealing anti-fibrotic effects as well as a potential role in oncological treatment.The updated paradigm is that hAEC do not necessarily need to mature into adult cells, but they can rescue native parenchymal cells via indirect paracrine. The ability to treat most common (chronic/congenital) diseases with allogeneic stem cells without the administration of immunosuppressive drugs will greatly expand the number of patients who could receive cellular therapy. Immune evasive capacity could be a “game changer”, and the modulation, rather than suppression, of innate and adaptive immune cells may result in enhanced cell treatments for regenerative purposes, autoimmune disorders, and tumors treated with augmented immune response

MitoCeption and G-force assist in the uptake of nanorods into amniotic-derived cells.

Metabolic dysfunction-associated steatohepatitis (MASH) is a leading cause of chronic liver disease with few therapeutic options. To narrow the translational gap in the development of pharmacological MASH treatments, a 3D liver model from primary human hepatocytes and non-parenchymal cells derived from patients with histologically confirmed MASH was established. The model closely mirrors disease-relevant endpoints, such as steatosis, inflammation and fibrosis, and multi-omics analyses show excellent alignment with biopsy data from 306 MASH patients and 77 controls. By combining high-content imaging with scalable biochemical assays and chemogenomic screening, multiple novel targets with anti-steatotic, anti-inflammatory, and anti-fibrotic effects are identified. Among these, activation of the muscarinic M1 receptor (CHRM1) and inhibition of the TRPM8 cation channel result in strong anti-fibrotic effects, which are confirmed using orthogonal genetic assays. Strikingly, using biosensors based on bioluminescence resonance energy transfer, a functional interaction along a novel MASH signaling axis in which CHRM1 inhibits TRPM8 via Gq/11 and phospholipase C-mediated depletion of phosphatidylinositol 4,5-bisphosphate can be demonstrated. Combined, this study presents the first patient-derived 3D MASH model, identifies a novel signaling module with anti-fibrotic effects, and highlights the potential of organotypic culture systems for phenotype-based chemogenomic drug target identification at scale.

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.

The potential of extracellular vesicles (EVs) isolated from mesenchymal stromal cells in guiding macrophages toward anti-inflammatory immunophenotypes, has been reported in several studies. In our study, we provided experimental evidence of a distinctive effect played by Wharton Jelly mesenchymal stromal cell-derived EVs (WJ-EVs) on human macrophages. We particularly analyzed their anti-inflammatory effects on macrophages by evaluating their interactions with stellate cells, and their protective role in liver fibrosis. A three-step gradient method was used to isolate monocytes from umbilical cord blood (UCB). Two subpopulations of WJ-EVs were isolated by high-speed (20,000 g ) and differential ultracentrifugation (110,000 g ). Further to their characterization, they were designated as EV20K and EV110K and incubated at different concentrations with UCB-derived monocytes for 7 days. Their anti-fibrotic effect was assessed by studying the differentiation and functional levels of generated macrophages and their potential to modulate the survival and activity of LX2 stellate cells. The EV20K triggers the polarization of UCB-derived monocytes towards a peculiar M2-like functional phenotype more effectively than the M-CSF positive control. The EV20K treated macrophages were characterized by a higher expression of scavenger receptors, increased phagocytic capacity and production level of interleukin-10 and transforming growth factor-β. Conditioned medium from those polarized macrophages attenuated the proliferation, contractility and activation of LX2 stellate cells. Our data show that EV20K derived from WJ-MSCs induces activated macrophages to suppress immune responses and potentially play a protective role in the pathogenesis of liver fibrosis by directly inhibiting HSC’s activation.

