No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
A Method for Encapsulation and Transplantation into Diabetic Mice of Human Induced Pluripotent Stem Cells (hiPSC)-Derived Pancreatic Progenitors
Abstract
Pancreatic islet endocrine cells generated from patient-derived induced pluripotent stem cells represent a great strategy for both disease modeling and regenerative medicine. Nevertheless, these cells inherently miss the effects of the intricate network of systemic signals characterizing the living organisms. Xenotransplantation of in vitro differentiating cells into murine hosts substantially compensates for this drawback.Here we describe our transplantation strategy of encapsulated differentiating pancreatic progenitors into diabetic immunosuppressed (NSG) overtly diabetic mice generated by the total ablation of insulin-producing cells following diphtheria toxin administration. We will detail the differentiation protocol employed, the alginate encapsulation procedure, and the xenotransplantation steps required for a successful and reproducible experiment.
Non-obese diabetes (NOD) mice are an established, spontaneous model of type 1 diabetes in which diabetes develops through insulitis. Using next-generation sequencing, coupled with pathway analysis, the molecular fingerprint of early insulitis was mapped in a cohort of mice ranging from 4 to 12 weeks of age. The resulting dynamic timeline revealed an initial decrease in proliferative capacity followed by the emergence of an inflammatory signature between 6 and 8 weeks that increased to a regulatory plateau between 10 and 12 weeks. The inflammatory signature is identified by the activation of central immunogenic factors such as Infg, Il1b, and Tnfa, and activation of canonical inflammatory signaling. Analysis of the regulatory landscape revealed the transcription factor Atf3 as a potential novel modulator of inflammatory signaling in the NOD islets. Furthermore, the Hedgehog signaling pathway correlated with Atf3 regulation, suggesting that the two play a role in regulating islet inflammation; however, further studies are needed to establish the nature of this connection.
Abbreviations used in this paper: GLI, glioma-associated oncogene; HH, hedgehog ; hiPSC, human induced pluripotent stem cell; HNF, hepatocyte nuclear factor. Int. J. Dev. Biol. In Press ABSTRACT Differentiation of human induced pluripotent stem cells towards pancreatic islet endocrine cells is a complex process, involving the stepwise modulation of key developmental pathways, such as the Hedgehog signaling inhibition during early differentiation stages. In tandem with this active inhibition, key transcription factors for the islet endocrine cell fate, such as HNF1A, show specific changes in their expression patterns. Here we designed a pilot study aimed at investigating the potential interconnection between HH-signaling inhibition and the increase in the HNF1A expression during early regeneration, by inducing changes in the GLI code. This unveiled a link between the two, where GLI3-R mediated Hedgehog target genes inhibition is apparently required for HNF1A efficient expression.
Aim:
The variation in quality between the human islet samples represents a major problem for research, especially when used as control material. The assays assessing the quality of human islets used in research are non-standardized and limited, with many important parameters not being consistently assessed. High-throughput studies aimed at characterizing the diversity and segregation markers among apparently functionally healthy islet preps are thus a requirement. Here, we designed a pilot study to comprehensively identify the diversity of global proteome signatures and the deviation from normal homeostasis in randomly selected human-isolated islet samples.
Methods:
By using Tandem Mass Tag 16-plex proteomics, we focused on the recurrently observed disparity in the detected insulin abundance between the samples, used it as a segregating parameter, and analyzed the correlated changes in the proteome signature and homeostasis by pathway analysis.
Results:
In this pilot study, we showed that insulin protein abundance is a predictor of human islet homeostasis and quality. This parameter is independent of other quality predictors within their acceptable range, thus being able to further stratify islets samples of apparent good quality. Human islets with low amounts of insulin displayed changes in their metabolic and signaling profile, especially in regard to energy homeostasis and cell identity maintenance. We further showed that xenotransplantation into diabetic hosts is not expected to improve the pre-transplantation signature, as it has a negative effect on energy balance, antioxidant activity, and islet cell identity.
Conclusions:
Insulin protein abundance predicts significant changes in human islet homeostasis among random samples of apparently good quality.
Induced pluripotent stem cells as a source for generating pancreatic islet endocrine cells represent a great research tool for deciphering the molecular mechanisms of lineage commitment, a layered multi-step process. Additionally, targeted gene silencing by using GapmeRs, short antisense oligonucleotides, proved instrumental in studying the role of different developmental genes. Here we describe our approach to induce mTOR silencing by using specific GapmeRs during the differentiation of induced pluripotent stem cells toward pancreatic progenitors. We will describe our current differentiation protocol, the transfection procedure, and the quality control steps required for a successful experiment.Key wordsGapmeRGene silencingHuman iPSCIn vitro differentiationIslet endocrine cellsPancreatic endocrine progenitors
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.
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.
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.
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 hyperglycemia 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 hyperglycemia exposure on the cell fate acquisition and maintenance of transplanted human pancreatic progenitors.
Methods:
To rigorously study the effect of hyperglycemia, in vitro differentiated human induced pluripotent stem cells (hiPSC)-derived pancreatic progenitors were xenotransplanted in normoglycemic and diabetic NSG RIP-DTR mice. The transplants were retrieved after one-week or one-month exposure to overt hyperglycemia and analyzed 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 hyperglycemia was different, involving largely different mechanisms, according to the period spent in the hyperglycemic environment. Surprisingly, brief hyperglycemia 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:
Hyperglycemia exposure induced distinct, period-dependent, negative effects on xenotransplanted human pancreatic progenitor, affecting their energy homeostasis, cell fate acquisition and survival.
