No full-text available
To read the full-text of this research,
you can request a copy directly from the author.
In-vitro differentiation of pancreatic beta-cells
Abstract
Stem cell biology is a new field that holds promise for in-vitro mass production of pancreatic beta-cells, which are responsible for insulin synthesis, storage, and release. Lack or defect of insulin produces diabetes mellitus, a devastating disease suffered by 150 million people in the world. Transplantation of insulin-producing cells could be a cure for type 1 and some cases of type 2 diabetes, however this procedure is limited by the scarcity of material. Obtaining pancreatic beta-cells from embryonic stem cells would overcome this problem. We have derived insulin-producing cells from mouse embryonic stem cells by a 3-step in-vitro differentiation method consisting of directed differentiation, cell-lineage selection, and maturation. These insulin-producing cells normalize blood glucose when transplanted into streptozotocin-diabetic mice. Strategies to increase islet precursor cells from embryonic stem cells include the expression of relevant transcription factors (Pdx1, Ngn3, Isl-1, etc), together with the use of extracellular factors. Once a high enough proportion of islet precursors has been obtained there is a need for cell-lineage selection in order to purify the desired cell population. For this purpose, we designed a cell-trapping method based on a chimeric gene that fuses the human insulin gene regulatory region with the structural gene that confers resistance to neomycin. When incorporated into embryonic stem cells, this fusion gene will generate neomycin resistance in those cells that initiate the synthesis of insulin. Not only embryonic, but also adult stem cells are potential sources for insulin-containing cells. Duct cells from the adult pancreas are committed to differentiate into the four islet cell types; other possibilities may include nestin-positive cells from islets and adult pluripotent stem cells from other origins. Whilst the former are committed to be islet cells but have a reduced capacity to expand, the latter are more pluripotent and more expandable, but a longer pathway separates them from the insulin-producing stage. The aim of this review is to discuss the different strategies that may be followed to in-vitro differentiate pancreatic beta-cells from stem cells.
... The Hnf3β gene is a critical factor in the endodermal cell lineage development. In fact, Hnf3β is a transcriptional regulator of Pdx1, which is important in the regulation of insulin gene expression and also required for the differentiation of the mature pancreas (Soria, 2001). Our study demonstrated that defined condition and activin A were necessary to generate DE in a hypoxic atmosphere. ...
... Interestingly, Nkx6.1 is also restricted to β-cells and some neurons (Docherty et al., 2007). This finding suggests the presence of a common progenitor of neurons and insulin-positive cells during in vitro differentiation (Soria, 2001). Moreover, the IPC derived from induced pluripotent stem cells could be enhanced by overexpression of Pdx1 and Nkx6.1 factors (Walczak et al., 2016). ...
... Similarly, it has been demonstrated that Ngn3 and NeuroD1/BETA2 can drive the early differentiation of islet cells (Schwitzgebel et al., 2000). Ngn3 has been shown to induce the expression of BETA2 (NeuroD), a transcription factor implicated in the insulin gene expression and in islet cells differentiation (Soria, 2001). However, the Nestin and Hnf3β expressions were also observed in stage III cells representing the presence of the immature stage of pancreatic development. ...
Diabetes mellitus (DM) is a group of diseases characterized by abnormally high levels of glucose in the blood stream. In developing a potential therapy for diabetic patients, pancreatic cells transplantation has drawn great attention. However, the hinder of cell transplantation for diabetes treatment is insufficient sources of insulin-producing cells. Therefore, new cell based therapy need to be developed. In this regard, human embryonic stem cells (hESCs) may serve as good candidates for this based on their capability of differentiation into various cell types. In this study, we designed a new differentiation protocol that can generate hESC-derived insulin-producing cells (hES-DIPCs) in a hypoxic condition. We also emphasized on the induction of definitive endoderm during embryoid bodies (EBs) formation. After induction of hESCs differentiation into insulin-producing cells (IPCs), the cells obtained from the cultures exhibited pancreas-related genes such as Pdx1, Ngn3, Nkx6.1, GLUT2, and insulin. These cells also showed positive for DTZ-stained cellular clusters and contained ability of insulin secretion in a glucose-dependent manner. After achievement to generated functional hES-DIPCs in vitro, some of the hES-DIPCs were then encapsulated named encapsulated hES-DIPCs. The data showed that the encapsulated cells could possess the function of insulin secretion in a time-dependent manner. The hES-DIPCs and encapsulated hES-DIPCs were then separately transplanted into STZ-induced diabetic mice. The findings showed the significant blood glucose levels regulation capacity and declination of IL-1β concentration in all transplanted mice. These results indicated that both hES-DIPCs and encapsulated hES-DIPCs contained the ability to sustain hyperglycemia condition as well as decrease inflammatory cytokine level in vivo. The findings of this study may apply for generation of a large number of hES-DIPCs in vitro. In addition, the implication of this work is therapeutic value in type I diabetes treatment in the future. The application for type II diabetes treatment remain to be investigated.
... PCMOs were cultured with either hepatocyte conditioning medium (HCM; 3 ng/mL fibroblast growth factor [FGF]-4; Sigma, Madrid, Spain) for differentiation into neohepatocytes 5 or islet cell conditioning medium (ICM; 10 ng/mL epidermal growth factor and 20 ng/mL hepatocyte growth factor [both from Calbiochem, Munich, Germany], 10 mmol/L nicotinamide [Sigma], and 5 mmol/L glucose) for differentiation into pancreatic islet-like cells. 6 After 7, 14, and 21 days, differentiated PCMOs were subjected to immunohistochemistry with antibodies against connexin 32, pan-actin, cytokeratin 18, and albumin (Dako, Hamburg, Germany) for neohepatocytes and insulin (Dako), glucagon (Biomeda, Foster City, CA), and Pdx-1 (CeMines, Evergreen, CO) for neoislet cells. ...
... Strikingly, several transcription factors involved in early -cell differentiation were strongly induced in neoislet cells (HNF3, Ngn3, Beta2/NeuroD, Nkx2.2, and Nkx6.1). 6,26 Moreover, neoislet cells express MafA, a nuclear protein that is absolutely required for transcription of the insulin gene. 27 High expression of Beta2/NeuroD and Ngn3 and low expression of Pdx-1 in neoislets is reminiscent of the common ␣ and  progenitor cell. ...
... 27 High expression of Beta2/NeuroD and Ngn3 and low expression of Pdx-1 in neoislets is reminiscent of the common ␣ and  progenitor cell. 6 De novo expression of glucagon, together with enhanced expression of transcription factors involved in the generation of ␣ cells and/or regulation of the proglucagon gene (Cdx2, Nkx2.2, c-Maf, and Pax6), provides further evidence for pancreatic islet differentiation. The still-immature phenotype of these cells in vitro may explain both the lower expression and secretion of insulin relative to that of isolated human islets. ...
... Die beiden wichtigsten Eigenschaften, die ESCs zu einer vielversprechenden Wahl für die Stammzelltherapie machen, sind ihr unbegrenztes undifferenziertes Selbsterneuerungspotenzial in vitro und ihre Pluripotenz, die es ihnen ermöglicht, sich in Zelltypen der drei Keimblätter zu differenzieren. Frühere Studien haben gezeigt, dass undifferenzierte ESCs unter geeigneten Kulturbedingungen von Natur aus Insulin sezernieren können (Soria et al. 2000;Soria 2001). Die neuartige, Insulin sezernierende ESC-Population wurde erfolgreich durch die Zellfangtechnik isoliert und in vitro charakterisiert. ...
... Diese Ergebnisse machen eine Manipulation der Spenderzellen erforderlich, um eine stabilere Insulinexpression zu erreichen. Verschiedene Forschungsgruppen haben die ESC-Differenzierungsprotokolle optimiert, um die Rate der β-Zell-Differenzierung zu erhöhen (Soria 2001;Leon-Quinto et al. 2004;Bruin et al. 2014). Nach der Differenzierung ordnen sich die ESCs von selbst zu 3D-Clustern an, die in ihrer Topologie den Inselzellen in der Bauchspeicheldrüse ähneln, und reagieren auf Glukose, um Insulin freizusetzen (Lumelsky et al. 2001). ...
... Die beiden wichtigsten Eigenschaften, die ESCs zu einer vielversprechenden Wahl für die Stammzelltherapie machen, sind ihr unbegrenztes undifferenziertes Selbsterneuerungspotenzial in vitro und ihre Pluripotenz, die es ihnen ermöglicht, sich in Zelltypen der drei Keimblätter zu differenzieren. Frühere Studien haben gezeigt, dass undifferenzierte ESCs unter geeigneten Kulturbedingungen von Natur aus Insulin sezernieren können (Soria et al. 2000;Soria 2001). Die neuartige, Insulin sezernierende ESC-Population wurde erfolgreich durch die Zellfangtechnik isoliert und in vitro charakterisiert. ...
... Diese Ergebnisse machen eine Manipulation der Spenderzellen erforderlich, um eine stabilere Insulinexpression zu erreichen. Verschiedene Forschungsgruppen haben die ESC-Differenzierungsprotokolle optimiert, um die Rate der β-Zell-Differenzierung zu erhöhen (Soria 2001;Leon-Quinto et al. 2004;Bruin et al. 2014). Nach der Differenzierung ordnen sich die ESCs von selbst zu 3D-Clustern an, die in ihrer Topologie den Inselzellen in der Bauchspeicheldrüse ähneln, und reagieren auf Glukose, um Insulin freizusetzen (Lumelsky et al. 2001). ...
... Die beiden wichtigsten Eigenschaften, die ESCs zu einer vielversprechenden Wahl für die Stammzelltherapie machen, sind ihr unbegrenztes undifferenziertes Selbsterneuerungspotenzial in vitro und ihre Pluripotenz, die es ihnen ermöglicht, sich in Zelltypen der drei Keimblätter zu differenzieren. Frühere Studien haben gezeigt, dass undifferenzierte ESCs unter geeigneten Kulturbedingungen von Natur aus Insulin sezernieren können (Soria et al. 2000;Soria 2001). Die neuartige, Insulin sezernierende ESC-Population wurde erfolgreich durch die Zellfangtechnik isoliert und in vitro charakterisiert. ...
... Diese Ergebnisse machen eine Manipulation der Spenderzellen erforderlich, um eine stabilere Insulinexpression zu erreichen. Verschiedene Forschungsgruppen haben die ESC-Differenzierungsprotokolle optimiert, um die Rate der β-Zell-Differenzierung zu erhöhen (Soria 2001;Leon-Quinto et al. 2004;Bruin et al. 2014). Nach der Differenzierung ordnen sich die ESCs von selbst zu 3D-Clustern an, die in ihrer Topologie den Inselzellen in der Bauchspeicheldrüse ähneln, und reagieren auf Glukose, um Insulin freizusetzen (Lumelsky et al. 2001). ...
... Die beiden wichtigsten Eigenschaften, die ESCs zu einer vielversprechenden Wahl für die Stammzelltherapie machen, sind ihr unbegrenztes undifferenziertes Selbsterneuerungspotenzial in vitro und ihre Pluripotenz, die es ihnen ermöglicht, sich in Zelltypen der drei Keimblätter zu differenzieren. Frühere Studien haben gezeigt, dass undifferenzierte ESCs unter geeigneten Kulturbedingungen von Natur aus Insulin sezernieren können (Soria et al. 2000;Soria 2001). Die neuartige, Insulin sezernierende ESC-Population wurde erfolgreich durch die Zellfangtechnik isoliert und in vitro charakterisiert. ...
... Diese Ergebnisse machen eine Manipulation der Spenderzellen erforderlich, um eine stabilere Insulinexpression zu erreichen. Verschiedene Forschungsgruppen haben die ESC-Differenzierungsprotokolle optimiert, um die Rate der β-Zell-Differenzierung zu erhöhen (Soria 2001;Leon-Quinto et al. 2004;Bruin et al. 2014). Nach der Differenzierung ordnen sich die ESCs von selbst zu 3D-Clustern an, die in ihrer Topologie den Inselzellen in der Bauchspeicheldrüse ähneln, und reagieren auf Glukose, um Insulin freizusetzen (Lumelsky et al. 2001). ...
... The two main properties that make ESCs a promising choice for stem cell therapy are their unlimited undifferentiated self-renewal potential in vitro and pluripotency that enables them to differentiate into cell types of the three germ layers. The earlier studies have shown that undifferentiated ESCs can inherently secrete insulin under appropriate culture conditions (Soria et al. 2000;Soria 2001). The novel insulin-secreting ESCs population was successfully isolated by the celltrapping technique and characterized in vitro. ...
