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Organoids, i.e. laboratory-grown organ models developed from stem cells, are emerging tools for studying organ physiology, disease modeling and drug development. On-line analysis of organoids with mass spectrometry would provide analytical versatility and automation. To achieve these features with robust hardware, we have loaded liquid chromatography column housings with induced pluripotent stem cell (iPSC) derived liver organoids and coupled the “organ-in-a-column” units on-line with liquid chromatography-mass spectrometry (LC-MS). Liver organoids were co-loaded with glass beads to achieve an even distribution of organoids throughout the column while preventing clogging. The liver organoids were interrogated “on column” with heroin, followed by on-line monitoring of the drug’s phase 1 metabolism. Enzymatic metabolism of heroin produced in the “organ-in-a-column” units was detected and monitored using a triple quadrupole MS instrument, serving as a proof-of-concept for on-line coupling of liver organoids and mass spectrometry. Taken together, the technology allows direct integration of liver organoids with LC-MS, allowing selective and automated tracking of drug metabolism over time.

Supplementary Figure S1. Chemical structures of selected tankyrase inhibitors. Supplementary Figure S2. OM-153 specifically inhibits TNKS1/2 and WNT/β-catenin signaling. Supplementary Figure S3. OM-153 specifically inhibits cell growth of an APC-mutated colon carcinoma cell line. Supplementary Figure S4. OM-153 shows an anti-proliferative effect in human cancer cell lines. Supplementary Figure S5. OM-153 inhibits WNT/β-catenin, YAP and MYC signaling in human cancer cell lines. Supplementary Figure S6. OM-153 inhibits the WNT/β-catenin signaling pathway and shows anti-tumor effect in a human colon carcinoma xenograft model. Supplementary Figure S7. Combined OM-153 and anti-PD-1 treatment confers anti-tumor effect in mouse melanoma. Supplementary Figure S8. PO-BID treatment with 10 mg/kg OM-153 does not reduce body weight and food consumption or induce toxicity in mice.

The catalytic enzymes tankyrase 1 and 2 (TNKS1/2) alter protein turnover by poly-ADP-ribosylating target proteins, which earmark them for degradation by the ubiquitin–proteasomal system. Prominent targets of the catalytic activity of TNKS1/2 include AXIN proteins, resulting in TNKS1/2 being attractive biotargets for addressing of oncogenic WNT/β-catenin signaling. Although several potent small molecules have been developed to inhibit TNKS1/2, there are currently no TNKS1/2 inhibitors available in clinical practice. The development of tankyrase inhibitors has mainly been disadvantaged by concerns over biotarget-dependent intestinal toxicity and a deficient therapeutic window. Here we show that the novel, potent, and selective 1,2,4-triazole–based TNKS1/2 inhibitor OM-153 reduces WNT/β-catenin signaling and tumor progression in COLO 320DM colon carcinoma xenografts upon oral administration of 0.33–10 mg/kg twice daily. In addition, OM-153 potentiates anti–programmed cell death protein 1 (anti–PD-1) immune checkpoint inhibition and antitumor effect in a B16-F10 mouse melanoma model. A 28-day repeated dose mouse toxicity study documents body weight loss, intestinal damage, and tubular damage in the kidney after oral–twice daily administration of 100 mg/kg. In contrast, mice treated oral–twice daily with 10 mg/kg show an intact intestinal architecture and no atypical histopathologic changes in other organs. In addition, clinical biochemistry and hematologic analyses do not identify changes indicating substantial toxicity. The results demonstrate OM-153–mediated antitumor effects and a therapeutic window in a colon carcinoma mouse model ranging from 0.33 to at least 10 mg/kg, and provide a framework for using OM-153 for further preclinical evaluations.Significance:This study uncovers the effectiveness and therapeutic window for a novel tankyrase inhibitor in mouse tumor models.

Confocal Raman spectral imaging (RSI) enables high-content, label-free visualization of a wide range of molecules in biological specimens without sample preparation. However, reliable quantification of the deconvoluted spectra is needed. Here we develop an integrated bioanalytical methodology, qRamanomics, to qualify RSI as a tissue phantom calibrated tool for quantitative spatial chemotyping of major classes of biomolecules. Next, we apply qRamanomics to fixed 3D liver organoids generated from stem-cell-derived or primary hepatocytes to assess specimen variation and maturity. We then demonstrate the utility of qRamanomics for identifying biomolecular response signatures from a panel of liver-altering drugs, probing drug-induced compositional changes in 3D organoids followed by in situ monitoring of drug metabolism and accumulation. Quantitative chemometric phenotyping constitutes an important step in developing quantitative label-free interrogation of 3D biological specimens.