Cell-identity switches, in which terminally differentiated cells are converted into different cell types when stressed, represent a widespread regenerative strategy in animals, yet they are poorly documented in mammals. In mice, some glucagon-producing pancreatic α-cells and somatostatin-producing δ-cells become insulin-expressing cells after the ablation of insulin-secreting β-cells, thus promoting diabetes recovery. Whether human islets also display this plasticity, especially in diabetic conditions, remains unknown. Here we show that islet non-β-cells, namely α-cells and pancreatic polypeptide (PPY)-producing γ-cells, obtained from deceased non-diabetic or diabetic human donors, can be lineage-traced and reprogrammed by the transcription factors PDX1 and MAFA to produce and secrete insulin in response to glucose. When transplanted into diabetic mice, converted human α-cells reverse diabetes and continue to produce insulin even after six months. Notably, insulin-producing α-cells maintain expression of α-cell markers, as seen by deep transcriptomic and proteomic characterization. These observations provide conceptual evidence and a molecular framework for a mechanistic understanding of in situ cell plasticity as a treatment for diabetes and other degenerative diseases.
The mechanisms that restrict regeneration and maintain cell identity following injury are poorly characterized in higher vertebrates. Following β-cell loss, 1–2% of the glucagon-producing α-cells spontaneously engage in insulin production in mice. Here we explore the mechanisms inhibiting α-cell plasticity. We show that adaptive α-cell identity changes are constrained by intra-islet insulin- and Smoothened-mediated signalling, among others. The combination of β-cell loss or insulin-signalling inhibition, with Smoothened inactivation in α- or δ-cells, stimulates insulin production in more α-cells. These findings suggest that the removal of constitutive ‘brake signals’ is crucial to neutralize the refractoriness to adaptive cell-fate changes. It appears that the maintenance of cell identity is an active process mediated by repressive signals, which are released by neighbouring cells and curb an intrinsic trend of differentiated cells to change.
The pancreas is made from two distinct components: the exocrine pancreas, a reservoir of digestive enzymes, and the endocrine islets, the source of the vital metabolic hormone insulin. Human islets possess limited regenerative ability; loss of islet β-cells in diseases such as type 1 diabetes requires therapeutic intervention. The leading strategy for restoration of β-cell mass is through the generation and transplantation of new β-cells derived from human pluripotent stem cells. Other approaches include stimulating endogenous β-cell proliferation, reprogramming non-β-cells to β-like cells, and harvesting islets from genetically engineered animals. Together these approaches form a rich pipeline of therapeutic development for pancreatic regeneration.
Axolotl salamanders are powerful models for understanding how regeneration of complex body parts can be achieved, whereas mammals are severely limited in this ability. Factors that promote normal axolotl regeneration can be examined in mammals to determine if they exhibit altered activity in this context. Furthermore, factors prohibiting axolotl regeneration can offer key insight into the mechanisms present in regeneration-incompetent species. We sought to determine if we could experimentally compromise the axolotl’s ability to regenerate limbs and, if so, discover the molecular changes that might underlie their inability to regenerate. We found that repeated limb amputation severely compromised axolotls’ ability to initiate limb regeneration. Using RNA-seq, we observed that a majority of differentially expressed transcripts were hyperactivated in limbs compromised by repeated amputation, suggesting that mis-regulation of these genes antagonizes regeneration. To confirm our findings, we additionally assayed the role of amphiregulin, an EGF-like ligand, which is aberrantly upregulated in compromised animals. During normal limb regeneration, amphiregulin is expressed by the early wound epidermis, and mis-expressing this factor lead to thickened wound epithelium, delayed initiation of regeneration, and severe regenerative defects. Collectively, our results suggest that repeatedly amputated limbs may undergo a persistent wound healing response, which interferes with their ability to initiate the regenerative program. These findings have important implications for human regenerative medicine.
Key points:
We used a mouse expressing a light-sensitive ion channel in β-cells to understand how α-cell activity is regulated by β-cells. Light activation of β-cells triggered a suppression of α-cell activity via gap junction-dependent activation of δ-cells. Mathematical modelling of human islets suggests that 23% of the inhibitory effect of glucose on glucagon secretion is mediated by β-cells via gap junction-dependent activation of δ-cells/somatostatin secretion.
Abstract:
Glucagon, the body's principal hyperglycaemic hormone, is released from α-cells of the pancreatic islet. Secretion of this hormone is dysregulated in type 2 diabetes mellitus but the mechanisms controlling secretion are not well understood. Regulation of glucagon secretion by factors secreted by neighbouring β- and δ-cells (paracrine regulation) have been proposed to be important. In this study, we explored the importance of paracrine regulation by using an optogenetic strategy. Specific light-induced activation of β-cells in mouse islets expressing the light-gated channelrhodopsin-2 resulted in stimulation of electrical activity in δ-cells but suppression of α-cell activity. Activation of the δ-cells was rapid and sensitive to the gap junction inhibitor carbenoxolone, whereas the effect on electrical activity in α-cells was blocked by CYN 154806, an antagonist of the somatostatin-2 receptor. These observations indicate that optogenetic activation of the β-cells propagates to the δ-cells via gap junctions, and the consequential stimulation of somatostatin secretion inhibits α-cell electrical activity by a paracrine mechanism. To explore whether this pathway is important for regulating α-cell activity and glucagon secretion in human islets, we constructed computational models of human islets. These models had detailed architectures based on human islets and consisted of a collection of >500 α-, β- and δ-cells. Simulations of these models revealed that this gap junctional/paracrine mechanism accounts for up to 23% of the suppression of glucagon secretion by high glucose.