... These results necessitate manipulation of the donor cells to achieve a more stable expression of insulin. Various research groups have optimized the ESCs differentiation protocols to enhance the rate of β-cell differentiation (Soria 2001;Leon-Quinto et al. 2004;Bruin et al. 2014). Upon differentiation, ESCs self-assemble to form 3D clusters similar in topology to the islets in the pancreas and display glucoseresponsiveness to release insulin (Lumelsky et al. 2001). ...
Dental mesenchymal stem/progenitor cells (MSCs) have unique biological criteria in terms of genes and protein expression. They possess the common characteristics of non-dental MSCs that include the self-renewal ability, differentiation potential into variable cell lineages, and immunomodulatory properties. In addition, they reveal superior regenerative potential as compared to MSCs derived from other body tissues. Being easily acquired with minimally invasive procedures, make them promising resources in tissue engineering and regenerative therapies. This chapter presents a brief review of different sources of dental stem/progenitor cells, the specific criteria of dental MSCs and their advantages over non-dental MSCs. Moreover, this chapter reviews the osteogenic, hepatogenic, neurogenic, immunomodulatory, and dental tissue regenerative potential of these cells. A comparison between dental stem/progenitor cells from different sources regarding their proliferative potential and regenerative potential is discussed. Furthermore, this chapter explores the growth factors and cytokines expressed or induced by these cells, the effect of these signaling molecules on the diversity of their differentiation and regeneration potential. Since maintaining the genetic integrity against DNA damage and other challenges concerning cell-based therapy hinder the utility of dental MSCs in clinical implementation, this chapter displays the major limitations concerning their clinical translation and suggests possible solutions.
... The two main properties that make ESCs a promising choice for stem cell therapy are their unlimited undifferentiated self-renewal potential in vitro and pluripotency that enables them to differentiate into cell types of the three germ layers. The earlier studies have shown that undifferentiated ESCs can inherently secrete insulin under appropriate culture conditions (Soria et al. 2000;Soria 2001). The novel insulin-secreting ESCs population was successfully isolated by the celltrapping technique and characterized in vitro. ...
... These results necessitate manipulation of the donor cells to achieve a more stable expression of insulin. Various research groups have optimized the ESCs differentiation protocols to enhance the rate of β-cell differentiation (Soria 2001;Leon-Quinto et al. 2004;Bruin et al. 2014). Upon differentiation, ESCs self-assemble to form 3D clusters similar in topology to the islets in the pancreas and display glucoseresponsiveness to release insulin (Lumelsky et al. 2001). ...
Regenerative medicine research provides a new hope in treatment of patients suffering from severe injuries or chronic diseases. The increased interest in stem/progenitor cells is encouraged by their biological nature for self-renewal and multilineage differentiation potential. Cornerstones for tissue engineering are stem/progenitor cells, scaffolds, and morphogenic signals, the dynamic actors that induce cells for tissue or organ regeneration. The dental medicine field was recently tempted to test the possible applications of tissue engineering in regenerating dental structures. Dental caries which is a common worldwide health problem, causing enamel and dentin destruction, besides if not treated progresses to the dental pulp with loss of its vitality. As the available artificial filling materials used to restore the affected dental tissues have many inherent deficiencies, many recent approaches have been proposed to regenerate the hard dental tissues. There has been an increasing demand to regenerate enamel, dentin, and maintain pulp vitality. Stem/Progenitor cell therapy could aid in the regeneration of enamel and dentin/pulp complex as a more conservative replacement to conventional restorative therapies. In view of these facts, this chapter will discuss the recent approaches in tissue engineering for enamel, dentin, and pulp regeneration.
... Mesoderm gives rise to muscles (skeletal, cardiac, visceral and vascular smooth muscles), bone, cartilage, blood and connective tissue [160]. Pancreas and liver cells derive from the endoderm layer [3,161,162]. ...
... Within pancreatic islets and in non-endocrine compartments of the pancreas, some multipotent stem cells have been described [105,[420][421][422] and have the capacity to differentiate into pancreatic islet-like structures [423,424]. Furthermore, cells that do not reside within the pancreas have been differentiated into pancreatic endocrine hormoneproducing cells in vitro and in vivo, including embryonic stem cells [161,423,425], hepatic oval cells [426,427], spleen-derived cells [428] or the central nervous system [429]. BMderived stem cells could also play a supportive role in pancreas regeneration in vivo rather than participate in the differentiation of endocrine cells themselves [430]. ...
Mesenchymal stem cells (MSCs) constitute a heterogeneous population ofundifferentiated and committed multipotential cells. They serve in vitro and in vivo asprecursors for bone marrow stroma, bone, fat, cartilage, muscle (smooth, cardiac andskeletal) and neural cells. They are able to home upon engraftment to a number ofmicroenvironments, capable of extensive proliferation and of producing large number ofdifferentiated progenitors to repair functionally tissue after injury. MSCs are usuallyisolated from adult bone marrow but can also be isolated from several other tissues, suchas fetal liver, adult circulating blood, umbilical cord blood, placenta or adipose tissue.Furthermore, MSCs present several immuno-regulatory characteristics with immunosuppressiveeffects that induce a tolerance and could be therapeutic for reduction ofgraft-versus-host disease, rejection and modulation of inflammation. Since MSCs followa vascular smooth muscle differentiation pathway, their therapeutic potential has beenwidely investigated in the treatment of vascular diseases. Indeed, MSCs can participate inthe development of new vessels from pre-existing vascular walls (angiogenesis), in theinduction of new vascular networks (vasculogenesis), in the collateral artery growth(arteriogenesis) or in plastic and reconstructive surgery applications. These cells canpromote vascular growth by incorporating into vessels' wall, but they also may functionas supporting factors by producing paracrine vascular promoting factors. In this chapter,the mesenchymal stem cells isolation, culture and characterization, but also their potential use in vascular therapy, in both human and animal models, are reported anddiscussed, whereas perspectives in angiogenesis, vasculogenesis, arteriogenesis orvascular engineering are explored.
... The initial step in directed differentiation to form IPCs is to induce expression of PDX-1. PDX-1 is one of the well-studied transcription factors that are critical to both cell development and function [17]. PDX-1 controls the maturation of pancreatic islet cells by regulating insulin 1 and other downstream genes. ...
... Evidences were provided that TSA has the potential of chromatin remodeling and can allow differentiation of bone marrow cells into IPCs under appropriated culture conditions in the presence of high glucose concentrations and GLP-1 [11]. Glucose is well known as a growth factor for cells [17]. It promotes cells replication in vitro as well as in vivo at concentrations of 20-30 mmol/L [25]. ...
... The initial step in directed differentiation to form IPCs is to induce expression of PDX-1. PDX-1 is one of the well-studied transcription factors that are critical to both í µí»½ cell development and function [17]. PDX-1 controls the maturation of pancreatic islet cells by regulating insulin 1 and other downstream genes. ...
... Evidences were provided that TSA has the potential of chromatin remodeling and can allow differentiation of bone marrow cells into IPCs under appropriated culture conditions in the presence of high glucose concentrations and GLP-1 [11]. Glucose is well known as a growth factor for í µí»½ cells [17]. It promotes í µí»½ cells replication in vitro as well as in vivo at concentrations of 20-30 mmol/L [25]. ...
... There is a temporal sequence of genes during activation for the development of α-and β-cells (17). PDX-1 is one of the well studied transcription factors that are critical to both beta cell development and function (38). It controls the maturation of pancreatic islet cells by regulating insulin 1 and other down stream genes. ...
Harvesting, expansion and directed differentiation of human bone marrow-derived mesenchymal stem cells (BM-MSCs) could provide an autologous source of surrogate β-cells that would alleviate the limitations of availability and/or allogenic rejection following pancreatic or islet transplantation. Bone marrow cells were obtained from three adult type 2 diabetic volunteers and 3 non-diabetic donors. After 3 days in culture, adherent MSCs were expanded for 2 passages. At passage 3, differentiation was carried out in a 3-staged procedure. Cells were cultured in a glucose-rich medium containing several activation and growth factors. Cells were evaluated in-vitro by flow cytometry, immunolabelling, Rt-PCR and human insulin and c-peptide release in responses to increasing glucose concentrations. One thousand cell-clusters were inserted under the renal capsule of diabetic nude mice followed by monitoring of their diabetic status. At the end of differentiation, ~5-10% of cells were immunofluorescent for insulin, c-peptide or glucagon; insulin and c-peptide were co-expressed. Nanogold immunolabelling for electron microscopy demonstrated the presence of c-peptide in the rough endoplasmic reticulum. Insulin-producing cells (IPCs) expressed transcription factors and genes of pancreatic hormones similar to those expressed by pancreatic islets. There was a stepwise increase in human insulin and c-peptide release by IPCs in response to increasing glucose concentrations. Transplantation of IPCs into nude diabetic mice resulted in control of their diabetic status for 3 months. The sera of IPC-transplanted mice contained human insulin and c-peptide but negligible levels of mouse insulin. When the IPCs-bearing kidneys were removed, rapid return of diabetic state was noted. BM-MSCs from diabetic and non-diabetic human subjects could be differentiated without genetic manipulation to form IPCs which, when transplanted, could maintain euglycaemia in diabetic mice for 3 months. Optimization of the culture conditions are required to improve the yield of IPCs and their functional performance.
... The PDBs in the forms of cytokines and signaling molecules could enhance the differentiation potential of stem cells into insulin-secreting cells, which may inhibit insulin resistance. The b-FGF and EGF may promote differentiation of stem cells into islet-like cells and proliferation of Pdx1-positive pancreatic progenitors' cells, respectively, and eventually increase insulin levels [147,148]. The β-cell proliferation, islet number, β-cell mass, and total insulin secretion (2-fold) could be increased by overexpression of HGF [149], whereas, VEGF-A and islet vascular structure are correlated and important for the expansion of beta-cell mass [150]. ...
Reports indicate the increasing prevalence of liver disorders in diabetes mellitus (DM) patients. Clinically, it has also been revealed that the existence of nonalcoholic fatty liver disease (NAFLD) enhances the incidence of type 2 diabetes mellitus (T2DM), while T2DM exacerbates NAFLD to extremely severe forms of steatohepatitis, cirrhosis, and hepatocellular carcinoma. This implies the coexistence and bidirectional nature of NAFLD and T2DM, which function synergistically to drive adverse consequences in clinical practice. For treatment of such comorbid state, though the existing practices such as lifestyle management, traditional Chinese medicines (TCM), and pharmaceuticals have offered somewhat relief, the debate continues about the optimal therapeutic impacts. Recent developments in the field of tissue engineering have led to a renewed interest in novel biomaterial alternatives such as stem cells. This might be attributable to their differentiation potential towards hepatic and pancreatic lineage. These cellular therapies could be further complemented by platelet-derived biomaterials, TCM formulations, or any specific drug. Based on these abovementioned approaches, we aimed to comprehensively analyze various preclinical and clinical studies from traditional to regenerative therapeutic approaches in managing concomitant NAFLD and T2DM.
... Nevertheless, in the group exposed to B27 and N2, the cells could express mature and immature pancreatic genes. B27 and N2 enhance the differentiation of embryonic stem cells into IPCs [34,35]. According to the findings, while B27 and N2 cause differentiation, their effect is significantly weaker than that observed in the group subjected to activin-A and retinoic acid. ...
This study aimed toengineer a pancreatic tissue. Intact rat pancreases were successfully decellularized, and were reseeded with human-induced pluripotent stem cells using different 2D and 3D culture growth factors. The differentiation process was assessed for the presence of a pancreas-like tissue. The histology and SEM analysis revealed cell attachment in all samples, except for the Exp4, and the Flow-cytometry provided 87% viability for the differentiated cells. In Exp1, PDX1 with the positive expression of 2.87±0.06 was dramatically higher than Exp2 with a 2.44±0.06 reaction. NGN3-reactions were 8±0.1 and 6.6±0.2 in Exp1 and Exp2 at P<0.05, respectively. C-peptide with the expression of 7.5±0.7 in Exp3 was almost equal to that in Exp1 and Exp2. Glucagon (5.1±1) and PDX1 (3.2±0.82) in Exp3 indicated no significant difference. The significant upregulations of pancreatic endocrine markers (PDX1 and NGN3), and the cell-specific glucose transporter (GLUT2) were observed in the differentiated IPCs in the 3D culture of Exp2 after 21 days. The highest insulin and C-peptide concentrations were observed in Exp2. In Exp3, insulin secretion in response to high glucose and 10 mM arginine was 42.43 ±6.34 μU/ml. A decellularized pancreas in the presence of hiPSCs and growth factors could be efficiently used as a natural scaffold.
... Furthermore, our results agreed with Soria (2001) who mentioned that the initial step in directed differentiation to form IPCs is to induce expression of PDX-1, which is one of the well-studied transcription factors that are critical to both β-cell development and function. ...