Human duodenal submucosal glands contain a defined stem/progenitor subpopulation with liver-specific regenerative potentialJournal of HepatologyVol. 78Issue 1PreviewCommon precursors for the liver, biliary tree, and pancreas exist at an early stage of development in the definitive endoderm forming the foregut. We have identified and characterised endodermal stem/progenitor cells with regenerative potential persisting in the adult human duodenum. Full-Text PDF See Article, pages 165–179 See Article, pages 165–179 The year 1992 is widely considered “year zero” for liver cell-based treatments in humans. It was December 1992 when the first clinical report was published, describing an autologous cell-based approach for 10 individuals with end-stage cirrhosis in Japan.[1]Mito M. Kusano M. Kawaura Y. Hepatocyte transplantation in man.Transpl Proc. 1992; 24: 3052-3053PubMed Google Scholar It was still 1992 when Drs Strom and Fisher used allogeneic hepatocytes to rescue a patient with fulminant hepatitis for the first time.[2]Strom S.C. Fisher R.A. Thompson M.T. Sanyal A.J. Cole P.E. Ham J.M. et al.Hepatocyte transplantation as a bridge to orthotopic liver transplantation in terminal liver failure.Transplantation. 1997; 63: 559-569Crossref PubMed Scopus (468) Google Scholar This early success led to the development of similar treatments for both acute and congenital liver disorders in subsequent years.[3]Gramignoli R. Vosough M. Kannisto K. Srinivasan R.C. Strom S.C. Clinical hepatocyte transplantation: practical limits and possible solutions.Eur Surg Res. 2015; 54: 162-177Crossref PubMed Scopus (78) Google Scholar Drs Strom and Fisher's seminal transplants paved the way for further clinical studies and for the compassionate use of such treatments by several groups and transplant centers around the globe. The major indications remain treatment for patients with acquired or inherited liver disease, where the long-term acceptance and metabolic support offered by allogenic proficient cells can correct metabolic defects. Additionally, cell therapies have gained recognition as important supportive treatments to rescue patients with fulminant hepatitis or acute liver failure, an alternative to solid organ transplantation, or temporary support for patients waiting for a matched donor. Today, thirty years later, hepatocyte transplantation has been offered to no more than 150 patients. Several roadblocks have limited the widespread application of hepatocyte transplantation, and it took several years to solve most of them.[4]Gramignoli R. Tahan V. Dorko K. Venkataramanan R. Fox I.J. Ellis E.C. et al.Rapid and sensitive assessment of human hepatocyte functions.Cel Transpl. 2014; 23: 1545-1556Crossref PubMed Scopus (36) Google Scholar The obstacles of significance included the lack of clinical-grade reagents and procedure; the limited number and quality of donor tissues (as a cell source); short- and long-term storage of cells prior to transplant; tracking or monitoring cells after transplantation; preconditioning treatments to enhance engraftment and proliferation of donor cells. Although major accomplishments have been made, there are still hurdles limiting the widespread application of hepatocyte transplantation, or cellular therapies in general, for liver disorders. Apart from the initial success, the past decades have witnessed several attempts with sometimes modest and short-term effects, where allogeneic cells have been administered or implanted. The reported differences in the level of corrections or outcomes have been largely imputed to quality in donor cells. The isolation procedure to generate primary hepatocytes has been optimized according to Good Manufacturing Practice (GMP) requirements, and the final cell product has been extensively studied to generate hepato-specific release criteria to validate the final product before infusion.[5]Tolosa L. Bonora-Centelles A. Teresa Donato M. Pareja E. Negro A. Lopez S. et al.Steatotic liver: a suitable source for the isolation of hepatic progenitor cells.Liver Int : official J Int Assoc Study Liver. 2011; 31: 1231-1238Crossref PubMed Scopus (11) Google Scholar But the limited availability of primary hepatocytes for clinical approaches is still recognized as a major burden. Livers rejected from organ transplantation have represented the primary source of hepatocytes for a long time. But the yield and quality of human hepatocytes isolated from rejected organs is severely hampered by prolonged ischemia and elevated micro-/macro-steatosis. Furthermore, hepatocytes isolated from such tissues exhibit reduced hepatic functions, and are sometimes insufficient in number to reach an adequate cell dose for a single adult patient.[6]Gramignoli R. Green M.L. Tahan V. Dorko K. Skvorak K.J. Marongiu F. et al.Development and application of purified tissue dissociation enzyme mixtures for human hepatocyte isolation.Cel Transpl. 2012; 21: 1245-1260Crossref PubMed Scopus (54) Google Scholar,[7]Gramignoli R. Tahan V. Dorko K. Skvorak K.J. Hansel M.C. Zhao W. et al.New potential cell source for hepatocyte transplantation: discarded livers from metabolic disease liver transplants.Stem Cel Res. 2013; 11: 563-573Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar Alternatively, explanted organs from patients undergoing orthotopic liver transplantation have been investigated and validated.