MODY1 is a maturity-onset monogenic diabetes, caused by heterozygous mutations of the HNF4A gene. To date the cellular and molecular mechanisms leading to disease onset remain largely unknown. In this study, we demonstrate that insulin-positive cells can be generated in vitro from human induced pluripotent stem cells (hiPSCs) derived from patients carrying a non-sense HNF4A mutation, proving for the first time, that a human HNF4A mutation is neither blocking the expression of the insulin genes nor the development of insulin-producing cells in vitro. However, regardless of the mutation or diabetes status, these insulin-producing cells are immature, a common downfall off most current β-cell differentiation protocols. To further address the immature state of the cells, in vitro differentiated cells and adult human islets were compared by global proteomic analysis. We report the predicted upstream regulators and signalling pathways characterizing the proteome landscape of each entity. Subsequently, we focused on the molecular components absent or misregulated in the in vitro differentiated cells, to probe the components involved in the deficient in vitro maturation towards fully functional β-cells. This analysis identified the modulation of key developmental signalling pathways representing potential targets for improving the efficiency of the current differentiation protocols.
Pluripotent human embryonic stem cells (hESCs) are a potential source of transplantable cells for treating patients with diabetes. To investigate the impact of the host recipient on hESC-derived pancreatic progenitor cell maturation, cells were transplanted into immunodeficient SCID-beige mice or nude rats. Following the transplant, basal human C-peptide levels were consistently higher in mice compared with rats, but only rats showed robust meal- and glucose-responsive human C-peptide secretion by 19–21 weeks. Grafts from rats contained a higher proportion of insulin:glucagon immunoreactivity, fewer exocrine cells, and improved expression of mature β cell markers compared with mice. Moreover, ECM-related genes were enriched, the collagen network was denser, and blood vessels were more intricately integrated into the engrafted endocrine tissue in rats relative to mice. Overall, hESC-derived pancreatic progenitor cells matured faster in nude rats compared with SCID-beige mice, indicating that the host recipient can greatly influence the fate of immature pancreatic progenitor cells post-transplantation.
Objective
Glucagon-like peptide-1 induces glucose-dependent insulin secretion and, in rodents, increases proliferation and survival of pancreatic beta cells. To investigate the effects on human beta cells, we used immunodeficient mice transplanted with human islets. The goal was to determine whether lixisenatide, a glucagon-like peptide-1 receptor agonist, improves human islet function and survival in vivo.
Methods
Five independent transplant studies were conducted with human islets from five individual donors. Diabetic human islet-engrafted immunodeficient mice were treated with lixisenatide (50, 150, and 500 µg/kg) or vehicle. Islet function was determined by blood glucose, plasma human insulin/C-peptide, and glucose tolerance tests. Grafts were analyzed for total beta- and alpha-cell number, percent proliferation, and levels of apoptosis.
Results
Diabetic mice transplanted with marginal human islet mass and treated with lixisenatide were restored to euglycemia more rapidly than vehicle-treated mice. Glucose tolerance tests, human plasma insulin, and glucose-stimulation indices of lixisenatide-treated mice were significantly improved compared to vehicle-treated mice. The percentages of proliferating or apoptotic beta cells at graft recovery were not different between lixisenatide-treated and vehicle-treated mice. Nevertheless, in one experiment we found a significant twofold to threefold increase in human beta-cell numbers in lixisenatide-treated compared to vehicle-treated mice.
Conclusion
Diabetic human islet-engrafted immunodeficient mice treated with lixisenatide show improved restoration of normoglycemia, human plasma insulin, and glucose tolerance compared to vehicle-treated mice engrafted with the same donor islets. Because the proliferative capacity of human beta cells is limited, improved beta-cell survival coupled with enhanced beta-cell function following lixisenatide treatment may provide the greatest benefit for diabetic patients with reduced functional islet mass.
This review compiles information regarding the use of alginate, and in particular alginate hydrogels, in culturing cells in 3D. Knowledge of alginate chemical structure and functionality are shown to be important parameters in design of alginate-based matrices for cell culture. Gel elasticity as well as hydrogel stability can be impacted by the type of alginate used, its concentration, the choice of gelation technique (ionic or covalent), and divalent cation chosen as the gel inducing ion. The use of peptide-coupled alginate can control cell–matrix interactions. Gelation of alginate with concomitant immobilization of cells can take various forms. Droplets or beads have been utilized since the 1980s for immobilizing cells. Newer matrices such as macroporous scaffolds are now entering the 3D cell culture product market. Finally, delayed gelling, injectable, alginate systems show utility in the translation of in vitro cell culture to in vivo tissue engineering applications. Alginate has a history and a future in 3D cell culture. Historically, cells were encapsulated in alginate droplets cross-linked with calcium for the development of artificial organs. Now, several commercial products based on alginate are being used as 3D cell culture systems that also demonstrate the possibility of replacing or regenerating tissue.
Directed differentiation of human pluripotent stem cells into functional insulin-producing beta-like cells holds great promise for cell replacement therapy for patients suffering from diabetes. This approach also offers the unique opportunity to study otherwise inaccessible aspects of human beta cell development and function in vitro. Here, we show that current pancreatic progenitor differentiation protocols promote precocious endocrine commitment, ultimately resulting in the generation of non-functional polyhormonal cells. Omission of commonly used BMP inhibitors during pancreatic specification prevents precocious endocrine formation while treatment with retinoic acid followed by combined EGF/KGF efficiently generates both PDX1(+) and subsequent PDX1(+)/NKX6.1(+) pancreatic progenitor populations, respectively. Precise temporal activation of endocrine differentiation in PDX1(+)/NKX6.1(+) progenitors produces glucose-responsive beta-like cells in vitro that exhibit key features of bona fide human beta cells, remain functional after short-term transplantation, and reduce blood glucose levels in diabetic mice. Thus, our simplified and scalable system accurately recapitulates key steps of human pancreas development and provides a fast and reproducible supply of functional human beta-like cells.