Background:
Diabetes mellitus has become the third human killer following cancer and cardiovascular disease. Millions of patients, often children, suffer from type 1 diabetes (T1D). Stem cells created hopes to regenerate damaged body tissues and restore their function.
Aim:
This work aimed at clarifying and comparing the therapeutic potential of differentiated and non-differentiated mesenchymal stem cells (MSCs) as a new line of therapy for T1D.
Methods:
40 female albino rats divided into Group I (control): 10 rats and group II (diabetic), III and IV, 10 rats in each, were injected with streptozotocin (50mg/kg body weight). Group III (MSCs) were transplanted with bone marrow derived MSCs from male rats and group IV (IPCs) with differentiated insulin producing cells. Blood and pancreatic tissue samples were taken from all rats for biochemical and histological studies.
Results:
MSCs reduced hyperglycemia in diabetic rats on day 15 while IPCs normalizes blood glucose level on day 7. Histological and morphometric analysis of pancreas of experimental diabetic rats showed improvement in MSCs-treated group but in IPCs-treated group, β-cells insulin immunoreactions were obviously returned to normal, with normal distribution of β-cells in the centre and other cells at the periphery. Meanwhile, most of the pathological lesions were still detected in diabetic rats.
Conclusion:
MSCs transplantation can reduce blood glucose level in recipient diabetic rats. IPCs initiate endogenous pancreatic regeneration by neogenesis of islets. IPCs are better than MSCs in regeneration of β-cells. So, IPCs therapy can be considered clinically to offer a hope for patients suffering from T1D.
... When the PRP is activated, it releases a lot of growth factors and adhesive molecules implicated in the differentiation of stem cells to IPCs such as bFGF, EGF, HGF, VEGF-A, and fibronectin (Burnouf et al., 2016). The bFGF were found previously to enhance the degeneration of islet-like cells from pluripotent stem cells (Lumelsky et al., 2001;Soria, 2001). The EGF has facilitated the expansion of Pdx1-positive pancreatic progenitors and supported the final stages of insulin expression (Jiang et al., 2008;Zhang et al., 2009). ...
Studies on patient-specific human-induced pluripotent stem cells (hiPSCs) as well as a series of autologous growth factors presumably will reveal their many benefits for cell base replacement therapy in type 1 diabetes mellitus (TIDM) patients in the future. For this purpose, we established a multistep protocol by adding platelet-rich plasma (PRP) that induce the hiPSCs into insulin-producing cells (IPCs). We present here a differentiation protocol consisting of five stages in two groups including protocol with PRP and without PRP. Characteristics of derived IPCs in both groups were evaluated at the mRNA and protein levels, cell cycle and viability in the end stage of cell differentiation. The in vitro studies indicated the treatment of hiPSCs in the protocol with PRP resulting in differentiated cells with strong characteristics of IPCs including islet-like cells, the expression of mature and functional pancreatic beta cell specific marker genes. In addition to these pancreatic specific markers were detected by immunochemistry and Western blot. Our differentiated cells in two groups secreted insulin and C-peptide in a glucose stimulation test by ELISA showing in vitro functional. The results of the cell cycle assay confirmed that differentiation has been done. We reported for the first time that PRP might be ideal additive in the culture medium to induce pancreatic differentiation in the hiPSCs. This study provides a new approach to investigate the role of PRP in pancreatic differentiation protocols and enhance the feasibility of using patient-specific iPSCs and autologous PRP for future beta cells replacement therapies for T1DM. This article is protected by copyright. All rights reserved
... Количество островковых клеток в кластерах и нестин-позитивных клеток в монослое также было несопоставимо. Возможно, неогенез островковых клеток происходит как в результате дифференцировки нестин-позитивных клеток, так и путем трансдифференцировки типичных протоковых клеток [11,14]. Поскольку мы наблюдали тотальное окрашивание монослоя антителами к цитокератину-19 (все клетки монослоя, в том числе и фибробластоподобные, экспрессировали этот специальный маркер протокового эпителия) и лишь незначительная часть фибробластоподобных клеток была при этом иммунопозитивна к нестину, можно предположить, что нестин-позитивные клетки в нашем случае представляли некую субпопуляцию клеток протокового эпителия. ...
Newborn rabbit pancreatic cell monolayer was obtained as we described earlier.The cultivated epithelial cells were shown by immunofluorescence to express special ductal marker CK19 and were insulin-and glucagon- negative for 10–15 days. A few fusiforms of nestin-positive cells were found in monolayer. Over 2 weeks in serum-free medium the plaques of epithelial cells became crowded and formed 3-dimentional structures – islet- like clusters. Islet-like clusters contain some insulin- and glucagon-positive cells recognized by immunohysto- chemistry staining. Pancreatic endocrine cell generation in 3-dimentional structures is discussed.
... It has been over 15 years since the first reports of efforts to generate beta cells from stem cells. The field was initiated with attempts using mouse stem cells and cellselection approaches [16][17][18], as well as spontaneous differentiation and identification of insulin-producing cells with approaches that included staining with the zinc-binding dye dithizone [19]. As an example of the former approach, Soria et al characterised cells from mouse embryonic stem cell cultures isolated via a neomycin-resistance cassette under the control of human INS regulatory elements [20]. ...
The production of fully functional insulin-secreting cells to treat diabetes is a major goal of regenerative medicine. In this article, I review progress towards this goal over the last 15 years from the perspective of a beta cell biologist. I describe the current state-of-the-art, and speculate on the general approaches that will be required to identify and achieve our ultimate goal of producing functional beta cells. The need for deeper phenotyping of heterogeneous cultures of stem cell derived islet-like cells in parallel with a better understanding of the heterogeneity of the target cell type(s) is emphasised. This deep phenotyping should include high-throughput single-cell analysis, as well as comprehensive ’omics technologies to provide unbiased characterisation of cell products and human beta cells. There are justified calls for more detailed and well-powered studies of primary human pancreatic beta cell physiology, and I propose online databases of standardised human beta cell responses to physiological stimuli, including both functional and metabolomic/proteomic/transcriptomic profiles. With a concerted, community-wide effort, including both basic and applied scientists, beta cell replacement will become a clinical reality for patients with diabetes.
... The development of the endocrine pancreas is under multifactorial control, and many of the key proteins or transcription factors involved in each developmental step have now been identified [59]. Expression of the transcription factor PDX-1/IDX-1 is essential for development and regeneration of the endocrine pancreas, and stimulation of PDX-1 may be one of the intracellular mechanisms by which exendin-4 and GLP-1 can enhance h-cell mass leading to long-term improvement in glucose control. ...
... Differentiation of ESCs occurs spontaneously upon the removal of feeder cells and LIF from the culture. Directing differentiation in a more controlled manner is most often achieved either by inducing formation of embryoid bodies (Boheler et al. 2002), adding certain growth factors to the cultures, nutrient restriction during ESC culture, or by the engineered expression of certain genes (Soria, 2001). Given their differentiation capabilities, ESCs are often used as the starting material by which to obtain cells of multiple lineages in vitro. ...
In mammalian development, primordial germ cells (PGCs) represent the initial population of cells that are committed to the germ cell lineage. PGCs segregate early in development, triggered by signals from the extra-embryonic ectoderm. They are distinguished from surrounding cells by their unique gene expression patterns. Some of the more common genes used to identify them are Blimp1, Oct3/4, Fragilis, Stella, c-Kit, Mvh, Dazl and Gcna1. These genes are involved in regulating their migration and differentiation, and in maintaining the pluripotency of these cells.
Recent research has demonstrated the possibility of obtaining PGCs, and subsequently, mature germ cells from a starting population of embryonic stem cells (ESCs) in culture. This phenomenon has been investigated using a variety of methods, and ESC lines of both mouse and human origin. Embryonic stem cells can differentiate into germ cells of both the male and female phenotype and in one case has resulted in the birth of live pups from the fertilization of oocytes with ESC derived sperm. This finding leads to the prospect of using ESC derived germ cells as a treatment for sterility. This review outlines the evolvement of germ cells from ESCs in vitro in relation to in vivo events.
... These results are promising, but a shortage of donors has lead to an explosion in the number of investigations of methods to generate islets surrogates suitable for transplantation. Some of the roads taken so far have been expansion of existing -cell lines, encapsulated islet xenografts, conversion of a stem cell population (either pancreatic or non pancreatic adult stem cells) to -cell, in vitro differentiation of embryonic stem cells to -cells (Soria 2001) or transdifferentiation from a different cell type (Nygaard Jensen and Jensen 2004). ...
... Da in jüngster Zeit adulte Stammzellen neben dem Knochenmark auch in vielen anderen Organen nachgewiesen wurden, u.a. im Gehirn [15], Leber [16,17], Muskulatur [18], Haut [19], Gefäßen [20] und auch in der Bauchspeicheldrüse [21], ist es bis heute völlig unklar, in welcher Beziehung diese adulten Stammzellen zueinander stehen. Es gibt Hinweise, daß diese Zellen gewebsspezifische Vorläufer sind, die sich unter bestimmten Bedingungen auch in andere Zelltypen "trans-" differenzieren können. ...
... Nicotinamide could protect differentiated cells from differentiation into other cell types. Exendin-4, a potent agonist of GLP-1 [4] and activin A was used to promote islet differentiation, maturation, increase insulin content in various cell lines [30,31], and inhibit apoptosis of b cells [32]. In this study activin A with exendin-4 up regulated the expressions of pdx1, ngn3, nkx2.2, insulin, glucagon, somatostatin, pancreatic polypeptide and glut2 genes. ...
Background:
MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs that regulate gene expression at the post-transcriptional level. A number of studies have led to the notion that some miRNAs have key roles in control of pancreatic islet development and insulin secretion. Based on some studies on miRNAs pattern, the researchers in this paper investigated the pancreatic differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) by up-regulation of miR-375 and down-regulation of miR-9 by lentiviruses containing miR-375 and anti-miR-9.
Methodology:
After 21 days of induction, islet-like clusters containing insulin producing cells (IPCs) were confirmed by dithizone (DTZ) staining. The IPCs and β cell specific related genes and proteins were detected using qRT-PCR and immunofluorescence on days 7, 14 and 21 of differentiation. Glucose challenge test was performed at different concentrations of glucose so extracellular and intracellular insulin and C-peptide were assayed using ELISA kit. Although derived IPCs by miR-375 alone were capable to express insulin and other endocrine specific transcription factors, the cells lacked the machinery to respond to glucose.
Conclusion:
It was found that over-expression of miR-375 led to a reduction in levels of Mtpn protein in derived IPCs, while treatment with anti-miR-9 following miR-375 over-expression had synergistic effects on MSCs differentiation and insulin secretion in a glucose-regulated manner. The researchers reported that silencing of miR-9 increased OC-2 protein in IPCs that may contribute to the observed glucose-regulated insulin secretion. Although the roles of miR-375 and miR-9 are well known in pancreatic development and insulin secretion, the use of these miRNAs in transdifferentiation was never demonstrated. These findings highlight miRNAs functions in stem cells differentiation and suggest that they could be used as therapeutic tools for gene-based therapy in diabetes mellitus.
... Improvement of the in vitro differentiation process has benefited from a deeper knowledge of islet development. Sequential expression of the transcription factors [7][8][9] and signaling pathways [10] involved in human β-cell genesis are instrumental to achieve in vitro differentiation processes. Hence, the common approach to differentiate hESCs is based on a multi-stages protocol attempting to reproduce in vivo pancreas development aiming to induce hESCs to follow a sequential transition through mesendoderm, definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor stages, finally obtaining functional insulin-expressing cells [11][12][13]. ...
Human embryonic stem cells (hESCs) retain the extraordinary capacity to differentiate into different cell types of an adult organism, including pancreatic β-cells. For this particular lineage, although a lot of effort has been made in the last ten years to achieve an efficient and reproducible differentiation protocol, it was not until recently that this aim was roughly accomplished. Besides, several studies evidenced the impact of resveratrol (RSV) on insulin secretion, even though the mechanism by which this polyphenol potentiates glucose-stimulated insulin secretion (GSIS) is still not clear. The aim of this study was to optimize an efficient differentiation protocol that mimics in vivo pancreatic organogenesis and to investigate whether RSV may improve the final maturation step to obtain functional insulin-secreting cells. Our results indicate that treatment of hESCs (HS-181) with activin-A induced definitive endoderm differentiation as detected by the expression of SOX17 and FOXA2. Addition of retinoic acid (RA), Noggin and Cyclopamine promoted pancreatic differentiation as indicated by the expression of the early pancreatic progenitor markers ISL1, NGN3 and PDX1. Moreover, during maturation in suspension culture, differentiating cells assembled in islet-like clusters, which expressed specific endocrine markers such as PDX1, SST, GCG and INS. Similar results were confirmed with the human induced Pluripotent Stem Cell (hiPSC) line MSUH-001. Finally, differentiation protocols incorporating RSV treatment yielded numerous insulin-positive cells, induced significantly higher PDX1 expression and were able to transiently normalize glycaemia when transplanted in streptozotocin (STZ) induced diabetic mice thus promoting its survival. In conclusion, our strategy allows the efficient differentiation of hESCs into pancreatic endoderm capable of generating β-cell-like cells and demonstrates that RSV improves the maturation process.