[8]Giancotti A. Monti M. Nevi L. Safarikia S. D’Ambrosio V. Brunelli R. et al.Functions and the emerging role of the foetal liver into regenerative medicine.Cells. 2019; 8: 914Crossref PubMed Scopus (21) Google Scholar The livers of many individuals with inborn errors of metabolism are morphologically and biochemically normal, except for the impaired function that characterizes that disease, and these livers rarely suffer from ischemia or steatosis. Importantly, the cells isolated from explanted livers and infused into patients are not sufficient in number to transfer the donor’s disease. But explanted livers are not an abundant source of hepatocytes either and are often insufficient to meet clinical needs. Thus, prenatal (fetal) and neonatal donors have been investigated.[9]Bluhme E. Henckel E. Gramignoli R. Kjellin T. Hammarstedt C. Nowak G. et al.Procurement and evaluation of hepatocytes for transplantation from neonatal donors after circulatory death.Cel Transpl. 2022; 319636897211069900PubMed Google Scholar,[10]Zabulica M. Srinivasan R.C. Vosough M. Hammarstedt C. Wu T. Gramignoli R. et al.Guide to the assessment of mature liver gene expression in stem cell-derived hepatocytes.Stem Cell Dev. 2019; 28: 907-919Crossref PubMed Scopus (39) Google Scholar Unfortunately, such young or progenitor cells, despite undoubted resistance to cryogenic preservation and poor immunological profile, are limited in number and introduce another important risk in cellular therapy: donor cells may not be able to mature into functional hepatocytes and supply the recipient with the required synthetic or enzymatic activities. Similar considerations need to be kept in mind when hepatic or ectopic progenitor/stem cells are proposed as alternatives to mature liver cells. Cell transplantation is a platform technology in rapid and continuous development. The generation of stem cell-derived hepatocyte-like cells could potentially offer an almost unlimited cell source that could be quickly incorporated into existing procedures if cell products are proven safe and functional (in terms of hepatic secretive and metabolic pathways). Not all the proposed progenitor cells or multipotent stem cells described so far have been eligible and efficient in managing (or curing) patients on the waiting list. Over the past years, several groups and companies have proposed stem cells as an alternative solution, bounded by genetic and epigenetic instabilities and, more importantly, limited in hepatic maturation level (Fig. 1).[11]Turner R. Lozoya O. Wang Y. Cardinale V. Gaudio E. Alpini G. et al.Human hepatic stem cell and maturational liver lineage biology.Hepatology. 2011; 53: 1035-1045Crossref PubMed Scopus (243) Google Scholar While all the stem and progenitor cells deserve to be studied, progenitors derived from foregut endoderm may represent the first line of investigation. Such cells may be reasonably equipped with molecules and transporters, as well as phenotypically closer to liver parenchymal cells (or hypothetically able to reach full maturation in a shorter time). Cardinale and co-authors have large experience in biliary tract analysis and progenitor cell extraction,[12]Cooper S. Bennett W. Andrade J. Reubinoff B.E. Thomson J. Pera M.F. Biochemical properties of a keratan sulphate/chondroitin sulphate proteoglycan expressed in primate pluripotent stem cells.J Anat. 2002; 200: 259-265Crossref PubMed Scopus (32) Google Scholar and in the present work, they exploited such expertise to investigate another tissue characterized by foregut endoderm origin: duodenal submucosal glands.[13]Cardinale V. Carpino G. Overi D. Safarikia S. Zhang W. Kanke M. et al.Human duodenal submucosal glands contain a defined stem/progenitor subpopulation with liver-specific regenerative potential.J Hepatol. 2022; 78https://doi.org/10.1016/j.jhep.2022.08.037Abstract Full Text Full Text PDF Scopus (3) Google Scholar The authors developed a method to isolate EpCAM-positive cells and coupled isolation with immunological analysis to determine if such progenitor cells may be able to acquire a hepatocyte-like phenotype. The extrahepatic progenitor cells isolated by Cardinale and coworkers have been described as possessing some level of homogeneity (determined by static surface markers) and inherited capacity to progress toward hepatic maturation. Interestingly, keratan sulfate-associated antigens (tumor rejection antigen [TRA] 1-60) have been found expressed on such progenitor cells. Notably, TRA-1-60 and TRA-1-81 have been largely described on pluripotent stem cells, such as embryonic or induced pluripotent cells, but also on perinatal stem cells.[14]Gupta S. Rajvanshi P. Lee C.D. Integration of transplanted hepatocytes into host liver plates demonstrated with dipeptidyl peptidase iv-deficient rats.Proc Natl Acad Sci United States America. 