Purpose of Review
During the last decades, the field of regenerative medicine has been rapidly evolving. Major progress has been made in the development of biological substitutes applying the ...principles of cell transplantation, material science, and bioengineering.
Recent Findings
Among other sources, amniotic-derived products have been used for decades in various fields of medicine as a biomaterial for the wound care and tissue replacement. Moreover, human amniotic epithelial and mesenchymal cells have been intensively studied for their immunomodulatory capacities.
Summary
Amniotic cells possess two major characteristics that have already been widely exploited. The first is their ability to modulate and suppress the innate and adaptive immunities, making them a true asset for chronic inflammatory disorders and for the induction of tolerance in transplantation models. The second is their multilineage differentiation capacity, offering a source of cells for tissue engineering. The latter combined with the use of amniotic membrane as a scaffold offers all components necessary to create an optimal environment for cell and tissue regeneration. This review summarizes beneficial properties of hAM and its derivatives and discusses their potential in regenerative medicine.
The arrangement of β cells within islets of Langerhans is critical for insulin release through the generation of rhythmic activity. A privileged role for individual β cells in orchestrating these ...responses has long been suspected, but not directly demonstrated. We show here that the β cell population in situ is operationally heterogeneous. Mapping of islet functional architecture revealed the presence of hub cells with pacemaker properties, which remain stable over recording periods of 2 to 3 hr. Using a dual optogenetic/photopharmacological strategy, silencing of hubs abolished coordinated islet responses to glucose, whereas specific stimulation restored communication patterns. Hubs were metabolically adapted and targeted by both pro-inflammatory and glucolipotoxic insults to induce widespread β cell dysfunction. Thus, the islet is wired by hubs, whose failure may contribute to type 2 diabetes mellitus.
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•Optogenetic targeting reveals a pacemaker-like β cell subpopulation•These cells, termed hubs, are required for normal insulin release•Hubs are highly metabolic and transcriptionally immature•Hubs are targeted by a diabetic milieu to induce islet failure
Combining optogenetics and photopharmacology, Johnston et al. show that a few (1%–10%) β cells exert disproportionate control over islet responses to glucose. These specialized cells, called hubs, are transcriptionally immature and highly metabolic. Their failure during type 2 diabetes mellitus may lead to reduced insulin secretion and impaired glucose homeostasis.
Maintaining long-term euglycemia after intraportal islet transplantation is hampered by the considerable islet loss in the peri-transplant period attributed to inflammation, ischemia and poor ...angiogenesis. Here, we show that viable and functional islet organoids can be successfully generated from dissociated islet cells (ICs) and human amniotic epithelial cells (hAECs). Incorporation of hAECs into islet organoids markedly enhances engraftment, viability and graft function in a mouse type 1 diabetes model. Our results demonstrate that the integration of hAECs into islet cell organoids has great potential in the development of cell-based therapies for type 1 diabetes. Engineering of functional mini-organs using this strategy will allow the exploration of more favorable implantation sites, and can be expanded to unlimited (stem-cell-derived or xenogeneic) sources of insulin-producing cells.
Chronic inflammation impairs insulin secretion and sensitivity. β-cell dedifferentiation has recently been proposed as a mechanism underlying β-cell failure in T2D. Yet the effect of inflammation on ...β-cell identity in T2D has not been studied. Therefore, we investigated whether pro-inflammatory cytokines induce β-cell dedifferentiation and whether anti-inflammatory treatments improve insulin secretion via β-cell redifferentiation. We observed that IL-1β, IL-6 and TNFα promote β-cell dedifferentiation in cultured human and mouse islets, with IL-1β being the most potent one of them. In particular, β-cell identity maintaining transcription factor Foxo1 was downregulated upon IL-1β exposure. In vivo, anti-IL-1β, anti-TNFα or NF-kB inhibiting sodium salicylate treatment improved insulin secretion of isolated islets. However, only TNFα antagonism partially prevented the loss of β-cell identity gene expression. Finally, the combination of IL-1β and TNFα antagonism improved insulin secretion of ex vivo isolated islets in a synergistic manner. Thus, while inflammation triggered β-cell dedifferentiation and dysfunction in vitro, this mechanism seems to be only partly responsible for the observed in vivo improvements in insulin secretion.