... In fact, the expression of Pdx1 increased markedly (Fig. 3a). This gene is well understood to be a master gene for the generation of early pancreatic development (Soria 2001) as well as of b-cells. ...
In order to produce insulin-secreting cells with a high value of glucose-stimulated insulin secretion (GSIS) from mouse embryonic stem cells, we have developed an optimized 5-stage protocol by referring to culture conditions so far reported elsewhere. This protocol is characterized by 4 points: (1) use of an activin-free medium in the first stage, (2) use of gelatin/fibronectin coated culture dishes in 1–4 stages throughout, (3) removal of undifferentiated cells by cell sorter at the end of 4th stage, and (4) sedimental culture in the 5th stage.
GSIS value of the produced cells reached 2.4, that was at a higher rank of those so far reported. The produced cells were transplanted in diabetes model mice but no remedy effect was observed. Then transplantation was conducted in pre-diabetes model mice, in which GSIS was impaired without affecting insulin producing function. The transplantation of 5 × 106 cells resulted in a marked improvement of glucose tolerance within 20 days. This effect decreased but was still observed at 120 days post-transplantation. This demonstrates the feasibility of the novel optimized protocol.
... Una opción muy prometedora es la diferenciación artificial de células beta para ser utilizadas como terapia celular sustitutiva de la diabetes tipo 1 (36). Este tipo de célula artificial debe ser capaz de producir y segregar suficiente cantidad de insulina, contando al mismo tiempo con un sistema regulador FNH 1 alfa/FNH 4 alfa intacto (37). Por otra parte, las mutaciones leves en FNH 1alfa, FNH 4 alfa e IPF 1 asociadas a diabetes tipo 2 de aparición tardía indican que algunos de los componentes de la red transcripcional ligada a la diabetes MODY pueden estar implicados en la fisiopatología de formas de diabetes tipo 2 clásicas (38). ...
... RA and cAMP have been shown in several studies to be essential for inducing ESC differentiation into IPCs, which occurs by inhibiting cell proliferation and promoting the differentiation direction toward endocrine cells (Grover and Adamson 1986;Globus et al. 1994;Stafford and Prince 2002;Tulachan et al. 2003). Moreover, it has been shown that nicotinamide preserves islet cell viability and function, and activin-A promotes islet differentiation and maturation or increase the insulin content in various cell lines (Soria 2001;D'Ippolito et al. 2004;Tang et al. 2004). IGF-1 may promote insulin secretion and prevent apoptosis of β cells in vivo (Kulkarni et al. 2002). ...
Advances in cell-replacement strategies for diabetes have focused on renewable sources of glucose-responsive, insulin-producing cells (IPCs). One of the most proper alternatives is multipotent skin-derived precursors cells (SKPs), which can be differentiated into IPCs. In this study, we reported the isolation and expansion of human skin-derived precursors (hSKPs) followed by their differentiation into IPCs in vitro, through exposure to suitable differentiation factors. The gene expression of endocrine β cell markers was analyzed by reverse transcriptase-polymerase chain reaction. In addition, insulin production was examined immunocytochemically, and insulin and C-peptide secretion were examined using enzyme-linked immunosorbent assay. Dithizone-stained cellular clusters were observed after approximately 20 d. The clusters were found to be immunoreactive to insulin and expressed multiple genes related to pancreatic β cell development and function: insulin, Pdx-1, Islet-1, NeuroD, Glut-2, Pax-4, Ngn-3, and Nkx2.2, but not to other pancreas-specific hormones such as glucagon and somatostatin. Cellular clusters were also able to secrete detectable amounts of insulin and C-peptide in a glucose dose-dependent manner. These findings suggest that human SKPs can differentiate into functional IPCs. This may offer a safer cell source for future stem cell-based therapies.
Type 1 diabetes (T1D) is a degenerative disease affecting 8.5% of the world population. Treatments for T1D are currently limited to insulin injections, to control hyperglycemia, and in severe cases transplantation of the whole pancreas or islets of Langerhans. Unfortunately, these therapies are not free of side effects, nor do they represent a definitive solution for T1D cure. Exogenous stem cell transplantation is a promising strategy to promote tissue replacement. Creating pancreatic organoids from differentiated human-induced pluripotent stem cells (hiPSCs) will overcome the shortage of usable donor pancreas with a personalized regenerative medicine approach. This chapter provides an insight into the most influential factors that may enable successful transplantation using stem cell-derived pancreatic organoids and the current technologies available to be able to do this.
According to the figures released by the International Diabetes Federation (IDF), there were more than 4.8 million diabetes-related deaths in 2012 alone, and a total of 371 million people were diagnosed with diabetes. Incidentally, with the number of undiagnosed and diagnosed diabetic patients increasing globally, the Kingdom of Saudi Arabia is rated as one of the top few countries with a high prevalence rate estimated at 23.9%. Insufficient availability of insulin remains the hallmark of diabetes type 1 due to autoimmune loss of functionally competent β-cells, whereas diabetes type 2 may result from a combination of insufficiency of insulin and tissue resistance to the available insulin. Islet transplantation is a gold-standard treatment for diabetic patients, but poor availability of donor islets and concerns regarding long-term survival and functional ineffectiveness of the graft have hampered the progress of this treatment strategy for diabetic patients as a routine therapeutic option. Current advances in pharmacological intervention to maintain glucose homeostasis and preserve insulin-secreting β-cells can merely provide symptomatic relief. More than a decade of research has shown that stem/progenitor cells can cross lineage restriction and differentiate to adopt morphofunctionally competent β-cells. In vitro protocols have been devised based on optimization of culture conditions or genetic modification of stem cells for their efficient differentiation to mature β-cells which can secrete insulin in a glucose-responsive manner. There is a recent surge in the interest to use pluripotent stem cells following the new less stringent legislation regarding the use of embryonic stem cells (ESCs). This has also led to the advent of protocols for the reprogramming of somatic cells to achieve pluripotency reminiscence of ESCs. Induced pluripotent stem cells (iPSCs) may provide an alternative renewable source of patient-specific β-cells for transplantation to support an inefficient intrinsic repair mechanism. Additionally, given that cellular reprogramming is a multistep event and entails diverse epigenetically unstable intermediate stages, morphofunctionally competent surrogate progenitors with β-cell characteristics without iPSC generation will provide a renewable source of progenitors for tumor-free de novo regeneration of islets. This book chapter explicitly focuses on the effect of diabetes on the functionality of the intrinsic pool of stem/progenitor cells and critically appreciates the published data defining the possibility to exploit exogenous stem cells for replacement of nonfunctional pancreatic β-cells to normalize insulin production.
Type 2 diabetes more often develops in obese persons. Impaired β cell precursor proliferation, β cell maturation, glucose uptake, and insulin secretion in response to high glucose result in mitochondrial and endoplasmic reticulum stress associated with ROS release, inflammation, and β cell death. The multiple-hit pathogenesis models of obesity and type 2 diabetes share an imbalance in the expression of let-7, miR-9, miR-17, miR-21, miR-26a, miR-27, miR-29, miR-30, miR-34a, miR-124, miR-130, miR-138, miR-145, miR-146a, miR-150, miR-181, miR-221–222, miR-223, miR-375, miR-378 and miR-455, and lncRNAs βLINC, GAS5, HOTAIR, H19, MEG3, NEAT1 and TINCR, and circ-RNA PVT1. Typically for the pancreas, the up-regulation of miR-7 due to loss of silencing circular RNA ciRs-7 protects against diabetes by inducing differentiation of stem cells to β cells but exacerbates the diabetic phenotype by decreasing β cell mass and impairing insulin signaling. High glucose-induced miR-375 is essential for retaining β cell mass but inhibited PI3K and thereby insulin signaling. The PDX1 associated lncRNA, up-regulator of transcription (PLUTO), is a tissue-specific inhibitor of β cell maturation and insulin production.
The feasibility of isolating and manipulating mesenchymal stem cells (MSCs) from human patients provides hope for curing numerous diseases and disorders. Recent phenotypic analysis has shown heterogeneity of MSCs. Nestin progenitor cell is a subpopulation within MSCs which plays a role in pancreas regeneration during embryogenesis. This study aimed to separate nestin (+) cells from human bone marrow MSCs, and differentiate these cells into functional insulin producing cells (IPCs) compared with nestin (-) cells. Manual magnetic separation was performed to obtain nestin (+) cells from MSCs. Approximately 91±3.3% of nestin (+) cells were positive for anti-nestin antibody. Pluripotent genes were overexpressed in nestin (+) cells compared with nestin (-) cells as revealed by quantitative real time-PCR (qRT-PCR). Following in vitro differentiation, flow cytometric analysis showed that 2.7±0.5% of differentiated nestin (+) cells were positive for anti-insulin antibody in comparison with 0.08±0.02% of nestin (-) cells. QRT-PCR showed higher expression of insulin and other endocrine genes in comparison with nestin (-) cells. While immunofluorescence technique showed the presence of insulin and C-peptide granules in nestin (+) cells. Therefore, our results introduced nestin (+) cells as a pluripotent subpopulation within human MSCs which is capable to differentiate and produce functional IPCs.
The studies have been done on patient-specific human adipose-derived from mesenchymal stem cells (hADSCs) like a series of autologous growth factors and nanofibrous scaffolds (3D culture) will probably have many benefits for regenerative medicine in type 1 diabetes mellitus (TIDM) patients in the future. For this purpose, we established a polyvinyl alcohol (PVA) scaffold and a differentiation protocol by adding platelet-rich plasma (PRP) that induces the hADSCs into insulin-producing cells (IPCs). The Characteristics of the derived IPCs in 3D culture were compared with conventional culture (2D) groups evaluated at the mRNA and protein levels. The viability of induced pancreatic cells was 14 days. The in vitro studies showed that the treatment of hADSCs in the 3D culture resulted in differentiated cells with strong characteristics of IPCs including pancreatic-like cells, the expression of the islet-associated genes at the mRNA and protein levels in comparison of 2D culture group. Furthermore, the immunoassay tests showed that these differentiated cells in these two groups are functional and secreted C-peptide and insulin in a glucose stimulation challenge. The results of our study for the first time demonstrated that the PVA nanofibrous scaffolds along with the optimized differentiation protocol with PRP can enhance the differentiation of IPCs from hADSCs. In conclusion, this study provides a new approach to the future pancreatic tissue engineering and beta cell replacement therapies for T1DM. This article is protected by copyright. All rights reserved
Methods for differentiating human pluripotent stem cells into islet-like cells are provided. In certain embodiments, the methods utilize sequential culturing of the human pluripotent stem Cells With certain factors to produce islet-like cells. In certain embodiments, the population of cells produced by the methods is further enriched for islet-like cells.
Diabetes mellitus (DM) is reported as the most common metabolic disorder and considered major causes of morbidity and mortality. Type 1 DM (T1DM) is considered an autoimmune disease characterized by deficiency of the insulin secretion due to destruction of specific insulin-producing β-cells. Type 2 DM (T2DM) is defined as an endocrine disease associated with predominantly insulin resistance/metabolically active obese, adipocytokines abnormalities, and secondary development of β-cell dysfunction. With growing understanding of the pathogenesis of DM, alternative approaches aiming at repair and restoration of endogenous insulin production, regulation of metabolic processes with stem cell transplantation are increasingly considered as complements to current diabetes therapy strategies. However, the data on regenerative care in DM are not uniform. There are discrepancies between results received from the animal studies and data obtained by clinical investigations in this field. On the one hand, such inconsistencies have accompanied the fact that several types of stem cells were tested as perspective for regenerative strategy and nor all of stem cells were available in routine clinical practice. On the other hand, patients with different types of diabetes at several stages of evolution of the disease are failed to uniform considered candidates for stem cells transplantation and they probably are required a controversial approaches. Given the conflicting evidence concerning stem cell replacement in DM, the aim of the chapter is to explore, analyze, and summarize the data to clarify current knowledge and identify the future perspectives for regenerative care among DM patients. The present chapter accumulated contemporary knowledge regarding a paradigm of the regenerative therapy in DM. It is discussed the role of use of reprogramming stem cells, bone marrow-derived mononuclear cells, and lineage-specified progenitor cells in modern approaches of DM care.