The catalytic enzymes tankyrase 1 and 2 (TNKS1/2) alter protein turnover by poly-ADP-ribosylating target proteins, which earmark them for degradation by the ubiquitin–proteasomal system. Prominent targets of the catalytic activity of TNKS1/2 include AXIN proteins, resulting in TNKS1/2 being attractive biotargets for addressing of oncogenic WNT/β-catenin signaling. Although several potent small molecules have been developed to inhibit TNKS1/2, there are currently no TNKS1/2 inhibitors available in clinical practice. The development of tankyrase inhibitors has mainly been disadvantaged by concerns over biotarget-dependent intestinal toxicity and a deficient therapeutic window. Here we show that the novel, potent, and selective 1,2,4-triazole–based TNKS1/2 inhibitor OM-153 reduces WNT/β-catenin signaling and tumor progression in COLO 320DM colon carcinoma xenografts upon oral administration of 0.33–10 mg/kg twice daily. In addition, OM-153 potentiates anti–programmed cell death protein 1 (anti–PD-1) immune checkpoint inhibition and antitumor effect in a B16-F10 mouse melanoma model. A 28-day repeated dose mouse toxicity study documents body weight loss, intestinal damage, and tubular damage in the kidney after oral–twice daily administration of 100 mg/kg. In contrast, mice treated oral–twice daily with 10 mg/kg show an intact intestinal architecture and no atypical histopathologic changes in other organs. In addition, clinical biochemistry and hematologic analyses do not identify changes indicating substantial toxicity. The results demonstrate OM-153–mediated antitumor effects and a therapeutic window in a colon carcinoma mouse model ranging from 0.33 to at least 10 mg/kg, and provide a framework for using OM-153 for further preclinical evaluations. Significance: This study uncovers the effectiveness and therapeutic window for a novel tankyrase inhibitor in mouse tumor models.

Studies of monogenic diabetes are particularly useful because we can gain insight into the molecular events of pancreatic β-cell failure. Maturity-onset diabetes of the young 1 (MODY1) is a form of monogenic diabetes caused by a mutation in the HNF4A gene. Human-induced pluripotent stem cells (hiPSCs) provide an excellent tool for disease modeling by subsequently directing differentiation toward desired pancreatic islet cells, but cellular phenotypes in terminally differentiated cells are notoriously difficult to detect. Re-creating a spatial (three-dimensional [3D]) environment may facilitate phenotype detection. We studied MODY1 by using hiPSC-derived pancreatic β-like patient and isogenic control cell lines in two different 3D contexts. Using size-adjusted cell aggregates and alginate capsules, we show that the 3D context is critical to facilitating the detection of mutation-specific phenotypes. In 3D cell aggregates, we identified irregular cell clusters and lower levels of structural proteins by proteome analysis, whereas in 3D alginate capsules, we identified altered levels of glycolytic proteins in the glucose sensing apparatus by proteome analysis. Our study provides novel knowledge on normal and abnormal function of HNF4A, paving the way for translational studies of new drug targets that can be used in precision diabetes medicine in MODY.

Intra-portal islet transplantation is the method of choice for treatment of insulin dependent type 1 diabetes, but its outcome is hindered by limited islet survival due to the immunological and metabolic stress post transplantation. Adipose-derived stromal cells (ASCs) promise to improve significantly the islet micro-environment but an efficient long-term delivery method has not been achieved. We therefore explore the potential of generating ASC enriched islet transplant structure by 3D bioprinting. Here, we fabricate a double-layered 3D bioprinted scaffold for islets and ASCs by using alginate-nanofibrillated cellulose bioink. We demonstrate the diffusion properties of the scaffold and report that human ASCs increase the islet viability, preserve the endocrine function, and reduce pro-inflammatory cytokines secretion in vitro. Intraperitoneal implantation of the ASCs and islets in 3D bioprinted scaffold improve the long-term function of islets in diabetic mice. Our data reveals an important role for ASCs on the islet micro-environment. We suggest a novel cell therapy approach of ASCs combined with islets in a 3D structure with a potential for clinical beta cell replacement therapies at extrahepatic sites.

The discovery that long noncoding RNA TUNAR regulates β-cell proliferation may be important in designing new treatments for diabetes.