© 2015 The Authors.
Transplantation of pancreatic progenitors or insulin-secreting cells derived from human embryonic stem cells (hESCs) has been proposed as a therapy for diabetes. We describe a seven-stage protocol that efficiently converts hESCs into insulin-producing cells. Stage (S) 7 cells expressed key markers of mature pancreatic beta cells, including MAFA, and displayed glucose-stimulated insulin secretion similar to that of human islets during static incubations in vitro. Additional characterization using single-cell imaging and dynamic glucose stimulation assays revealed similarities but also notable differences between S7 insulin-secreting cells and primary human beta cells. Nevertheless, S7 cells rapidly reversed diabetes in mice within 40 days, roughly four times faster than pancreatic progenitors. Therefore, although S7 cells are not fully equivalent to mature beta cells, their capacity for glucose-responsive insulin secretion and rapid reversal of diabetes in vitro makes them a promising alternative to pancreatic progenitor cells or cadaveric islets for the treatment of diabetes.
Total or near-total loss of insulin-producing β-cells occurs in type 1 diabetes. Restoration of insulin production in type 1 diabetes is thus a major medical challenge. We previously observed in mice in which β-cells are completely ablated that the pancreas reconstitutes new insulin-producing cells in the absence of autoimmunity. The process involves the contribution of islet non-β-cells; specifically, glucagon-producing α-cells begin producing insulin by a process of reprogramming (transdifferentiation) without proliferation. Here we show the influence of age on β-cell reconstitution from heterologous islet cells after near-total β-cell loss in mice. We found that senescence does not alter α-cell plasticity: α-cells can reprogram to produce insulin from puberty through to adulthood, and also in aged individuals, even a long time after β-cell loss. In contrast, before puberty there is no detectable α-cell conversion, although β-cell reconstitution after injury is more efficient, always leading to diabetes recovery. This process occurs through a newly discovered mechanism: the spontaneous en masse reprogramming of somatostatin-producing δ-cells. The juveniles display 'somatostatin-to-insulin' δ-cell conversion, involving dedifferentiation, proliferation and re-expression of islet developmental regulators. This juvenile adaptability relies, at least in part, upon the combined action of FoxO1 and downstream effectors. Restoration of insulin producing-cells from non-β-cell origins is thus enabled throughout life via δ- or α-cell spontaneous reprogramming. A landscape with multiple intra-islet cell interconversion events is emerging, offering new perspectives for therapy.
Diabetes is characterized by "glucotoxic" loss of pancreatic β cell function and insulin content, but underlying mechanisms remain unclear. A mouse model of insulin-secretory deficiency induced by β cell inexcitability (KATP gain of function) demonstrates development of diabetes and reiterates the features of human neonatal diabetes. In the diabetic state, β cells lose their mature identity and dedifferentiate to neurogenin3-positive and insulin-negative cells. Lineage-tracing experiments show that dedifferentiated cells can subsequently redifferentiate to mature neurogenin3-negative, insulin-positive β cells after lowering of blood glucose by insulin therapy. We demonstrate here that β cell dedifferentiation, rather than apoptosis, is the main mechanism of loss of insulin-positive cells, and redifferentiation accounts for restoration of insulin content and antidiabetic drug responsivity in these animals. These results may help explain gradual decrease in β cell mass in long-standing diabetes and recovery of β cell function and drug responsivity in type 2 diabetic patients following insulin therapy, and they suggest an approach to rescuing "exhausted" β cells in diabetes.
Human embryonic stem cells (hESCs) were used as a model system of human pancreas development to study characteristics of the polyhormonal cells that arise during fetal pancreas development. HESCs were differentiated into fetal-like pancreatic cells in vitro using a 33-day, 7-stage protocol. Cultures were ~90-95% PDX1-positive by day (d) 11 and 70-75% NKX6.1-positive by d17. Polyhormonal cells were scattered at d17, but developed into islet-like clusters that expressed key transcription factors by d33. Human C-peptide and glucagon secretion were first detected at d17 and increased thereafter in parallel with INS and GCG transcript levels. HESC-derived cells were responsive to KCl and arginine, but not glucose in perifusion studies. Compared to adult human islets, hESC-derived cells expressed ~10-fold higher levels of glucose transporter 1 (GLUT1) mRNA, but similar levels of glucokinase (GCK). In situ hybridization confirmed the presence of GLUT1 transcript within endocrine cells. However, GLUT1 protein was excluded from this population and was instead observed predominantly in non-endocrine cells, whereas GCK was co-expressed in insulin-positive cells. In rubidium efflux assays, hESC-derived cells displayed mild potassium channel activity, but no responsiveness to glucose, metabolic inhibitors or glibenclamide. Western blotting experiments revealed that the higher molecular weight SUR1 band was absent in hESC-derived cells, suggesting a lack of functional KATP channels at the cell surface. In addition, KATP channel subunit transcript levels were not at a 1:1 ratio, as would be expected (SUR1 levels were ~5-fold lower than KIR6.2). Various ratios of SUR1:KIR6.2 plasmids were transfected into COSM6 cells and rubidium efflux was found to be particularly sensitive to a reduction in SUR1. These data suggest that an impaired ratio of SUR1:KIR6.2 may contribute to the observed KATP channel defects in hESC-derived islet endocrine cells, and along with lack of GLUT1, may explain the absence of glucose-stimulated insulin secretion.