Methods for differentiating human pluripotent stem cells into islet-like cells are provided. In certain embodiments, the methods utilize sequential culturing of the human pluripotent stem cells with certain factors to produce islet-like cells. In certain embodiments, the population of cells produced by the methods is further enriched for islet-like cells.
Haematopoietic stem cells (HSCs) possess multipotent ability to differentiate into various types of cells on providing appropriate niche. In the present study, the differentiating potential of human HSCs into β-cells of islets of langerhans was explored. Human HSCs were apheretically isolated from a donor and cultured. Phenotypic characterization of CD34 glycoprotein in the growing monolayer HSCs was confirmed by immunocytochemistry and flow cytometry techniques. HSCs were induced by selection with beta cell differentiating medium (BDM), which consists of epidermal growth factor (EGF), fibroblast growth factor (FGF), transferrin, Triiodo-l-Tyronine, nicotinamide and activin A. Distinct morphological changes of differentiated cells were observed on staining with dithizone (DTZ) and expression of PDX1, insulin and synaptophysin was confirmed by immunocytochemistry. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed distinct expression of specific β-cell markers, pancreatic and duodenal homeobox-1 (PDX1), glucose transporter-2 (GLUT-2), synaptophysin (SYP) and insulin (INS) in these differentiated cells compared to HSCs. Further, these cells exhibited elevated expression of INS gene at 10 mM glucose upon inducing with different glucose concentrations. The prominent feature of the obtained β-cells was the presence of glucose sensors, which was determined by glucokinase activity and high glucokinase activity compared with CD34+ stem cells. These findings illustrate the differentiation of CD34+ HSCs into β-cells of islets of langerhans.
Allogeneic islet cell transplantation is currently the only curative treatment of diabetes mellitus. However due to organ shortage islet transplantation is limited and immunsupressive protocols are hazardous and complicated side effects. The incidence of diabetes is constantly growing and a treatment with autologous insulin producing cells would hold great promise for the therapy of diabetes. We herein describe the generation of insulin producing »Neo-islet cells« from human peripheral blood monocytes, which are able to secret insulin in a glucose dependent fashion and normalize blood glucose levels after transplantation into diabetic mice for a period of ten days. In comparison with pancreatic islet cells »Neo-islet cells« exhibit a gene expression pattern which is comparable to an endocrine precursor cell as known from embryonic pancreas developmental studies.
Human monocytes were treated with MCSF and IL-3 in order to undergo a step of dedifferentiation. Further culture in EGF, HGF and nicotinamide for 4 to 8 days resulted in the formation of cell aggregates expressing insulin, somatostatin and glucagon. Insulin and C-peptide secretion was shown to be glucose dependent after incubation with 22mM glucose whereas 3mM glucose did not induce insulin secretion. MRNA was isolated from »Neo-islet cells« at different times and analyzed for expression of insulin, somatostatin, glucagon, PDX-1, Ngn3, carboxypeptidase, Nkx6.1, NeuroD, Glut2, Ngn3, MAFa and Pax4. Stable expression of insulin, carboxypeptidase, glucagon, Maffa, Nkn6.1, Ngn3 and Neuro D was found. The expression of somatostatin, Isl-1, pro-hormone-convertase, Glut2, Pax 4 and PDX 1 was not reproducible in all experiments and so far not stable. Although »Neoislet cells« are not fully differentiated they were able to normalize blood glucose in diabetic mice transiently. We hope to characterize these cells in more detail in order to optimize the differentiation protocol as a perspective for autologous cell therapy of diabetes.
Since their derivation in 1999, human pluripotent embryonic stem (ES) and embryonic germ (EG) cells have attracted considerable attention (1–3) Mainly this is the result of the promise of application of these cells to the treatment of a variety of diseases that involve tissue and organ degeneration (4, 5) There is no doubt that even with the best organ procurement programs in place, the supply of cadaveric organs needed for transplantation would never satisfy the demand. Type I and type II diabetes mellitus, which affect 16 million people in the United States alone, are among the most promising targets for pluripotent-stem-cell-based applications (6,7) Although they are different diseases, type I and type II diabetes both involve inadequate mass of insulin-producing β-cells, a condition resulting in elevated blood glucose level (hyperglycemia). Insulin injections alleviate hyperglycemia in most patients, but they do not provide a stable finely tuned control of glucose homeostasis that is provided by the healthy pancreas. This lack of fine control in turn leads to numerous complications, including cardiovascular and kidney disease, neuropathy, and blindness.
Cell lineage development is a finely tuned process of proliferation and differentiation, survival and apoptosis, that is regulated by numerous extracellular signals. Here we review some of the extracellular signals-including insoluble cell-cell and extracellular matrix-cell interactions, as well as soluble factors-that appear critical for pancreatic beta-cell development. Knowledge of how these signals control the development of pancreatic endocrine stem/precursor cells into fully functional insulin-secreting beta cells is a platform for the restoration of beta-cell function and the cure therapy of type 1 diabetes.
Background: Islet and islet cell transplantation for the treatment of diabetes has achieved effect, but the research is limited dut to the shortage of islet and immune rejection. Objective: To observe the effect of transplantation of islet-like cells that in vitro differentiated from bone marrow mesenchymal stem cells on the treatment of diabetes in rats. Methods: The rat bone marrow mesenchymal stem cells were induced with basic fibroblast growth factors and hepatocyte growth factors, and then received immunocytochemistry staining to detect the induction. The Sprague Dawley rats received intraperitoneal injection of streptozotocin to establish the diabetes models. After modeling, the rats were randomly divided into control group and experimental group (transplanted with induced islet-like cells). The experimental group was transplanted with the induced islet-like cells through renal capsule, and the control group was transplanted with normal saline in the same dose. The blood glucose and body mass of the diabetes rats were observed after transplantation. Results and Conclusion: The bone marrow mesenchymal stem cells could differentiate into islet-like cells after in vitro induced with basic fibroblast growth factors and hepatocyte growth factors. There was no significant change in blood glucose of the control group after transplantation (P > 0.05), and the blood glucose of the rats in the experimental group was significantly decreased compared with the control group (P < 0.05). The bone marrow mesenchymal stem cells can differentiate into islet-like cells after in vitro induced with the induction system containing basic fibroblast growth factors and hepatocyte growth factors, and the islet-like cells have a certain ability of insulin secretion. The transplantation of induced islet-like cells after transplanted into the diabetes rats through renal capsule can decrease the blood glucose level of the rats.
Shortage in the number of available pancreas and isolated islets has forced researchers to study and develop different cell types that can be potentially used for cell replacement therapy in diabetes. The major question is the choice of such a cell type. Ideally, an islet progenitor cell should meet two criteria: (1) ability to proliferate while retaining its stem-cell properties under defined conditions, in vitro, and (2) to be able to efficiently differentiate into insulin-producing cells that can release physiologically significant amount of insulin in response to glucose. Although cell types such as embryonic stem cells satisfy the former criteria, none of the studies carried out until now have been able to meet the latter criteria. We proposed earlier that precursor/progenitor cells generated from human insulin-producing islet of Langerhans would be ideal candidates for clinical use in diabetes. One of the major reasons to propose this hypothesis is that epigenetic marks that characterize insulin promoter region in these islet cells are heritable and are retained in proliferating progenitor cells obtained from islets. There is now a significant amount of data to believe that human pancreatic islet-derived cells are better progenitors for differentiation into insulin-producing cells. In this chapter, we discuss the major cell types that have been proposed and assessed for potential use in cell replacement therapy for diabetes. Understanding the potential and safety of such cell types will help in considering their suitability for clinical use in diabetes.
Embryonic stem cells (ESC) have potential to be used in future therapeutic applications due to their unlimited self-renewal capabilities coupled with their ability to differentiate into any cell type. Mathematical models of ESC have gained much attention in recent years for their ability to extract information and insight from this self-renewal and differentiation system which might be otherwise elusive when using experimental data alone. In this chapter, we first present a brief review of previous efforts to model the ESC system, including foci on single cells, populations, self-renewal and differentiation mechanisms, and signaling and gene regulatory networks (GRN). GRN identification in ESC is invaluable, as proper information on network connections can give insight on how stem cells differentiate and can help in the development of efficient differentiation to specific cellular phenotypes. Although there has been considerable work on network identification of bacteria and the ESC self-renewal circuitry, work is still limited on differentiation. We therefore present our work on reverse engineering the gene regulatory network in differentiating ESC. In our network identification algorithm, we incorporate the inherent biological feature of sparsity, the notion that a network favors as few connections as possible. Our algorithm consists of a bi-level formulation, in which the upper level predicts the network topology and minimizes the number of connections, while the bottom level estimates the kinetic parameters and minimizes the error between predicted and experimental profiles. We apply our bi-level formulation to the system of mouse ESC differentiating towards pancreatic lineage. The input to the algorithm was the expression dynamics of relevant transcription factors. We show that the predicted gene behavior is in very good agreement with the in vitro experimental data, and that many of the interactions in the reconstructed network and predicted effects of external perturbations have been reported in literature, even though this information was not used to train the model a priori. The predictive capability of the algorithm was further substantiated by modeling the effect of Foxa2 silencing on differentiation outcome and validating it experimentally by gene silencing experiments.
Mesenchymal stem cells(MSCs) are multipotent cells that may serve as a source of cells for generation of surrogate β cell. The objectives of this study are to generate insulin-producing cells in human mesenchymal stem cells(hMSCs) using PDX1(pancreatic duodenal homeobox 1) lentiviral vectors. PDX1 is essential for normal of pancreatic islet function as suggested by its regulatory action on the expression of a number of pancreatic genes, including insulin, somatostatin, islet amyoid polypeptide, glucagon. Thus, human mesenchymal stem cells(hMSCs) were exposed to a recombinant lentivirus expressing human PDX1 under control of EF-1á promoter. To induce a pancreatic fate in the PDX1 population, it was isolated by cell sorting, cultured in the presence of factors known to promote pancreatic development. Differentiated cells were analyzed by qRT-PCR and immunocytochemistry were further characterized for markers specific to mature pancreatic cells. In PDX1-expressing hMSCs, PAX4, Nkx6.1, insulin, glucagon mRNA levels increase significantly. Insulin-immunoreactivity was examined in PDX1-expressing hMSCs with DTZ-staining. In this study, we demonstrated that constitutive overexpression of exogenous PDX1 in hMSCs cells might have potentials to induce pancreatic lineage differentiation.
Diabetes mellitus is one of the most prevalent chronic diseases. Glucose homeostasis disruption occurs when β-cells fail to secrete the insulin necessary to maintain the homeostasis of glucose in the blood flow. Over time, diabetes can lead to the rise of different long-term complications, such as diabetic foot, retinopathy, neuropathy, nephropathy and arteriosclerosis. Nowadays, the only treatments for diabetes consist of exogenous insulin supply or pancreas/islet transplantation, but the inability to achieve a tight control over glucose regulation by exogenous insulin administration and the shortage of pancreatic islets donors have motivated recent efforts to develop renewable sources and protocols for effective β-cell replacement.
Introduction:
Tremendous progress has been made in generating insulin-producing cells from pluripotent stem cells. The best outcome of the refined protocols became apparent in the first clinical trial announced by ViaCyte, based on the implantation of pancreatic progenitors that would further mature into functional insulin-producing cells inside the patient's body. Areas covered: Several groups, including ours, have contributed to improve strategies to generate insulin-producing cells. Of note, the latest results have gained a substantial amount of interest as a method to create a potentially functional and limitless supply of β-cell to revert diabetes mellitus. This review analyzes the accomplishments that have taken place over the last few decades, summarizes the state-of-art methods for β-cell replacement therapies based on the differentiation of embryonic stem cells into glucose-responsive and insulin-producing cells in a dish and discusses alternative approaches to obtain new sources of insulin-producing cells. Expert opinion: Undoubtedly, recent events preface the beginning of a new era in diabetes therapy. However, in our opinion, a number of significant hurdles still stand in the way of clinical application. We believe that the combination of the private and public sectors will accelerate the process of obtaining the desired safe and functional β-cell surrogates.
Diabetes mellitus is a metabolic disease characterized by elevated blood glucose levels due to absent (juvenile or type 1 diabetes) or insufficient insulin production (type 2 diabetes). In the light of the actual worldwide diabetes epidemic, the generation of insulin producing cells represents an attractive alternative treatment option for patients who have lost their residual insulin production. Mesenchymal stem cells (MSC) from mouse bone marrow were recently shown to harbor the potential to differentiate into insulin secreting cells in vitro and to reverse hyperglycemia in an animal model of diabetes [Tang et al., 2004]. MSC from human bone marrow and adipose tissue represent very similar cell populations with comparable phenotypes. Adipose tissue and bone marrow from human adults are abundant and easily accessible and could also harbor cells with the potential to differentiate in insulin producing cells.