Tissue engineering with scaffolds to form transplantable organs is of wide interest. Decellularized tissues have been tested for this purpose, although supplies of healthy donor tissues, vascular ...recellularization for perfusion, and tissue homeostasis in engineered organs pose challenges. We hypothesized that decellularized human placenta will be suitable for tissue engineering. The universal availability and unique structures of placenta for accommodating tissue, including presence of embedded vessels, were major attractions. We found decellularized placental vessels were reendothelialized by adjacent native cells and bridged vessel defects in rats. In addition, implantation of liver fragments containing all cell types successfully hepatized placenta with maintenance of albumin and urea synthesis, as well as hepatobiliary transport of 99mTc‐mebrofenin, up to 3 days in vitro. After hepatized placenta containing autologous liver was transplanted into sheep, tissue units were well‐perfused and self‐assembled. Histological examination indicated transplanted tissue retained hepatic cord structures with characteristic hepatic organelles, such as gap junctions, and hepatic sinusoids lined by endothelial cells, Kupffer cells, and other cell types. Hepatocytes in this neo‐organ expressed albumin and contained glycogen. Moreover, transplantation of hepatized placenta containing autologous tissue rescued sheep in extended partial hepatectomy‐induced acute liver failure. This rescue concerned amelioration of injury and induction of regeneration in native liver. The grafted hepatized placenta was intact with healthy tissue that neither proliferated nor was otherwise altered. Conclusion: The unique anatomic structure and matrix of human placenta were effective for hepatic tissue engineering. This will advance applications ranging from biological studies, drug development, and toxicology to patient therapies. (Hepatology 2018;67:1956‐1969).
Three-dimensional (3D) cell culture by engineering spheroids has gained increasing attention in recent years because of the potential advantages of such systems over conventional two-dimensional (2D) ...tissue culture. Benefits include the ability of 3D to provide a more physiologically relevant environment, for the generation of uniform, size-controlled spheroids with organ-like microarchitecture and morphology. In recent years, different techniques have been described for the generation of cellular spheroids. Here, we have compared the efficiency of four different methods of islet cell aggregation. Rat pancreatic islets were dissociated into single cells before reaggregation. Spheroids were generated either by (i) self-aggregation in nonadherent petri dishes, (ii) in 3D hanging drop culture, (iii) in agarose microwell plates or (iv) using the Sphericalplate 5D™. Generated spheroids consisted of 250 cells, except for the self-aggregation method, where the number of cells per spheroid cannot be controlled. Cell function and morphology were assessed by glucose stimulated insulin secretion (GSIS) test and histology, respectively. The quantity of material, labor intensity, and time necessary for spheroid production were compared between the different techniques. Results were also compared with native islets. Native islets and self-aggregated spheroids showed an important heterogeneity in terms of size and shape and were larger than spheroids generated with the other methods. Spheroids generated in hanging drops, in the Sphericalplate 5D™, and in agarose microwell plates were homogeneous, with well-defined round shape and a mean diameter of 90 µm. GSIS results showed improved insulin secretion in response to glucose in comparison with native islets and self-aggregated spheroids. Spheroids can be generated using different techniques and each of them present advantages and inconveniences. For islet cell aggregation, we recommend, based on our results, to use the hanging drop technique, the agarose microwell plates, or the Sphericalplate 5D™ depending on the experiments, the latter being the only option available for large-scale spheroids production.