Conversion of one terminally differentiated cell type into another (or transdifferentiation) usually requires the forced expression of key transcription factors. We examined the plasticity of human insulin-producing β-cells in a model of islet cell aggregate formation. Here we show that primary human β-cells can undergo a conversion into glucagon-producing α-cells without introduction of any genetic modification. The process occurs within days as revealed by lentivirus-mediated β-cell lineage tracing. Converted cells are indistinguishable from native α-cells based on ultrastructural morphology and maintain their α-cell phenotype after transplantation in vivo. Transition of β-cells into α-cells occurs following β-cell degranulation and is characterized by the presence of β-cell specific transcription factors Pdx1 and Nkx6.1 in glucagon(+)-cells. Finally, we show that lentivirus-mediated knockdown of Arx, a determinant of the α-cell lineage, inhibits the conversion. Our findings reveal an unknown plasticity of human adult endocrine cells that can be modulated. This endocrine cell plasticity could have implications for islet development, (patho)physiology and regeneration.
Aristaless-related homeobox (Arx) was recently demonstrated to be involved in pancreatic alpha cell fate specification while simultaneously repressing the beta and delta cell lineages. To establish whether Arx is not only necessary, but also sufficient to instruct the alpha cell fate in endocrine progenitors, we used a gain-of-function approach to generate mice conditionally misexpressing this factor. Mice with forced Arx expression in the embryonic pancreas or in developing islet cells developed a dramatic hyperglycemia and eventually died. Further analysis demonstrated a drastic loss of beta and delta cells. Concurrently, a remarkable increase in the number of cells displaying alpha cell or, strikingly, pancreatic polypeptide (PP) cell features was observed. Notably, the ectopic expression of Arx induced in embryonic or adult beta cells led to a loss of the beta cell phenotype and a concomitant increase in a number of cells with alpha or PP cell characteristics. Combining quantitative real-time PCR and lineage-tracing experiments, we demonstrate that, in adult mice, the misexpression of Arx, rather than its overexpression, promotes a conversion of beta cells into glucagon- or PP-producing cells in vivo. These results provide important insights into the complex mechanisms underlying proper pancreatic endocrine cell allocation and cell identity acquisition.
Pancreatic insulin-producing beta-cells have a long lifespan, such that in healthy conditions they replicate little during a lifetime. Nevertheless, they show increased self-duplication after increased metabolic demand or after injury (that is, beta-cell loss). It is not known whether adult mammals can differentiate (regenerate) new beta-cells after extreme, total beta-cell loss, as in diabetes. This would indicate differentiation from precursors or another heterologous (non-beta-cell) source. Here we show beta-cell regeneration in a transgenic model of diphtheria-toxin-induced acute selective near-total beta-cell ablation. If given insulin, the mice survived and showed beta-cell mass augmentation with time. Lineage-tracing to label the glucagon-producing alpha-cells before beta-cell ablation tracked large fractions of regenerated beta-cells as deriving from alpha-cells, revealing a previously disregarded degree of pancreatic cell plasticity. Such inter-endocrine spontaneous adult cell conversion could be harnessed towards methods of producing beta-cells for diabetes therapies, either in differentiation settings in vitro or in induced regeneration.
Cell-cell coupling mediated by gap junctions formed from connexin (CX) contributes to the control of insulin secretion in the endocrine pancreas. We investigated the cellular production and localisation of CX36 and CX43, and gap junction-mediated beta cell coupling in pancreatic islets from rats of different ages, displaying different degrees of maturation of insulin secretion.
The presence and distribution of islet connexins were assessed by immunoblotting and immunofluorescence. The expression of connexin genes was evaluated by RT-PCR and quantitative real-time PCR. The ultrastructure of gap junctions and the function of connexin channels were assessed by freeze-fracture electron microscopy and tracer microinjection, respectively.
Young and adult beta cells, which respond to glucose, expressed significantly higher levels of Cx36 (also known as Gjd2) than fetal and newborn beta cells, which respond poorly to the sugar. Accordingly, adult beta cells also showed a significantly higher membrane density of gap junctions and greater intercellular exchange of ethidium bromide than newborn beta cells. Cx43 (also known as Gja1) was not expressed by beta cells, but was located in various cell types at the periphery of fetal and newborn islets.
These findings show that the pattern of connexins, gap junction membrane density and coupling changes in islets during the functional maturation of beta cells.
To date, most attention on tissue regeneration has focused on the exploration of positive cues promoting or allowing the engagement of natural cellular restoration upon injury. In contrast, the signals fostering cell identity maintenance in the vertebrate body have been poorly investigated, yet they are crucial, for their counteraction could become a powerful method to induce and modulate regeneration. Here we review the mechanisms inhibiting pro‐regenerative spontaneous adaptive cell responses in different model organisms and organs. The pharmacological or genetic/epigenetic modulation of such regenerative brakes could release a dormant but innate adaptive competence of certain cell types and therefore boost tissue regeneration in different situations. © AlphaMed Press 2019 The state of differentiation of a given cell is not carved in stone. Cell identity, at all stages of life, is modulated by inhibitory signals from the close cellular environment. Cell identity maintenance is therefore an active process of inhibition of a natural trend to change, and not an intrinsic or passive state of differentiation. Here we review some of the published knowledge regarding the brakes to these changes of cell identity, and thus to tissue regeneration, as described in different experimental models. The modulation of these inhibitory signals, by helping specialized cells to change their function, that is, by fostering regeneration, could prove crucial for treating pathologies due to massive or inappropriate cell death. Cell identity maintenance, at all stages of life, is modulated by inhibitory signals from the surrounding cellular environment. Stress‐mediated suppression of these signals, such as after a lesion, may lead fully mature differentiated specialized cells to change their function, thus fostering regeneration of injured tissue. Controlling this cellular plasticity could prove crucial to developing new therapies to treat degenerative diseases.