We isolated human adipose tissue-derived MSC from four and human bone marrow-derived MSC from five healthy donors. During the proliferation period, the cells expressed the stem cell markers nestin, ABCG2, SCF, Thy-1 as well as the pancreatic endocrine transcription factor Isl-1. The cells were induced to differentiate into a pancreatic endocrine phenotype by defined culture conditions within 3 days. Formation of islet like clusters was observed already 24 hours after initiation of the differentiation process. Using quantitative real-time PCR a down-regulation of ABCG2 and up-regulation of the pancreatic developmental transcription factors Isl-1, Ipf-1, and Ngn3 were observed together with induction of the islet hormones insulin, glucagon, and somatostatin. We also analyzed the islet proteins in differentiated MSC. C-peptide and glucagon positive cells were found in differentiated islet-like cluster. Although C-peptide or glucagon release was not found in differentiated islet like clusters we were able to measure somatostatin release into the culture medium.
In summary, Isl-1 positive MSC can be isolated from human bone marrow as well as from adipose tissue and are able to adopt a pancreatic endocrine phenotype. These cells could be used as a human model to develop stem cell based therapies for diabetes mellitus in the future.
Diabetes mellitus is a metabolic syndrome characterized by increased levels of blood glucose. Type 1 diabetic patients and patients with Type 2 diabetes suffering from defective insulin secretion rely on lifelong substitution with exogenous administration of insulin. Whole pancreas and purified pancreatic islet transplantation have offered the potential for independence from insulin injections. A major obstacle, however, is the limited supply of cadaveric human islets. Success in islet transplantation-based therapies for Type 1 diabetes, coupled with a worldwide shortage of transplant-ready islets, has motivated efforts to develop renewable sources of islet-replacement tissues. Stem cells offer the greatest potential for the development of an abundant source of pancreatic islets. Insulin-producing cells for transplantation can be generated from both embryonic and adult stem cells. Before stem cell therapeutic strategies for diabetes mellitus can be transferred to clinical application in humans, stem cell biologists have to address several pressing issues related to appropriate differentiation protocols, functional aspects of insulin secretion, its regulation, cell-maturation processes and control of proliferation, along with ethical norms and safety.
Background: Stem cell research holds a hope for better treatment of many diseases particularly those like diabetes. Mesenchymal stem cells
having the ability to differentiate into functionally insulin-producing cells
Aim of the work: Evaluation of the role of undifferentiated & differentiated mesenchymal stem cells (insulin producing cells IPCs) in diabetic dogs.
Materials and Methods: The present study was performed on two groups of nine dogs .Group I including one dog used as (–ve control). Group II formed of eight diabetic models received single intravenous (i.v) injection of alloxan (50 mg/kg) to induce diabetes ÷d into 3 subgroups. Subgroup IIA (two dogs) used as +ve control, subgroup IIB (two dogs) received undifferentiated mesenchymal stem cells in a dose of (5 ×106) intrahepatic percutaneously and subgroup IIC (four dogs) received differentiated MSCs (Insulin-Producing Cells IPCs) in a dose like subgroup IIB. Dogs were sacrificed with an overdose of sodium pentobarbital (100mg/kg) one week after transplantation. Fasting blood glucose level measurements, H&E and immunohistochemical staining were done.
Results: Significant increase in insulin immunoreactive cells of pancreas and liver of groups which received undifferentiated and differentiated mesenchymal stem cells (p<0.05) as compared to +ve control. Fasting blood glucose level improved in group which received mesenchymal stem cells.
Conclusion: Transplantation of autologus MSCs (Insulin-Producing Cells IPCs) can improve diabetes (improve blood glucose level).
Proper insulin secretion requires the coordinated functioning of the numerous beta cells that form pancreatic islets. This coordination depends on a network of communication mechanisms whereby beta cells interact with extracellular signals and adjacent cells via connexin channels. To assess whether connexin-dependent communication plays a role in vivo, we have developed transgenic mice in which connexin 32 (Cx32), one of the vertebrate connexins found in the pancreas, is expressed in beta cells. We show that the altered beta-cell coupling that results from this expression causes reduced insulin secretion in response to physiologically relevant concentrations of glucose and abnormal tolerance to the sugar. These alterations were observed in spite of normal numbers of islets, increased insulin content, and preserved secretory response to glucose by individual beta cells. Moreover, glucose-stimulated islets showed improved electrical synchronization of these cells and increased cytosolic levels of Ca(2+). The results show that connexins contribute to the control of beta cells in vivo and that their excess is detrimental for insulin secretion.
Human pluripotent stem cells (hPSCs) have been derived from the
inner cell mass cells of blastocysts (embryonic stem cells) and
primordial germ cells of the developing gonadal ridge (embryonic germ
cells). Like their mouse counterparts, hPSCs can be maintained in
culture in an undifferentiated state and, upon differentiation,
generate a wide variety of cell types. Embryoid body (EB) formation is
a requisite step in the process of in vitro
differentiation of these stem cells and has been used to derive neurons
and glia, vascular endothelium, hematopoietic cells, cardiomyocytes,
and glucose-responsive insulin-producing cells from mouse PSCs. EBs
generated from human embryonic germ cell cultures have also been found
to contain a wide variety of cell types, including neural cells,
vascular endothelium, muscle cells, and endodermal derivatives. Here,
we report the isolation and culture of cells from human EBs as well as
a characterization of their gene expression during growth in several
different culture environments. These heterogeneous cell cultures are
capable of robust and long-term [>70 population doublings (PD)]
proliferation in culture, have normal karyotypes, and can be
cryopreserved, clonally isolated, and stably transfected. Cell cultures
and clonal lines retain a broad pattern of gene expression including
simultaneous expression of markers normally associated with cells of
neural, vascular/hematopoietic, muscle, and endoderm lineages. The
growth and expression characteristics of these EB-derived cells suggest
that they are relatively uncommitted precursor or progenitor cells.
EB-derived cells may be suited to studies of human cell differentiation
and may play a role in future transplantation therapies.
Insulin gene expression is restricted to islet cells of the mammalian
pancreas through specific control mechanisms mediated in part by specific
transcription factors1,
2. The protein encoded by the pancreatic
and duodenal homeobox gene 1 (PDX-1) is central in regulating pancreatic development
and islet cell function3. PDX-1 regulates insulin gene expression
and is involved in islet cell-specific expression of various genes4,
5,
6,
7.
Involvement of PDX-1 in islet-cell differentiation and function has been demonstrated
mainly by 'loss-of-function' studies8,
9,
10,
11.
We used a 'gain-of-function' approach to test whether PDX-1 could
endow a non-islet tissue with pancreatic -cell characteristics in
vivo. Recombinant-adenovirus-mediated gene transfer of PDX-1 to the livers
of BALB/C and C57BL/6 mice activated expression of the endogenous, otherwise
silent, genes for mouse insulin 1 and 2 and prohormone convertase 1/3 (PC
1/3). Expression of PDX-1 resulted in a substantial increase in hepatic immunoreactive
insulin content and an increase of 300% in plasma immunoreactive insulin levels,
compared with that in mice treated with control adenovirus. Hepatic immunoreactive
insulin induced by PDX-1 was processed to mature mouse insulin 1 and 2 and
was biologically active; it ameliorated hyperglycemia in diabetic mice treated
with streptozotocin. These data indicate the capacity of PDX-1 to reprogram
extrapancreatic tissue towards a -cell phenotype, may provide a valuable
approach for generating 'self' surrogate cells, suitable
for replacing impaired islet-cell function in diabetics.
Pancreatic islets are neuroendocrine organs that control blood glucose homeostasis. The precise interplay of a heterogeneous group of cell populations (#, !, ' and PP cells) results in the fine-tuned release of counterbalanced hormones (insulin, glucagon, somatostatin and pancreatic polypeptide respectively). Under the premises of detailed knowledge of the physiological basis underlying this behaviour, two lines of investigation might be inferred: generating computational and operational models to explain and predict this behaviour and engineering islet cells to reconstruct pancreatic endocrine function. Whilst the former is being fuelled by new computational strategies, giving biophysicists the possibility of modelling a system in which new "emergent" properties appear, the latter is benefiting from the useful tools and strategic knowledge achieved by molecular, cell and developmental biologists. This includes using tumour cell lines, engineering islet cell precursors, knowledge of the mechanisms of differentiation, regeneration and growth and, finally, therapeutic cloning of human tissues. Gaining deep physiological understanding of the basis governing these processes is instrumental for engineering new pancreatic islets.
Development of endocrine cells in the endoderm involves Atonal and Achaete/Scute-related basic helix-loop-helix (bHLH) proteins. These proteins also serve as neuronal determination and differentiation factors, and are antagonized by the Notch pathway partly acting through Hairy and Enhancer-of-split (HES)-type proteins. Here we show that mice deficient in Hes1 (encoding Hes-1) display severe pancreatic hypoplasia caused by depletion of pancreatic epithelial precursors due to accelerated differentiation of post-mitotic endocrine cells expressing glucagon. Moreover, upregulation of several bHLH components is associated with precocious and excessive differentiation of multiple endocrine cell types in the developing stomach and gut, showing that Hes-1 operates as a general negative regulator of endodermal endocrine differentiation.
Totipotent murine ES cells h ave an enormous potential for the study of cell specification. He re we demonstrate that ES cells can diffe rentiate to hemopoietic cells th rough the proximal lateral mesoderm, me rely upon culturing in type IV collagen-coated dishes. Separation of the Flk1+ mesoderm f rom other cell lineages was critical for hemopoietic cell diffe rentiation, whe reas formation of the embryoid body was not. Since the two-dimensionally spreading cells can be monito red easily in real time, this culture system will g reatly facilitate the study of the mechanisms i nvolved in the cell specification to mesoderm, endothelial, and hemopoietic cells. In the cultu re of ES cells, how ever, lineages and stages of diffe rentiating cells can only be defined by their own characteristics. We showed that a combination of monoclonal antibodies against E-cadherin, Flk1/KDR, PDGF recepto rα, VEcadherin, CD45 and Ter119 was suf ficient to define most intermediate stages during diffe rentiation of ES cells to blood cells. Using this cultu re system and surface markers, we determined the following order for blood cell differentiation: ES cell (E-cadherin+Flk1™PDGFRα™), proximal lateral mesoderm (E-cadherin™ Flk1+VEcadherin™ ), progenitor with hemoangiogenic potential (Flk1+VE-cadherin+CD45™ ), hemopoietic p rogenitor (CD45+c-Kit+) and mature blood cells (c-Kit™ CD45+ or Ter119+), though direct differentiation of blood cells f rom the Flk1+VE-cadherin™ stage cannot be ruled out. Not only the VE-cadherin+CD45™ population generated f rom ES cells but also those di rectly sorted f rom the yolk sac of 9.5 dpc embryos h ave a potential to give rise to hemopoietic cells. P rogenitors
with hemoangiogenic potential were identified in both the Flk1+VE-cadherin™and Flk1+VEcadherin+populations by the single cell deposition experiment. This line of evidence implicates Flk1+VEcadherin+ cells as a diverging point of hemopoietic and endothelial cell lineages.
Cell adhesion molecules (CAMs) are important mediators of cell-cell interactions and regulate cell fate determination by influencing growth, differentiation, and organization within tissues. The human pancarcinoma antigen KSA is a glycoprotein of 40 kD originally identified as a marker of rapidly proliferating tumors of epithelial origin. Interestingly, most normal epithelia also express this antigen, although at lower levels, suggesting that a dynamic regulation of KSA may occur during cell growth and differentiation. Recently, evidence has been provided that this glycoprotein may function as an epithelial cell adhesion molecule (Ep-CAM). Here, we report that Ep-CAM exhibits the features of a morphoregulatory molecule involved in the development of human pancreatic islets. We demonstrate that Ep-CAM expression is targeted to the lateral domain of epithelial cells of the human fetal pancreas, and that it mediates calcium-independent cell-cell adhesion. Quantitative confocal immunofluorescence in fetal pancreata identified the highest levels of Ep-CAM expression in developing islet-like cell clusters budding from the ductal epithelium, a cell compartment thought to comprise endocrine progenitors. A surprisingly reversed pattern was observed in the human adult pancreas, displaying low levels of Ep-CAM in islet cells and high levels in ducts. We further demonstrate that culture conditions promoting epithelial cell growth induce upregulation of Ep-CAM, whereas endocrine differentiation of fetal pancreatic epithelial cells, transplanted in nude mice, is associated with a downregulation of Ep-CAM expression. In addition, a blockade of Ep-CAM function by KS1/4 mAb induced insulin and glucagon gene transcription and translation in fetal pancreatic cell clusters. These results indicate that developmentally regulated expression and function of Ep-CAM play a morphoregulatory role in pancreatic islet ontogeny.