Hypoxia, IL-1β production and oxidative stress are involved in islet graft dysfunction and destruction. However, the link between these events has not yet been determined in transplanted islets. The ...goal of this study was to determine whether NLRP3 inflammasome is responsible for IL-1β production and if it is activated by hypoxia-induced oxidative stress in transplanted islets. Rat islets were transplanted under the kidney capsule of immunodeficient mice. At different times post-transplantation, blood samples were collected and islet grafts harvested. Rat islets were also incubated in vitro either under normoxia or hypoxia for 24 h, in the absence or presence of inhibitors of NLRP3 inflammasome (CASP1 inhibitor) or oxidative stress (NAC). NLRP3, CASP1, IL1B, BBC3 pro-apoptotic and BCL2 anti-apoptotic genes in transplanted and in vitro incubated islets were then studied using real time PCR. IL-1β released in the blood and in the supernatant was quantified by ELISA. Cell death was analysed by propidium iodide and Annexin-V staining. NLRP3, CASP1 and BBC3 in transplanted rat islets and IL-1β in blood transiently increased during the first days after transplantation. In islets incubated under hypoxia, NRLP3, IL1B and CASP1 and IL-1β released in supernatant increased compared to islets incubated under normoxia. These effects were prevented by the inhibition of NLRP3 inflammasome by CASP1 or oxidative stress by NAC. However, these inhibitors did not prevent hypoxia-induced rat islet death. These data show that NLRP3 inflammasome in rat islets is transiently activated after their transplantation and induced through oxidative stress in vitro. However, NRLP3 inflammasome inhibition does not protect islet cells against hypoxia.
NRLP3 inflammasome is a protein complex involved in the maturation of IL1β. In the onset of type 1 diabetes as well as in islet transplantation, IL-1β is one of the cytokines involved in the ...recruitment of immune cells in islets and eventually in islet destruction. Whether IL-1β is produced by islet cells is still under debate and NLRP3 inflammasome-dependent IL-1β production has not been yet determined in human islets. The aim of this study was to determine the expression and the regulation of the NRLP3 inflammasome in human islets. Human islets were stimulated with LPS and successively with ATP (LPS + ATP) in the presence or absence of the inflammasome inhibitor glyburide. Islets were also incubated in hypoxic or normoxic conditions for 24 h in the presence or absence of glyburide. Then, IL1B and NLRP3 expression was studied by real time PCR, protein expression by western blot, protein localization by immunofluorescence and protein secretion by ELISA. LPS + ATP increased gene expression of NRLP3 and IL1B. Glyburide partially prevented this effect. IL-1β protein was localized in β and non-β cells. Moreover, LPS + ATP increased IL-1β protein expression and production, which were prevented by glyburide. Hypoxia increased gene expression of NRLP3 and IL1B and induced IL-1β and caspase-1 production. Finally, hypoxia-induced cell death which was not prevented by inhibition of NLRP3 inflammasome. NRLP3 inflammasome is expressed and plays a role in IL-1β production by human islets. By contrast, NRLP3 inflammasome activation is not involved in islet cell death induced by hypoxia.
The field of transplantation has witnessed the emergence of Advanced Therapy Medicinal Products (ATMPs) as highly promising solutions to address the challenges associated with organ and tissue ...transplantation. ATMPs encompass gene therapy, cell therapy, and tissue-engineered products, hold immense potential for breakthroughs in overcoming the obstacles of rejection and the limited availability of donor organs. However, the development and academic research access to ATMPs face significant bottlenecks that hinder progress. This opinion paper emphasizes the importance of addressing bottlenecks in the development and academic research access to ATMPs by implementing several key strategies. These include the establishment of streamlined regulatory processes, securing increased funding for ATMP research, fostering collaborations and partnerships, setting up centralized ATMP facilities, and actively engaging with patient groups. Advocacy at the policy level is essential to provide support for the development and accessibility of ATMPs, thereby driving advancements in transplantation and enhancing patient outcomes. By adopting these strategies, the field of transplantation can pave the way for the introduction of innovative and efficacious ATMP therapies, while simultaneously fostering a nurturing environment for academic research.
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