Objective β-cell replacement therapy (βCRT), including pancreas transplantation alone (PTA) and islet transplantation (ITX), is a treatment option for selected type 1 diabetes patients. All potential candidates for βCRT in Norway are referred to one national transplant centre for evaluation before any pre-transplant workup is started. This evaluation was performed by a transplant nephrologist alone prior to 2015 and by a multidisciplinary team (MDT) from 2015. We have reviewed the allocation of patients to treatment modality and the 1-year clinical outcome for the patients after transplantation. Research design and methods Medical charts of all patients evaluated for βCRT between 2010 and 2020 in Norway were retrospectively analysed and the outcome of patients receiving βCRT were studied. Results One hundred and forty-four patients were assessed for βCRT eligibility between 2010 and 2020. After MDT evaluation was introduced for βCRT eligibility in 2015, the percentage of referred patients accepted for the transplant waiting list fell from 84% to 40% (P < 0.005). One year after transplantation, 73% of the PTA and none of the ITX patients were independent of exogenous insulin, 8% of the PTA and 90% of the ITX patients had partial graft function while 19% of the PTA and 10% of the ITX patients suffered from graft loss. Conclusion The acceptance rate for βCRT was significantly reduced during a 10-year observation period and 81% of the PTA and 90% of the ITX patients had partial or normal graft function 1 year post-transplant.

Studies of monogenic diabetes are particularly useful as we can gain insight into the molecular events of pancreatic β-cell failure. Maturity-onset diabetes of the young 1 (MODY1) is a monogenic diabetes form, caused by a mutation in the HNF4A gene. Human induced pluripotent stem cells (hiPSC) provide an excellent tool for disease modelling by subsequent directed differentiation toward desired pancreatic islet cells, but cellular phenotypes in terminally differentiated cells are notoriously difficult to detect. Re-creating a spatial (3D) environment may facilitate phenotype detection. In this study, we studied MODY1 using hiPSC-derived pancreatic β-like patient and isogenic control cell lines in two different 3D contexts. Using size-adjusted cell aggregates and alginate capsules we showed that the 3D context was critical to facilitate the detection of mutation-specific phenotypes. In 3D cell aggregates we identified irregular cell clusters and lower levels of structural proteins by proteome analysis, whereas in 3D alginate capsules we identified altered levels of glycolytic proteins in the glucose sensing apparatus by proteome analysis. Our study provides novel knowledge on normal and abnormal function of HNF4A paving the way for translational studies of new drug targets that can be used in precision diabetes medicine of MODY.

Organoids are laboratory-grown 3D organ models, mimicking human organs for e.g. drug development and personalized therapy. Islet organoids (typically 100-200 μm), which can be grown from the patient’s own cells, are emerging as prototypes for transplantation-based therapy of diabetes. Selective methods for quantifying insulin production from islet organoids are needed, but sensitivity and carry-over have been major bottlenecks in previous efforts. We have developed a reverse phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) method for studying the insulin secretion of islet organoids. In contrast to our previous attempts using nano-scale LC columns, conventional 2.1 mm inner diameter LC column (combined with triple quadrupole mass spectrometry) was well suited for sensitive and selective measurements of insulin secreted from islet organoids with low microliter-scale samples. Insulin is highly prone to carry-over, so standard tubings and injector parts were replaced with shielded fused silica nanoViper™ connectors. As samples were expected to be very limited, an extended Box-Behnken experimental design for the MS settings was conducted to maximize performance. The finale method has excellent sensitivity, accuracy and precision (limit of detection: ≤ 0.2 pg/μL, relative error: ≤ ±10%, relative standard deviation: < 10%), and was well suited for measuring 20 μL amounts of Krebs buffer containing insulin secreted from islet organoids.

The generation of human mini-organs, the so-called organoids, is one of the biggest scientific advances in regenerative medicine. This technology exploits traditional three-dimensional culture techniques that support cell-autonomous self-organization responses of stem cells to derive micrometer to millimeter size versions of human organs. The convergence of the organoid technology with organ transplantation is still in its infancy but this alliance is expected to open new venues to change the way we conduct both transplant and organoid research. In this Forum we provide a summary on early achievements facilitating organoid derivation and culture. We further discuss on early advances of organoid transplantation also offering a comprehensive overview of current limitations and challenges to instruct organoid maturation. We expect that this Forum sets the ground for initial discussions between stem cell biologists, bioengineers, and the transplant community to better direct organoid basic research to advance the organ transplantation field.