Insulin-like growth factor I (IGF-I) mRNA expression was studied after 90% partial pancreatectomy in the rat to determine whether IGF-I was associated with pancreatic regeneration. The level of IGF-I mRNA was maximally increased (4-fold above control value) 3 days after pancreatectomy, but thereafter gradually decreased, returning to control levels by 14 days after surgery. By in situ hybridization, IGF-I mRNA in both pancreatectomized and sham-operated rats was localized to capillary endothelial cells, indicating that this is the site of IGF-I expression in the normal rat pancreas. However, enhanced IGF-I mRNA expression was localized to focal areas of regeneration unique to pancreatectomized rats. In these areas, epithelial cells of proliferating ductules and individual connective tissue cells expressed IGF-I, suggesting that IGF-I may play an important role in the growth or differentiation of pancreatic tissue.
Pancreatic beta cells, tightly organized in the islet of Langerhans, secrete insulin in response to glucose in a calcium-dependent manner. The calcium input required for this secretory activity is thought to be provided by an oscillatory electrical activity occurring in the form of "bursts" of calcium action potentials. The previous observation that islet intracellular free Ca2+ levels undergo spontaneous oscillations in the presence of glucose, together with the fact that islet cells are coupled through gap junctions, hinted at a highly effective co-ordination between individual islet cells. Through the use of simultaneous recordings of intracellular calcium and membrane potential it is now reported that the islet calcium waves are synchronized with the beta cell bursting electrical activity. This observation suggests that each calcium wave is due to Ca2+ entering the cells during a depolarized phase of electrical activity. Moreover, fura-2 fluorescence image analysis indicates that calcium oscillations occur synchronously across the whole islet tissue. The maximal phase shift between oscillations occurring in different islet cells is estimated as 2 s. This highly co-ordinated oscillatory calcium signalling system may underlie pulsatile insulin secretion and the islet behaviour as a secretory "syncytium". Since increasing glucose concentration lengthens calcium wave and burst duration without significantly affecting wave amplitude, we further propose that it is the fractional time at an enhanced Ca2+ level, rather than its amplitude, that encodes for the primary response of insulin-secreting cells to fuel secretagogues.
Growth factors have an important role in the regulation of cell growth, division and differentiation. They are also involved in the regulation of embryonic growth and differentiation. Insulin and insulin-like growth factor I (IGF I) play an important part in these events in the later stages of embryogenesis, when organogenesis is completed. In this study, we are presenting evidence that insulin and IGF I are also secreted by embryonic tissues during the prepancreatic stage of mouse development. We found measurable amounts of insulin and IGF I in 8- to 12-day-old mouse embryos. We also showed that embryonic cells derived from 8-, 9- and 10-day-old mouse embryos secrete insulin, IGF I and/or related molecules. Furthermore, the same growth factors, when added to the culture of 9-day-old mouse embryonic cells, stimulate their proliferation. These results lead to the conclusion that insulin can stimulate the growth of embryonic cells during the period when pancreas is not yet formed, which is indirect evidence for a paracrine (or autocrine) type of action.
Differentiation is the process by which multicellular organisms achieve the specialized functions necessary for adaptation and survival. An in vivo model in the Syrian golden hamster is described in which regenerating pancreatic cells are converted into hepatocyte-like cells, as evidenced by the presence of albumin, peroxisomes, and a variety of morphological markers. These cells are stable after the conversion is triggered by a single dose of the carcinogen N-nitrosobis(2-oxopropyl)amine administered during the S phase in regenerating pancreatic cells. This suggests that, given the proper stimulus, regenerating cells in adult pancreas can be redirected into a totally different pathway of differentiation.
Early neurogenesis progresses by an initial massive proliferation of neuroepithelial cells followed by a sequential differentiation of the various mature neural cell types. The regulation of these processes by growth factors is poorly understood. We intend to understand, in a well-defined biological system, the embryonic chicken retina, the role of the insulin-related growth factors in neurogenesis. We demonstrate the local presence of signaling elements together with a biological response to the factors. Neuroretina at days 6-8 of embryonic development (E6-E8) expressed proinsulin/insulin and insulin-like growth factor I (IGF-I) mRNAs as well as insulin receptor and IGF type I receptor mRNAs. In parallel with this in vivo gene expression, E5 cultured neuroretinas synthesized and released to the medium a metabolically radiolabeled immunoprecipitable insulin-related peptide. Furthermore, insulin-related immunoreactive material with a HPLC mobility close to that of proinsulin was found in the E6-E8 vitreous humor. Exogenous chicken IGF-I, human insulin, and human proinsulin added to E6 cultured neuroretinas showed relatively close potencies stimulating proliferation, as determined by [methyl-3H]thymidine incorporation, with a plateau reached at 10(-8) M. These factors also stimulated neuronal differentiation, indicated by the expression of the neuron-specific antigen G4. Thus, insulin-related growth factors, interestingly including proinsulin, are present in the developing chicken retina and appear to play an autocrine/paracrine stimulatory role in the progression of neurogenesis.
Growth factors have an important role in the regulation of cell growth, division and differentiation. They are also involved in the regulation of embryonic growth and differentiation. Insulin and insulin-like growth factor I (IGF I) play an important part in these events in the later stages of embryogenesis, when organogenesis is completed. In this study, we are presenting evidence that insulin and IGF I are also secreted by embryonic tissues during the prepancreatic stage of mouse development. We found measurable amounts of insulin and IGF I in 8- to 12-day-old mouse embryos. We also showed that embryonic cells derived from 8-, 9- and 10-day-old mouse embryos secrete insulin, IGF I and/or related molecules. Furthermore, the same growth factors, when added to the culture of 9-day-old mouse embryonic cells, stimulate their proliferation. These results lead to the conclusion that insulin can stimulate the growth of embryonic cells during the period when pancreas is not yet formed, which is indirect evidence for a paracrine (or autocrine) type of action.
The impact of nicotinamide on regulation of cell replication and hormone production by fetal porcine islet-like cell clusters (ICCs) containing a low fraction of beta-cells was investigated. For this purpose, ICCs were produced in tissue culture for 4 days in the presence or absence of nicotinamide (10 mM). ICCs formed in the presence of nicotinamide showed increased rates of (pro)insulin biosynthesis, contained more insulin, and displayed elevated contents of polyamines when compared with untreated ICCs. Immunocytochemical analyses of autoradiographed ICCs disclosed that nicotinamide expanded the relative size of the beta-cell population, and that the vitamin increased DNA synthesis of non-beta-cells only. The possibility that the effects of nicotinamide were conveyed by the increased polyamine content was explored by giving nicotinamide together with inhibitors of rate-limiting enzymes of polyamine biosynthesis, a maneuver that precluded the increases in polyamines of whole ICCs. Although this treatment prevented the increases in beta-cell population size, insulin production, and insulin content evoked by nicotinamide, the elevated cell replication nonetheless persisted. We conclude that the stimulatory effects of nicotinamide on insulin production and content by fetal porcine ICCs result from neoformation of beta-cells through differentiation. Since these effects were prevented by blocking the increased polyamine content, it is suggested that a stimulated synthesis of polyamines may be an important event in mediating the differentiating action of nicotinamide.
Isolation of endocrine cell precursors from the human fetal pancreas will be important to the study of islet cytodifferentiation and eventually for islet transplantation in insulin-dependent diabetes. These precursor cells, from which all four islet endocrine cell types arise, are present within fetal pancreatic ductal epithelium. After enzymatic digestion and culture of the fetal pancreas, we obtained cell clusters resembling islets, but with a high content of undifferentiated cells. Histochemical staining revealed very high acid beta-galactosidase activity in over 70% of cells within the clusters. After transplantation into athymic nude mice, the islet-like cell clusters gave rise to tissue rich in differentiated endocrine cells, but low in beta-galactosidase activity. The histochemical finding of high acid beta-galactosidase activity in endocrine precursor cells was confirmed by direct measurement of lysosomal enzyme activities. In addition, we found that the expression of acid beta-galactosidase was d...
We have recently discovered that, during prenatal development of rats and mice, the enzyme tyrosine hydroxylase (TH, E.C.1.14.3.x) which catalyzes the first step in the biosynthesis of catecholamines, appears in cells in the primordial gut (Gershon et al., 1978; Teitelman et al., 1978, 1979). The expression of the catecholaminergic phenotype is transient, for within 2 to 3 days the enzyme can no longer be detected in enteric cells (Cochard et al., 1978; Gershon et al., 1978; Teitelman et al., 1978, 1979). Since in adults catecholaminergic cells are not present in gut, the observation raises the question: Do the TH containing cells transform into gut cells, oossibly neurons, which contain other neurotransmitters and/or neuropeptides However, since the peptides are first visualized in cell of the gut after the disappearance of TH (Larsson, 1977), it is technically difficult, in vivo, to establish such a transition.
A major obstacle to successful islet transplantation for both type 1 and 2 diabetes is an inadequate supply of insulin-producing tissue. This need for transplantable human islets has stimulated efforts to expand existing pancreatic islets and/or grow new ones. To test the hypothesis that human adult duct tissue could be expanded and differentiated in vitro to form islet cells, digested pancreatic tissue that is normally discarded from eight human islet isolations was cultured under conditions that allowed expansion of the ductal cells as a monolayer whereupon the cells were overlaid with a thin layer of Matrigel. With this manipulation, the monolayer of epithelial cells formed three-dimensional structures of ductal cysts from which 50-to 150- micrometer diameter islet-like clusters of pancreatic endocrine cells budded. Over 3-4 weeks culture the insulin content per flask increased 10- to 15-fold as the DNA content increased up to 7-fold. The cultivated human islet buds were shown by immunofluorescence to consist of cytokeratin 19-positive duct cells and hormone-positive islet cells. Double staining of insulin and non-beta cell hormones in occasional cells indicated immature cells still in the process of differentiation. Insulin secretion studies were done over 24 h in culture. Compared with their basal secretion at 5 mM glucose, cysts/cultivated human islet buds exposed to stimulatory 20 mM glucose had a 2.3-fold increase in secreted insulin. Thus, duct tissue from human pancreas can be expanded in culture and then be directed to differentiate into glucose responsive islet tissue in vitro. This approach may provide a potential new source of pancreatic islet cells for transplantation.
BACKGROUND
Long-term microvascular and neurologic complications cause major morbidity and mortality in patients with insulin-dependent diabetes mellitus (IDDM). We examined whether intensive treatment with the goal of maintaining blood glucose concentrations close to the normal range could decrease the frequency and severity of these complications.
METHODS
A total of 1441 patients with IDDM -- 726 with no retinopathy at base line (the primary-prevention cohort) and 715 with mild retinopathy (the secondary-intervention cohort) were randomly assigned to intensive therapy administered either with an external insulin pump or by three or more daily insulin injections and guided by frequent blood glucose monitoring or to conventional therapy with one or two daily insulin injections. The patients were followed for a mean of 6.5 years, and the appearance and progression of retinopathy and other complications were assessed regularly.
RESULTS
In the primary-prevention cohort, intensive therapy reduced the adjusted mean risk for the development of retinopathy by 76 percent (95 percent confidence interval, 62 to 85 percent), as compared with conventional therapy. In the secondary-intervention cohort, intensive therapy slowed the progression of retinopathy by 54 percent (95 percent confidence interval, 39 to 66 percent) and reduced the development of proliferative or severe nonproliferative retinopathy by 47 percent (95 percent confidence interval, 14 to 67 percent). In the two cohorts combined, intensive therapy reduced the occurrence of microalbuminuria (urinary albumin excretion of ≥ 40 mg per 24 hours) by 39 percent (95 percent confidence interval, 21 to 52 percent), that of albuminuria (urinary albumin excretion of ≥ 300 mg per 24 hours) by 54 percent (95 percent confidence interval, 19 to 74 percent), and that of clinical neuropathy by 60 percent (95 percent confidence interval, 38 to 74 percent). The chief adverse event associated with intensive therapy was a two-to-threefold increase in severe hypoglycemia.
CONCLUSIONS
Intensive therapy effectively delays the onset and slows the progression of diabetic retinopathy, nephropathy, and neuropathy in patients with IDDM.