1995; 92: 5860-5864Crossref PubMed Scopus (122) Google Scholar However, the mechanism of action for such surface-bound molecules is still debated and their relevance in hepatic maturation is unclear. What is attractive is the ability of such duodenal submucosal gland-derived cells to offer support in reversing chronic (but not acute) liver injury. Correctly, the authors correlated such ability to potential paracrine mediators rather than direct maturation into a functional hepatic phenotype. Recent evidence suggests that the maturation of progenitor cells into a tissue-specific phenotype is not always required or strictly necessary. Paracrine effects, based on anti-inflammatory, extracellular matrix remodeling, anti-apoptotic and pro-angiogenic properties, may be sufficient to restore normal architecture and function in fibrotic tissues. Bridging new cell-based approaches into clinical practice depends on relevant preclinical validation. The use of relevant experimental models is of irreplaceable importance to evaluate the efficacy and safety of the product. And 15 years between the first preclinical test and the first-in-human hepatocyte transplantation affirm such experimental steps as crucial. The authors tested human duodenal progenitor cells in an experimental model of fatty liver, observing support in liver regeneration. Another critical factor, where preclinical analysis is instrumental to validate new products, is cell engraftment. Engraftment into a preclinical model has served as a valid predictor of the extravasation capacity and parenchymal integration characteristics of donor cells. Once in the portal blood, human epithelial cells, such as adult or pediatric hepatocytes, characterized by quite a large volume (approximately 20-40 μm in diameter), have been described to embolize into the terminal radicles of hepatic veins or arteries, resulting in transient portal hypertension and mild ischemia-reperfusion injury.[15]Bohnen N.I. Charron M. Reyes J. Rubinstein W. Strom S.C. Swanson D. et al.Use of indium-111-labeled hepatocytes to determine the biodistribution of transplanted hepatocytes through portal vein infusion.Clin Nucl Med. 2000; 25: 447-450Crossref PubMed Scopus (63) Google Scholar Such an effect on the microvasculature stimulates fenestration enlargement by the endothelial cells but also activates tissue-resident macrophages to perform a rapid and efficient phagocytosis of residual cells or cellular elements in the circulatory system. As part of the safety and preclinical validation, cell distribution and engraftment evaluation for the donor cells is commonly required. Indeed, several stem cells have been quite unsuccessful in engrafting into the liver parenchyma when injected through the blood system. Hepatocytes delivered via the portal vein efficiently engraft in the liver or are cleaved by Kupffer cells, while smaller cells pass through the organ and relocate to extrahepatic compartments.[16]Gramignoli R. Tahan V. Dorko K. Venkataramanan R. Fox I.J. Ellis E.C. et al.Rapid-and-sensitive assessment of human hepatocyte functions.Cel Transplant. 2014; 23: 1545-1556Crossref PubMed Scopus (0) Google Scholar Hence, it is critical to evaluate donor cell engraftment to determine the cell dose to be administered to the recipient. Clinical outcome is directly correlated to the administered cell dose. In clinical practice, such cell dose is conveniently calculated and commonly adjusted according to the patient’s size. During the past decades, doses ranging between 106 and 107 viable cells per kg of a patient’s weight have been used. Low cell dose has been used in patients affected by fulminant hepatitis, as well as end-stage cirrhosis. A great distinction, one or two log differences, needs to be made when inborn errors of metabolism need to be treated and pathophysiological effects need to be reversed. A liver mosaicism, where approximately 5-10% of the total parenchyma is replaced by proficient cells, is required to correct a metabolic imbalance. Considering an average liver organ contains approximately 1.5-2 billion hepatocytes, 8 to 15 billion proficient cells need to be implanted to reverse metabolic defects such as urea cycle defects or Crigler-Najjar (two of the major indications for allogenic hepatocyte transplantation, so far). Cardinale et al. extracted two to five hundred million duodenal progenitor cells from each donor. Such cell yield may be considered sufficient to infuse a patient with an acute or chronic disorder, but several donors will eventually be needed to grant long-term correction to a patient with a metabolic disorder. A burden shared with any other progenitor/stem cell sources, as mentioned. Notably, cell infusions are not limited to one or two procedures as commonly experienced with solid organ transplantation. The implantation of a large organ or tissue is an invasive procedure, requiring massive surgery, compared to cell infusion where donor cells are released into the circulatory torrent in close proximity to the target organ. Decades of hepatocyte transplantations have clearly shown that multiple cell infusions are not only possible, but preferable. Billions of donor(s) cells can be infused over a number of hours, days, weeks, or even months to achieve a physiological level of missing enzymes and reverse an inborn error of metabolism (Fig. 1). But as with any other cell therapies, compliance with Regulatory Agencies is required, including restrictions on the number of donors per recipient. Guidelines for GMP have been published and the vast majority of modern cell-based therapies (in particular allogeneic treatments) need to fulfill such criteria and respond to specific standard requirements. Liver cell-based therapies, where allogeneic progenitor or stem cells are infused into recipients, are classified as Advanced Therapy Medicinal Products. As an innovative medical