Pancreatic progenitors derived from human embryonic stem cells (hESCs) are now in clinical trials for insulin replacement in patients with type 1 diabetes. Animal studies indicate the cells can mature into a mixed population of pancreatic endocrine cells including glucose-responsive beta cells several months after implant, but it remains unclear how conditions in the recipient may influence the maturation and ultimately the function of these cells. Here, we investigated the effects of the host sex and pregnancy on the maturation of human Stage 4 (S4) pancreatic progenitor cells and more mature Stage 7 (S7) pancreatic endocrine cells implanted under the kidney capsule of immunodeficient SCID-Beige mice. Pregnancy led to increased proliferation of endogenous pancreatic beta cells, but did not appear to impact proliferation or maturation of S4 cells at mid-gestation. Interestingly, S4 and S7 cells both acquired glucose-stimulated C-peptide secretion in females before males. Moreover, S4 cells lowered fasting blood glucose levels in females sooner than in males whereas the responses with S7 cells were similar. These data indicate that the host sex may impact the maturation of hESC-derived cells in vivo and that this effect can be minimized by more advanced differentiation of the cells prior to implant.
Insulin-producing pancreatic β cells in mice can slowly regenerate from glucagon-producing α cells in settings like β cell loss, but the basis of this conversion is unknown. Moreover, it remains unclear if this intra-islet cell conversion is relevant to diseases like type 1 diabetes (T1D). We show that the α cell regulators Aristaless-related homeobox (Arx) and DNA methyltransferase 1 (Dnmt1) maintain α cell identity in mice. Within 3 months of Dnmt1 and Arx loss, lineage tracing and single-cell RNA sequencing revealed extensive α cell conversion into progeny resembling native β cells. Physiological studies demonstrated that converted α cells acquire hallmark β cell electrophysiology and show glucose-stimulated insulin secretion. In T1D patients, subsets of glucagon-expressing cells show loss of DNMT1 and ARX and produce insulin and other β cell factors, suggesting that DNMT1 and ARX maintain α cell identity in humans. Our work reveals pathways regulated by Arx and Dnmt1 that are sufficient for achieving targeted generation of β cells from adult pancreatic α cells.
The different forms of diabetes mellitus differ in their pathogenesis but, ultimately, they are all characterized by progressive islet β-cell loss. Restoring the β-cell mass is therefore a major goal for future therapeutic approaches. The number of β-cells found at birth is determined by proliferation and differentiation of pancreatic progenitor cells, and it has been considered to remain mostly unchanged throughout adult life. Recent studies in mice have revealed an unexpected plasticity in islet endocrine cells in response to stress; under certain conditions, islet non-β-cells have the potential to reprogram into insulin producers, thus contributing to restore the β-cell mass. Here, we discuss the latest findings on pancreas and islet cell plasticity upon physiological, pathological and experimental conditions of stress. Understanding the mechanisms involved in cell reprogramming in these models will allow the development of new strategies for the treatment of diabetes, by exploiting the intrinsic regeneration capacity of the pancreas.
The impaired ability to produce or respond to insulin, a hormone synthetized by the pancreatic β-cells, leads to diabetes. There is an excruciating need of finding new approaches to protect or restore these cells once they are lost. Replacement and ex vivo directed reprogramming methods have an undeniable therapeutic potential, yet they exhibit crucial flaws. The in vivo conversion of adult cells to functional insulin-producing cells is a promising alternative for regenerative treatments in diabetes. The stunning natural transdifferentiation potential of the adult endocrine pancreas was recently uncovered. Modulating molecular targets involved in β-cell fate maintenance or in general differentiation mechanisms can further potentiate this intrinsic cell plasticity, which leads to insulin production reconstitution.
Human embryonic stem cells (hESC) differentiated towards beta cells and fetal human pancreatic islet cells resemble each other transcriptionally and are characterized by immaturity with a lack of glucose responsiveness, low levels of insulin content and impaired pro-insulin-to-insulin processing. However, their response to stimuli that promote functionality have not been compared.
To evaluate the effects of our previous strategies for functional maturation developed in rodents in these two human models of beta-cell immaturity and compare their responses. Design, settings, participants and interventions: In proof-of-principle experiments using either adenoviral-mediated overexpression of MAFA or the physiologically driven path via thyroid hormone (T3), human fetal islet-like clusters (ICC) functional maturity was evaluated. Then, the effects of T3 were evaluated upon the functional maturation of hESC differentiated toward beta cells.
Functional maturation was evaluated by the following parameters: glucose responsiveness, insulin content, expression of the mature beta-cell transcription factor MAFA and proinsulin-to-insulin processing.
ICCs responded positively to MAFA overexpression and T3 treatment as assessed by two different maturation parameters: increased insulin secretion at 16.8 mM glucose and increased proinsulin-to-insulin processing. In hESC differentiated toward beta cells, T3 enhanced MAFA expression, increased insulin content (probably mediated by the increased MAFA) and increased insulin secretion at 16.8 mM glucose.