The catalytic enzymes tankyrase 1 and 2 (TNKS1/2) poly-ADP-ribosylate target proteins, including AXIN proteins, to earmark them for degradation by the ubiquitin-proteasomal system. Hence, inhibition of TNKS1/2 can stabilize AXIN proteins, and consequently β-catenin degradosomes, resulting in inhibition of WNT/β-catenin signaling. Although several potent small-molecules have been developed to inhibit TNKS1/2 and oncogenic WNT/β-catenin signaling, also showing convincing anti-tumor effects in numerous mouse cancer models, there are currently no TNKS1/2 inhibitors available in the clinic. The development has mainly been disadvantaged by concerns over previous reports unfolding intestinal toxicity, apparently caused by on-target and WNT/β-catenin signaling pathway-specific side effects, and a deficient therapeutic index. Here we show that the novel, highly potent and selective1,2,4-triazole-based TNKS1/2 inhibitor OM-153, displaying improved ADME-properties and mouse pharmacokinetics, reduced WNT/β-catenin signaling and tumor progression in COLO 320DM colon cancer xenografts upon peroral (PO) administration of 0.33-10 mg/kg twice daily (BID). In addition, combined OM-153 and anti-PD-1 treatment conferred a synergistic anti-tumor effect in a B16-F10 mouse melanoma model. In a 28-day repeated dose mouse toxicity study, body weight loss, intestinal damage and tubular damage in the kidney was documented after PO BID administration of 100 mg/kg. In contrast, in mice treated PO BID using 10 mg/kg, the intestinal architecture was intact and no atypical histopathological changes were observed in other organs, while clinical biochemistry and haematological analyses did not identify any changes indicating considerable toxicity. The results provide a scaffold for using OM-153 as a potential anti-cancer treatment and additional preclinical and clinical evaluations. Citation Format: Shoshy Alam Brinch, Enya Amundsen-Isaksen, Sandra Espada, Aleksandra Aizenshtadt, Lone Holmen, Clara Hammarström, Merete Høyem, Hanne Scholz, Gunnveig Grødeland, Sven T. Sowa, Albert Galera-Prat, Lari Lehtiö, Ilonka A.T.M. Meerts, Ruben G. G. Leenders, Anita Wegert, Stefan Krauss, Jo Waaler. The tankyrase inhibitor OM-153 demonstrates anti-tumor effect with a therapeutic index above 10 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2651.

Inflammasomes are multiprotein inflammatory platforms that induce caspase-1 activation and subsequently interleukin (IL)-1β and IL-18 processing. The NLRP3 inflammasome is activated by different forms of oxidative stress, and, based on the central role of IL-1β in the destruction of pancreatic islets, it could be related to the development of diabetes. We therefore investigated responses in wild-type C57Bl/6 (WT) mice, NLRP3−/− mice, and mice deficient in apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) after exposing islets to short-term hypoxia or alloxan-induced islet damage. NLRP3-deficient islets compared with WT islets had preserved function ex vivo and were protected against hypoxia-induced cell death. Furthermore, NLRP3 and ASC-deficient mice were protected against oxidative stress-induced diabetes caused by repetitive low-dose alloxan administration, and this was associated with reduced β-cell death and reduced macrophage infiltration. This suggests that the beneficial effect of NLRP3 inflammasome deficiency on oxidative stress-mediated β-cell damage could involve reduced macrophage infiltration and activation. To support the role of macrophage activation in alloxan-induced diabetes, we injected WT mice with liposomal clodronate, which causes macrophage depletion before induction of a diabetic phenotype by alloxan treatment, resulting in improved glucose homeostasis in WT mice. We show here that the NLRP3 inflammasome acts as a mediator of hypoxia and oxidative stress in insulin-producing cells, suggesting that inhibition of the NLRP3 inflammasome could have beneficial effects on β-cell preservation.