treatment, human cells may offer groundbreaking new opportunities for the treatment of diseases and injuries that are largely incurable today. However, such products need to respond to strict and specific release requirements. In published reports, cell viability is the sole evaluation for cell quality before infusion. Cell viability both before and after cell manipulation (i.e., isolation, selection, or cryogenic preservation) reflects cell integrity and characteristic, but common viability tests have little to no correlation with hepatic metabolic capabilities. Hepatic maturation and metabolism are critical parameters to assess liver therapies. During the past years, release criteria and functional tests have been developed and optimized to specifically evaluate donor liver or progenitor cells before infusion in patients.[16]Gramignoli R. Tahan V. Dorko K. Venkataramanan R. Fox I.J. Ellis E.C. et al.Rapid-and-sensitive assessment of human hepatocyte functions.Cel Transplant. 2014; 23: 1545-1556Crossref PubMed Scopus (0) Google Scholar,[17]Bonora-Centelles A. Donato M.T. Lahoz A. Pareja E. Mir J. Castell J.V. et al.Functional characterization of hepatocytes for cell transplantation: customized cell preparation for each receptor.Cel Transplant. 2010; 19: 21-28Crossref PubMed Scopus (33) Google Scholar As a result, cellular products can be qualified and customized to better match the recipient’s needs. Progenitors and stem cells are no exception, and their path to reach full maturity and recognition as new treatments will require in vitro and in vivo validations. We are very much looking forward to observing the progression in the described new source of hepatic progenitors generated by duodenal submucosal glands. These progenitor cells and other stemness sources may carve out a place as corrective cell therapies, or represent an adjunct product in support of more conventional treatments. In the medical community, we all contemplate similar new opportunities that can be brought to clinical practice, upon complete and rigorous validation analyses. The author did not receive any financial support. The author declares no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure form for further details. The following are the supplementary data to this article: Download .pdf (.93 MB) Help with pdf files Multimedia component 1

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.

Introduction: Despite 30 years since the first clinical infusion and more than 150 patients treated so far, Hepatocyte transplantation is still considered an experimental procedure, characterized by fluctuating outcomes in congenital or fulminant diseases. Our laboratory has always been active in the translation of hepatocyte transplant technology from bench to clinic. Our 30 years of experience on more than 2,000 human livers allowed us to standardize reagents and procedures in accordance with Good Manufacturing Practice (GMP). We performed hepatocyte isolation and cryopreservation on organs rejected from transplantation, explanted organs, fetal and neonatal liver tissues. We developed and validated rapid and sensitive functional assays to ensure freshly isolated hepatocytes, as well as cryopreserved products, function adequately. Methods: Different surgical procedures and enzymatic solutions have been tested during the past decades, since coming to a new collagenase-protease mixture specifically designed for the isolation of human hepatocytes. Both DMSO-supplemented and DMSO-free cryopreservation solutions were tested. Data from viability/apoptosis, recovery to plating adhesion, and hepatic function including basal and induced Cytochrome P450 (CYP) activities, phase II conjugation, and ammonia metabolism were collected on fetal, neonatal, pediatric, and adult hepatocytes.z Results: Human hepatocytes were isolated from 1315 post-natal and 706 fetal livers. Viability significantly improved from the first clinical applications in the early 90s (64±1%, mean±SEM) compared to the clinical transplants performed during the last decade (88±2%). The viability of fetal hepatocytes was significantly higher (91 + 9%), both immediately after isolation and after cryogenic procedure. High viability was also obtained from neonatal livers (83 + 16%), while adult liver have generated wide range of quality cells with an average value of 70 + 24%. Cryopreservation procedure was successfully performed on young cells, but poorly effective on adult and steatotic hepatocytes. CYP activity increased during gestational and postnatal age, but remained 1-10% of adult values. Phase II-conjugation and ammonia metabolism were null/low in fetal hepatocytes, but increased in vitro and in vivo. Conclusions: A large spectrum of primary cells has been analyzed to determine a normal range of activities to expect from isolated cells. Data collected on multiple hepatic functions worked as quality control and form the basis for patients’ need match criteria. Measurements on prenatal and postnatal liver cells allowed us to determine the normal range of metabolic activities and synthetic capacity for donor liver cells, guiding stem cell-derived hepatocyte-like validation. Our long-term database can serve as quality control and form the basis for an auxiliary strategy to validate clinical products but also evaluate stem cell sources proposed as hepatocyte replacement.