T3 is a useful in vitro stimulus to promote human beta-cell maturation as shown in both human fetal ICCs and differentiated hESCs. The degree of maturation induced varied in the two models, possibly due to the different developmental status at the beginning of the study.
The effect of alginate-based scaffolds with added basement membrane proteins on the in vitro development of hESC-derived pancreatic progenitors was investigated. Cell clusters were encapsulated in scaffolds containing the basement membrane proteins collagen IV, laminin, fibronectin, or extracellular matrix-derived peptides, and maintained in culture for up to 46 days. The cells remained viable throughout the experiment with no signs of central necrosis. Whereas non-encapsulated cells aggregated into larger clusters, some of which showed signs of morphological changes and tissue organization, the alginate matrix stabilized the cluster size and displayed more homogeneous cell morphologies, allowing culture for long periods of time. For all conditions tested, a stable or declining expression of insulin and PDX1 and an increase in glucagon and somatostatin over time indicated a progressive reduction in beta cell-related gene expression. Alginate scaffolds can provide a chemically defined, xeno-free and easily scalable alternative for culture of pancreatic progenitors. Although no increase in insulin and PDX1 gene expression after alginate-immobilized cell culture was seen in this study, further optimization of the matrix physicochemical and biological properties and of the medium composition may still be a relevant strategy to promote the stabilization or maturation of stem cell-derived beta cells. This article is protected by copyright. All rights reserved.
© 2015 Wiley Periodicals, Inc.
Loss of pancreatic islet β-cell mass and β-cell dysfunction are central in the development of type 2 diabetes (T2DM). We recently showed that mature human insulin-containing β-cells can convert into glucagon-containing α-cells ex vivo. This loss of β-cell identity was characterized by the presence of β-cell transcription factors (Nkx6.1, Pdx1) in glucagon(+) cells. Here, we investigated whether loss of β-cell identity also occurs in vivo and whether it is related to the presence of (pre)diabetes in humans and non-human primates. We observed an 8x increased frequency of insulin(+) cells co-expressing glucagon in diabetic donors. Up to 5% of the cells that were Nkx6.1(+) but insulin(-) co-expressed glucagon, which represents a 5x increased frequency compared to the control group. This increase in bihormonal and Nkx6.1(+)glucagon(+)insulin(-) cells was also found in islets of diabetic macaques. The higher proportion of bihormonal cells and Nkx6.1(+)glucagon(+)insulin(-) cells in diabetic macaques and humans was correlated with the presence and extent of islet amyloidosis. These data indicate that loss of β-cell identity occurs in T2DM and could contribute to the decrease of functional β-cell mass. Maintenance of β-cell identity is a potential novel strategy to preserve β-cell function in diabetes.
© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.
The generation of insulin-producing pancreatic β cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation therapy in diabetes. However, insulin-producing cells previously generated from human pluripotent stem cells (hPSC) lack many functional characteristics of bona fide β cells. Here, we report a scalable differentiation protocol that can generate hundreds of millions of glucose-responsive β cells from hPSC in vitro. These stem-cell-derived β cells (SC-β) express markers found in mature β cells, flux Ca(2+) in response to glucose, package insulin into secretory granules, and secrete quantities of insulin comparable to adult β cells in response to multiple sequential glucose challenges in vitro. Furthermore, these cells secrete human insulin into the serum of mice shortly after transplantation in a glucose-regulated manner, and transplantation of these cells ameliorates hyperglycemia in diabetic mice.
Pdx1 is a homeobox-containing transcription factor that plays a key role in pancreatic development and adult β cell function. In this study, we traced the fate of adult β cells after Pdx1 deletion. As expected, β-cell-specific removal of Pdx1 resulted in severe hyperglycemia within days. Surprisingly, a large fraction of Pdx1-deleted cells rapidly acquired ultrastructural and physiological features of α cells, indicating that a robust cellular reprogramming had occurred. Reprogrammed cells exhibited a global transcriptional shift that included derepression of the α cell transcription factor MafB, resulting in a transcriptional profile that closely resembled that of α cells. These findings indicate that Pdx1 acts as a master regulator of β cell fate by simultaneously activating genes essential for β cell identity and repressing those associated with α cell identity. We discuss the significance of these findings in the context of the emerging notion that loss of β cell identity contributes to the pathogenesis of type 2 diabetes.
Salamanders regenerate appendages via a progenitor pool called the blastema. The cellular mechanisms underlying regeneration of muscle have been much debated but have remained unclear. Here we applied Cre-loxP genetic fate mapping to skeletal muscle during limb regeneration in two salamander species, Notophthalmus viridescens (newt) and Ambystoma mexicanum (axolotl). Remarkably, we found that myofiber dedifferentiation is an integral part of limb regeneration in the newt, but not in axolotl. In the newt, myofiber fragmentation results in proliferating, PAX7(-) mononuclear cells in the blastema that give rise to the skeletal muscle in the new limb. In contrast, myofibers in axolotl do not generate proliferating cells, and do not contribute to newly regenerated muscle; instead, resident PAX7(+) cells provide the regeneration activity. Our results therefore show significant diversity in limb muscle regeneration mechanisms among salamanders and suggest that multiple strategies may be feasible for inducing regeneration in other species, including mammals.