The past decade revealed that cell identity changes, such as dedifferentiation or transdifferentiation, accompany the insulin-producing β-cell decay in most diabetes conditions. Mapping and controlling the mechanisms governing these processes is, thus, extremely valuable for managing the disease progression. Extracellular glucose is known to influence cell identity by impacting the redox balance. Here, we use global proteomics and pathway analysis to map the response of differentiating human pancreatic progenitors to chronically increased in vitro glucose levels. We show that exogenous high glucose levels impact different protein subsets in a concentration-dependent manner. In contrast, regardless of concentration, glucose elicits an antipodal effect on the proteome landscape, inducing both beneficial and detrimental changes in regard to achieving the desired islet cell fingerprint. Furthermore, we identified that only a subgroup of these effects and pathways are regulated by changes in redox balance. Our study highlights a complex effect of exogenous glucose on differentiating pancreas progenitors characterized by a distinct proteome signature. publishedVersion

Purpose of Review Beta cell replacement therapy as a form of islet transplantation is a promising alternative therapy with the possibility to make selected patients with type 1 diabetes (T1D) insulin independent. However, this technique faces challenges such as extensive activation of the host immune system post-transplantation, lifelong need for immunosuppression, and the scarcity of islet donor pancreas. Advancement in tissue engineering strategies can improve these challenges and allow for a more widespread application of this therapy. This review will discuss the recent development and clinical translation of tissue engineering strategies in beta cell replacement therapy. Recent Findings Tissue engineering offers innovative solutions for producing unlimited glucose responsive cells and fabrication of appropriate devices/scaffolds for transplantation applications. Generation of pancreatic organoids with supporting cells in biocompatible biomaterials is a powerful technique to improve the function of insulin-producing cell clusters. Fabrication of physical barriers such as encapsulation strategies can protect the cells from the host immune system and allow for graft retrieval, although this strategy still faces major challenges to fully restore physiological glucose regulation. Summary The three main components of tissue engineering strategies including the generation of stem cell-derived insulin-producing cells and organoids and the possibilities for therapeutic delivery of cell-seeded devices to extra-hepatic sites need to come together in order to provide safe and functional insulin-producing devices for clinical beta cell replacement therapy.

There have been several studies investigating whether musical sound can be used as cell stimuli in recent years. We systematically searched publications to get an overview of studies that have used audible sound played through speaker-based systems to induce mechanical perturbation in cell cultures. A total of 12 studies were identified. We focused on the experimental setups, the sound materials used as stimuli, and the outcomes. The stimuli were categorized into simple and complex sounds. The effects were reported as enhanced cell migration, proliferation, colony formation, and differentiation ability. However, there are significant differences in methodologies and cell type-specific outcomes, which made it difficult to find a systematic pattern in the results. We suggest that future experiments should consider using: 1) a more controlled acoustic environment), 2) standardized sound and noise measurement methods, and 3) a more comprehensive range of controlled sound stimuli.

Previous studies in rodents have indicated that function and survival of transplanted islets can be substantially improved by mesenchymal stem cells (MSC). The few human islet studies to date have confirmed these findings but have not determined whether physical contact between MSC and islets is required or whether the benefit to islets results from MSC-secreted proteins. This study aimed to investigate the protective capacity of MSC-preconditioned media for human islets. MSC were cultured for 2 or 5 days in normoxia or hypoxia before harvesting the cell-depleted media for human islet culture in normoxia or hypoxia for 6–8 or 3–4 days, respectively. To characterize MSC-preconditioned media, proteomic secretome profiling was performed to identify angiogenesis- and inflammation-related proteins. A protective effect of MSC-preconditioned media on survival and in vitro function of hypoxic human islets was observed irrespective of the atmosphere used for MSC preconditioning. Islet morphology changed markedly when media from hypoxic MSC were used for culture. However, PDX-1 and insulin gene expression did not confirm a change in the genetic phenotype of these islets. Proteomic profiling of preconditioned media revealed the heterogenicity of the secretome comprising angiogenic and antiapoptotic as well as angiostatic or proinflammatory mediators released at an identical pattern regardless whether MSC had been cultured in normoxic or hypoxic atmosphere. These findings do not allow a clear discrimination between normoxia and hypoxia as stimulus for protective MSC capabilities but indicate an ambivalent character of the MSC angiogenesis- and inflammation-related secretome. Nevertheless, culture of human islets in acellular MSC-preconditioned media resulted in improved morphological and functional islet integrity suggesting a disbalance in favor of protective factors. Further approaches should aim to eliminate potentially detrimental factors to enable the production of advanced clinical grade islet culture media with higher protective qualities.