The liver is a vital organ responsible for metabolic and digestive functions, protein synthesis, detoxification, and numerous other necessary functions. Various acute, chronic, and neoplastic disorders affect the liver and hamper its biological functions. Most of the untreated liver diseases lead to inflammation and fibrosis which develop into cirrhosis. The human amniotic membrane (hAM), the innermost layer of the fetal placenta, is composed of multiple layers that include growth-factor rich basement membrane, epithelial and mesenchymal stromal cell layers. hAM possesses distinct beneficial anti-fibrotic, anti-inflammatory and pro-regenerative properties via the secretion of multiple potent trophic factors and/or direct differentiation into hepatic cells which place hAM-based therapies as potential therapeutic strategies for the treatment of chronic liver diseases. Decellularized hAM is also an ideal scaffold for liver tissue engineering as this biocompatible niche provides an excellent milieu for cell proliferation and hepatocytic differentiation. Therefore, the current review discusses the therapeutic potential of hAM and its derivatives in providing therapeutic solutions for liver pathologies including acute liver failure, metabolic disorders, liver fibrosis as well as its application in liver tissue engineering.

Introduction: Liver diseases are associated with increased mortality and morbidity, with organ or hepatocyte transplantations as the sole treatment limited by the shortage of donors. Over the years, several groups/companies have proposed stem cells as an alternative solution, although limited by hepatic maturation capacity. Amnion epithelial stem cells (AESC), isolated from the full-term human placentae, have been proven able to mature into hepatocyte-like cells, rescuing preclinical models of congenital disorders or acute liver failure. Human AESCs represent a promising source of multipotent cells, supporting both regenerative strategies and drug development, if their hepatic maturation can be achieved ex vivo. Decellularized Liver extracellular matrix (dLECM) proteins and 3D architecture play a crucial role in supporting hepatic maturation. Thus, we combined the multipotency ability offered by human AESC seeded into rat dLECM constructs, and evaluated cell differentiation and functional properties in comparison with human fetal and adult hepatocytes. Methods: Fifty million human AESC were seeded into decellularized rat liver scaffolds and exposed for 40 days to repeated infusions or to endure perfusion of the hepatogenic medium. Initial mitogenic stimuli were replaced by hepatogenic culture conditions, and hepatic metabolism and synthetic activities were monitored at different time points. NMR, ELISA, and fluorescent probes were used to monitor hepatic maturation, and results were compared to primary human adult hepatocytes or fetal hepatoblasts. Transcriptome and proteomic analysis at intermediate and final time points supported functional analysis. Results: human AESCs were positive for epithelial markers but negative for hepatic enzymes. Once engrafted in rat dLECM, human AESC expressed some level of hepatic maturation within 2 weeks (AFP and Alb). Such hepatic maturation was maintained and enhanced in hepatic maturation medium, resulting in CK18+ hepatocyte-like cells, characterized by secretive and metabolic protein. Albumin and urea production increased in dynamic rather than static conditions, and NMR showed a shift in metabolite production. Transcriptome analysis expanded evaluation for hepatic characteristics in bioengineered liver tissue. Conclusion: dLECM-scaffold supports the maturation of human AESC in a 3D-whole liver model. Continuous perfusion resulted in remarkable cell distribution and hepato-specific activities compared to static conditions. The bioreactor technology provides enhanced distribution of oxygen and nutrients, leading to a more physiological condition in support of maturation and hepatic metabolism. Encouraged by safety and immune privilege characteristics of perinatal AESC, such technology may represent an effective method to evaluate hepatogenic capacities and provide functional bioartificial tissues instrumental for drug development as well as resolutive clinical applications.