Human embryonic stem cells (hESCs) are emerging as an attractive alternative source for cell replacement therapy since the cells can be expanded in culture indefinitely and differentiated into any cell types in the body. In order to optimize cell-to-cell interaction, cell proliferation and differentiation into specific lineages as well as tissue organization, it is important to provide a microenvironment for the hESCs which mimics the stem cell niche. One approach is to provide a three-dimensional (3D) environment such as encapsulation. We present an approach to culture and differentiate hESCs into midbrain dopamine (mdDA) neurons in a 3D microenvironment using alginate microcapsules for the first time. A detailed gene and protein expression analysis during neuronal differentiation showed an increased gene and protein expression of various specific DA neuronal markers, particularly tyrosine hydroxylase (TH) by >100 folds after 2weeks and at least 50% higher expression after 4weeks respectively, compared to cells differentiated under conventional two-dimensional (2D) platform. The encapsulated TH(+) cells co-expressed mdDA neuronal markers, forkhead box protein A-2 (FOXA2) and pituitary homeobox-3 (PITX3) after 4weeks and secreted approximately 60pg/ml/10(6) cells higher DA level when induced. We propose that the 3D platform facilitated an early onset of DA neuronal generation compared to that with conventional 2D system which also secretes more DA under potassium-induction. It is a very useful model to study the proliferation and directed differentiation of hESCs to various lineages, particularly to mdDA neurons. This 3D system also allows the separation of feeder cells from hESCs during the process of differentiation and also has potential for immune-isolation during transplantation studies.
Diabetes is associated with β cell failure. But it remains unclear whether the latter results from reduced β cell number or function. FoxO1 integrates β cell proliferation with adaptive β cell function. We interrogated the contribution of these two processes to β cell dysfunction, using mice lacking FoxO1 in β cells. FoxO1 ablation caused hyperglycemia with reduced β cell mass following physiologic stress, such as multiparity and aging. Surprisingly, lineage-tracing experiments demonstrated that loss of β cell mass was due to β cell dedifferentiation, not death. Dedifferentiated β cells reverted to progenitor-like cells expressing Neurogenin3, Oct4, Nanog, and L-Myc. A subset of FoxO1-deficient β cells adopted the α cell fate, resulting in hyperglucagonemia. Strikingly, we identify the same sequence of events as a feature of different models of murine diabetes. We propose that dedifferentiation trumps endocrine cell death in the natural history of β cell failure and suggest that treatment of β cell dysfunction should restore differentiation, rather than promoting β cell replication.
PaperFlick
eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIyMDFkZjRlNWVmYjhmYjQ4MzI4NTJiZTViMWM0NTU4NiIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjAwNDk2OTQ3fQ.aGCZ0uA2wVZHnHzYCsdkyUjIu-O7h3c2vI5LOG857S_Nkuu6FyaRk_ky2twea7ZQbd92AXkSrO4GJ5dha-w7zyoCdONgrFPXkJsHEXPS3ap2Ppk8BrAklNiS-31PtgdqEGsAEth9y5nIkJcRBiyiD6S-9ifX8888-SfjPbDq61nYxyEl9csKL01nHin6JaLaFq4aQvdAsrIMyu0s-E30rB0Y3fZ9yDyKC9V58tPBVJzOAXArM-z1jIUNqEN8WSymH_2PG71eP6LxelRjBD1k53Q3ydPZbwc2vQo_SpSQTjq-5i993G_GIW3s0_2yc6Em6DTXG3HG7JP6FzynqKrNYw
(mp4, (11.91 MB)
Download video
Our understanding of the basic biology of diabetes has been guided by observations made using animal models, particularly rodents. However, humans are not mice, and outcomes predicted by murine studies are not always representative of actual outcomes in the clinic. In particular, investigators studying diabetes have relied heavily on mouse and rat models of autoimmune type 1-like diabetes, and experimental results using these models have not been representative of many of the clinical trials in type 1 diabetes. In this article, we describe the availability of new models of humanized mice for the study of three areas of diabetes. These include the use of humanized mice for the study of (1) human islet stem and progenitor cells, (2) human islet allograft rejection, and (3) human immunity and autoimmunity. These humanized mouse models provide an important preclinical bridge between in vitro studies and rodent models and the translation of discoveries in these model systems to the clinic.
Single implantation of microencapsulated islets into rats with streptozotocin-induced diabetes corrected the diabetic state for 2 to 3 weeks. The microencapsulated islets remained morphologically and functionally intact throughout long-term culture studies lasting over 15 weeks.
Most but not all phyla include examples of species that are able to regenerate large sections of the body plan. The mechanisms underlying regeneration on this scale are currently being studied in a variety of contexts in both vertebrates and invertebrates. Regeneration generally involves the formation of a wound epithelium after transection or injury, followed by the generation of regenerative progenitor cells and morphogenesis to give the regenerate. Common mechanisms may exist in relation to each of these aspects. For example, the initial proliferation of progenitor cells often depends on the nerve supply, whereas morphogenesis reflects the generation of positional disparity between adjacent cells-the principle of intercalation. These mechanisms are reviewed here across a range of contexts. We also consider the evolutionary origins of regeneration and how regeneration may relate to both agametic reproduction and to ontogeny.
MAFA and T3 drive maturation of both fetal human islets Luiza Ghila et al. and insulin-producing cells differentiated from hESC
- C Aguayo-Mazzucato
- A Diienno
- J Hollister-Lock
- C Cahill
- A Sharma
- G Weir
- C Colton
- S Bonner-Weir
Aguayo-Mazzucato C, DiIenno A, Hollister-Lock J, Cahill C, Sharma A, Weir G,
Colton C, Bonner-Weir S (2015) MAFA and
T3 drive maturation of both fetal human islets
Luiza Ghila et al.
and insulin-producing cells differentiated from
hESC. J Clin Endocrinol Metab 100
(10):3651-3659. https://doi.org/10.1210/
jc.2015-2632