Generating insulin-producing β-cells from human induced pluripotent stem cells is a promising cell replacement therapy for improving or curing insulin-dependent diabetes. The transplantation of end-stages differentiating cells into living hosts was demonstrated to improve β-cell maturation. Nevertheless, the cellular and molecular mechanisms outlining the transplanted cells’ response to the in vivo environment are still to be properly characterized. Here we use global proteomics and large-scale imaging techniques to demultiplex and filter the cellular processes and molecular signatures modulated by the immediate in vivo effect. We show that in vivo exposure swiftly confines in vitro generated human pancreatic progenitors to single hormone expression. The global proteome landscape of the transplanted cells was closer to native human islets, especially in regard to energy metabolism and redox balance. Moreover, our study indicates a possible link between these processes and certain epigenetic regulators involved in cell identity. Pathway analysis predicted HNF1A and HNF4A as key regulators controlling the in vivo islet-promoting response, with experimental evidence suggesting their involvement in confining islet cell fate following xeno-transplantation. publishedVersion

Aim: The loss of insulin‐secreting β‐cells, ultimately characterizing most diabetes forms, demands the development of cell replacement therapies. The common endpoint for all ex vivo strategies is transplantation into diabetic patients. However, the effects of hyperglycaemia environment on the transplanted cells were not yet properly assessed. Thus, the main goal of this study was to characterize global effect of brief and prolonged in vivo hyperglycaemia exposure on the cell fate acquisition and maintenance of transplanted human pancreatic progenitors. Methods: To rigorously study the effect of hyperglycaemia, in vitro differentiated human‐induced pluripotent stem cells (hiPSC)‐derived pancreatic progenitors were xenotransplanted in normoglycaemic and diabetic NSG rat insulin promoter (RIP)‐diphtheria toxin receptor (DTR) mice. The transplants were retrieved after 1‐week or 1‐month exposure to overt hyperglycaemia and analysed by large‐scale microscopy or global proteomics. For this study we pioneer the use of the NSG RIP‐DTR system in the transplantation of hiPSC, making use of its highly reproducible specific and absolute β‐cell ablation property in the absence of inflammation or other organ toxicity. Results: Here we show for the first time that besides the presence of an induced oxidative stress signature, the cell fate and proteome landscape response to hyperglycaemia was different, involving largely different mechanisms, according to the period spent in the hyperglycaemic environment. Surprisingly, brief hyperglycaemia exposure increased the bihormonal cell number by impeding the activity of specific islet lineage determinants. Moreover, it activated antioxidant and inflammation protection mechanisms signatures in the transplanted cells. In contrast, the prolonged exposure was characterized by decreased numbers of hormone + cells, low/absent detoxification signature, augmented production of oxygen reactive species and increased apoptosis. Conclusion: Hyperglycaemia exposure induced distinct, period‐dependent, negative effects on xenotransplanted human pancreatic progenitor, affecting their energy homeostasis, cell fate acquisition and survival. publishedVersion

Despite increasing experience with islet allotransplantation for type 1 diabetes worldwide, outcomes remain variable and unpredictable. Mesenchymal stem cells (MSCs) have been shown to positively affect islets and improve transplantation outcomes in animal models and in a few clinical trials. MSCs are easily isolated from multiple tissues and expanded through ex vivo culture with preservation of their self-renewal capacity. The safety and feasibility of using MSCs-based cellular treatments for different conditions including diabetes have been confirmed in many clinical trials. With the favorable characteristics of MSCs that include both anti-inflammatory and immunomodulatory properties, the research interest of utilizing MSCs for improvement of a different aspect of the islet transplant procedure is increasing. We summarize the current knowledge available from MSCs-based therapy in preclinical and clinical islet transplantation, focus on the advantage for the islets, and how to ensure progress in this aspect of research, and finally discuss remaining questions and future perspectives.

Cell replacement therapies hold great therapeutic potential. Nevertheless, our knowledge of the mechanisms governing the developmental processes is limited, impeding the quality of differentiation protocols. Generating insulin-expressing cells in vitro is no exception, with the guided series of differentiation events producing heterogeneous cell populations that display mixed pancreatic islet phenotypes and immaturity. The achievement of terminal differentiation ultimately requires the in vivo transplantation of, usually, encapsulated cells. Here we show the impact of cell confinement on the pancreatic islet signature during the guided differentiation of alginate encapsulated human induced pluripotent stem cells (hiPSCs). Our results show that encapsulation improves differentiation by significantly reshaping the proteome landscape of the cells towards an islet-like signature. Pathway analysis is suggestive of integrins transducing the encapsulation effect into intracellular signalling cascades promoting differentiation. These analyses provide a molecular framework for understanding the confinement effects on hiPSCs differentiation while confirming its importance for this process. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.