The effects of GH, PRL, and placental lactogen (PL) on the proliferation of pancreatic β-cells in vitro were studied as well as the possible effect of insulin-like growth factor-I (IGF-I) in mediating this effect. Proliferating β-cells were identified by staining with a monoclonal antibody to bromode-oxyuridine (BrdU) after cells were incubated for 1 h in the presence of 10 μM BrdU. By double staining with insulin anti-bodies it was found that 6.3% of the β-cells had incorporated BrdU when cultured for 7 days in the presence of 1 μg/ml human GH (hGH) compared to 0.6% when cultured in the absence of hGH. Similar results were obtained using rat GH. The half-maximal effect of hGH on β-cell proliferation was observed at 10 ng/ml, and the maximal effect at 100 ng/ml. Islet cells cultured in the presence of PRL or PL caused a dose-dependent increase in β-cell proliferation similar to that caused by hGH. GH, PRL, and PL had no effect on the proliferation of glucagon- or somatostatin-producing cells. The addition of 100 ng/ml IGF-I to either control or GH-stimulated islet cells did not affect the labeling index. When GH-stimulated proliferation of β-cells was measured in the presence of neutralizing concentrations of a rabbit IGF-I antiserum, the percentage of β-cells incorporating BrdU was unaffected. Using Northern blot analysis, no IGF-I transcripts could be detected in RNA from GH-stimulated islets, whereas IGF-I transcripts were readily detected in RNA isolated from rat liver tissue. These data suggest that the stimulatory effect of GH, PRL, and PL on β-cell proliferation is not mediated by IGF-I, but, rather, is a direct mitogenic effect on the β-cell.
It is not clear which growth factors are crucial for the survival, proliferation, and differentiation of pancreatic b-cells. We used the relatively differentiated rat insulinoma cell line INS-1 to elucidate this issue. Responsiveness of the DNA synthesis of serum-starved cells was studied to a wide variety of growth factors. The most potent stimulators were PRL, GH, and betacellulin, a member of the epi- dermal growth factor (EGF) family that has not previously been shown to be mitogenic for b-cells. In addition to these, only vascular endothelial growth factor, insulin-like growth factor-1 and -2, had significant mitogenic activity, whereas hepatocyte growth factor, nerve growth factor-b, platelet-derived growth factors, basic fibro- blast growth factor, EGF, transforming growth factor-a (TGF-a), neu differentiation factor, and TGF-b were inactive. None of these factors affected the insulin content of INS-1 cells. In contrast, certain dif- ferentiation factors, including nicotinamide, sodium butyrate, activin A, and 1,25-dihydroxyvitamin D3 inhibited the DNA synthesis and increased the insulin content. Also all-trans-retinoic acid had an in- hibitory effect on cell DNA synthesis but no effect on insulin content. From these findings betacellulin emerges as a novel growth factor for the b-cell. Half-maximal stimulation of INS-1 DNA synthesis was obtained with 25 pM betacellulin. Interestingly, betacellulin had no effect on RINm5F cells, whereas both EGF and TGF-a were slightly mitogenic. These effects may possibly be explained by differential expression of the erbB receptor tyrosine kinases. In RINm5F cells a spectrum of erbB gene expression was detected (EGF receptor/erbB-1, erbB-2/neu, and erbB-3), whereas INS-1 cells showed only expression of EGF receptor. Expression of the erbB-4 gene was undetectable in these cell lines. In summary, our results suggest that the INS-1 cell line is a suitable model for the study of b-cell growth and differen- tiation because the responses to previously identified b-cell mitogens were essentially similar to those reported in primary cells. In addi- tion, we have identified betacellulin as a possible modulator of b-cell growth. (Endocrinology 139: 1494 -1499, 1998)
Nutrients and certain growth factors stimulate pancreatic β-cell mitogenesis, however, the appropriate mitogenic signal transduction
pathways have not been defined. In the glucose-sensitive pancreatic β-cell line, INS-1, it was found that glucose (6–18 mm) independently increased INS-1 cell proliferation (>20-fold at 15 mm glucose). Insulin-like growth factor I (IGF-I)-induced INS-1 cell proliferation was glucose-dependent only in the physiologically
relevant concentration range (6–18 mm glucose). The combination of IGF-I and glucose was synergistic, increasing INS-1 cell proliferation >50-fold at 15 mm glucose + 10 nm IGF-I. Glucose metabolism and phosphatidylinositol 3′-kinase (PI 3′-kinase) activation were necessary for both glucose and
IGF-I-stimulated INS-1 cell proliferation. IGF-I and 15 mm glucose increased tyrosine phosphorylation mediated recruitment of Grb2/mSOS and PI 3′-kinase to IRS-2 and pp60. Glucose
and IGF-I also induced Shc association with Grb2/mSOS. Glucose (3–18 mm) and IGF-I, independently of glucose, activated mitogen-activated protein kinase but this did not correlate with IGF-I-induced
β-cell proliferation. In contrast, p70S6K was activated with increasing glucose concentration (between 6 and 18 mm), and potentiated by IGF-I in the same glucose concentration range which correlated with INS-1 cell proliferation rate. Thus,
glucose and IGF-I-induced β-cell proliferation were mediated via a signaling mechanism that was facilitated by mitogen-activated
protein kinase but dependent on IRS-mediated induction of PI 3′-kinase activity and downstream activation of p70S6K. The glucose dependence of IGF-I mediated INS-1 cell proliferation emphasizes β-cell signaling mechanisms are rather unique
in being tightly linked to glycolytic metabolic flux.
• The properties of the calcium sensor for glucose-induced insulin secretion have been studied using cell-permeant Ca2+ buffers with distinct kinetics and affinities. In addition, submembrane cytosolic Ca2+ distribution has been modelled after trains of glucose-induced action potential-like depolarizations. • Slow Ca2+ buffers (around 1 mmol l−1 intracellular concentration) with different affinities (EGTA and Calcium Orange-5N) did not significantly affect glucose-induced insulin release. Modelling showed no effect on cytosolic Ca2+ concentrations at the outermost shell (0.05 m), their effects being observed in the innermost shells dependent on Ca2+ affinity. • In contrast, fast Ca2+ buffers (around 1 mmol l−1 intracellular concentration) with different affinities (BAPTA and Calcium Green-5N) caused a 50% inhibition of early insulin response and completely blocked the late phase of glucose-induced insulin response, their simulations showing a decrease of [Ca2+]i at both the inner and outermost shells. • These data are consistent with the existence in pancreatic -cells of a higher affinity Ca2+ sensor than that proposed for neurons. Moreover, these data are consistent with the proposed existence of two distinct pools of granules: (i) ‘primed’ vesicles, colocalized with Ca2+ channels and responsible of the first phase of insulin release; and (ii) ‘reserved pool’ vesicles, not colocalized and responsible for the second phase.
• The intracellular calcium concentration ([Ca2+]i) near the plasma membrane was measured in mouse pancreatic islet cells using confocal spot detection methods. • Whereas small cytosolic Ca2+ gradients were observed with 3 mM glucose, a steeper sustained gradient restricted to domains beneath the plasma membrane (space constant, 0.67 m) appeared with 16.7 mM glucose. • When the membrane potential was clamped with increasing K+ concentrations (5, 20 and 40 mM), no [Ca2+]i gradients were observed in any case. • Increasing glucose concentration (0, 5 and 16.7 mM) in the presence of 100 M diazoxide, a K+ channel opener, plus 40 mM K+ induced steeper [Ca2+]i gradients, confirming the role of membrane potential-independent effects of glucose. • Prevention of Ca2+ store refilling with 30 M cyclopiazonic acid (CPA) or blockade of uniporter-mediated Ca2+ influx into the mitochondria with 1 M carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or 1 M Ru-360 significantly reduced the steepness of the 16.7 mM glucose-induced [Ca2+]i gradients. • Measured values of [Ca2+]i reached 6.74 ± 0.67 M at a distance of 0.5 m from the plasma membrane and decayed to 0.27 ± 0.03 M at a distance of 2 m. Mathematically processed values at 0.25 and 0 m gave a higher [Ca2+]i, reaching 8.18 ± 0.86 and 10.05 ± 0.98 M, respectively. • The results presented indicate that glucose metabolism generates [Ca2+]i microgradients, which reach values of around 10 M, and whose regulation requires the involvement of both mitochondrial Ca2+ uptake and endoplasmic reticulum Ca2+ stores.
In this paper, some of our recent experiments concerning a novel second messenger in β-cells, cyclic ADP-ribose (cADPR), and Reg gene which is involved in β-cell regeneration will be discussed.
It is not clear which growth factors are crucial for the survival, proliferation, and differentiation of pancreatic beta-cells. We used the relatively differentiated rat insulinoma cell line INS-1 to elucidate this issue. Responsiveness of the DNA synthesis of serum-starved cells was studied to a wide variety of growth factors. The most potent stimulators were PRL, GH, and betacellulin, a member of the epidermal growth factor (EGF) family that has not previously been shown to be mitogenic for beta-cells. In addition to these, only vascular endothelial growth factor, insulin-like growth factor-1 and -2, had significant mitogenic activity, whereas hepatocyte growth factor, nerve growth factor-beta, platelet-derived growth factors, basic fibroblast growth factor, EGF, transforming growth factor-alpha (TGF-alpha), neu differentiation factor, and TGF-beta were inactive. None of these factors affected the insulin content of INS-1 cells. In contrast, certain differentiation factors, including nicotinamide, sodium butyrate, activin A, and 1,25-dihydroxyvitamin D3 inhibited the DNA synthesis and increased the insulin content. Also all-trans-retinoic acid had an inhibitory effect on cell DNA synthesis but no effect on insulin content. From these findings betacellulin emerges as a novel growth factor for the beta-cell. Half-maximal stimulation of INS-1 DNA synthesis was obtained with 25 pM betacellulin. Interestingly, betacellulin had no effect on RINm5F cells, whereas both EGF and TGF-alpha were slightly mitogenic. These effects may possibly be explained by differential expression of the erbB receptor tyrosine kinases. In RINm5F cells a spectrum of erbB gene expression was detected (EGF receptor/erbB-1, erbB-2/neu, and erbB-3), whereas INS-1 cells showed only expression of EGF receptor. Expression of the erbB-4 gene was undetectable in these cell lines. In summary, our results suggest that the INS-1 cell line is a suitable model for the study of beta-cell growth and differentiation because the responses to previously identified beta-cell mitogens were essentially similar to those reported in primary cells. In addition, we have identified betacellulin as a possible modulator of beta-cell growth.
Thesis (M.D.)--Yale University, 1998.
The embryonic rat pancreas, removed on the 14th day of gestation and cultivated in vitro, accumulates differentiated levels of exocrine enzymes and insulin. In the period corresponding to days 16–22 in vivo, 99% of the final insulin content accumulates. During this period we have studied the development of competence for insulin secretion, the regulation of this secretion by glucose and other secretatogues, and the rate of synthesis following a secretory challenge. Our results demonstrate that the capacity for insulin secretion develops in parallel with the accumulation of insulin in secretory granules since β granules appear at day 16. On day 16, after 48 hr of culture, both glucose and caffeine are required for detectable insulin secretion. At later stages, insulin release can be effectuated by glucose alone. In the fetal pancreas at day 20 of development, glucose is ten times more efficient than caffeine and fourfold more efficient than caffeine combined with either glucagon, cholera toxin or dibutyryl cyclic AMP. Glucagon, cholera toxin or cyclic AMP in the presence of caffeine increases equally (about tenfold) both the “basal” and the glucose-induced level of secretion. This suggests that glucose and caffeine act independently but synergistically. The integrity of the cells is maintained under the stimulation conditions, and there is a selective increase in insulin synthesis measured during 18 hr following stimulation of insulin release.
Nicotinamide, a poly (ADP-ribose) synthetase inhibitor, was administered intravenously to massive pancreatectomized dogs (NA group), whereas saline was administered intravenously to massive pancreatectomized dogs (saline group). Stathmokinetic studies were done to clarify the regenerating activities of B-cells. Nicotinamide significantly increased mitotic indices of B-cells after pancreatectomy (p less than 0.01). On the second day after pancreatectomy, mitotic index of B-cells in the NA group (2.00 +/- 0.20%) was higher than that in the saline group (0.25 +/- 0.10%). Though insulin content of pancreatic tissue decreased 2 d after pancreatectomy in the both groups, recovery of insulin content was significantly more immediate and more sufficient in the NA group. Intravenous glucose tolerance tests showed a significantly greater increase of insulin release on the 31st and 62nd day after pancreatectomy, and better glucose tolerance on the 62nd day in the NA group compared with the saline group. These results are considered to be attributed to the regenerating and protecting effects of nicotinamide on B-cells after pancreatectomy, and suggest a clinical value of nicotinamide also in man, especially for Sandmeyer's diabetes.
When embryonic stem cells are cultured directly in semisolid media (methyl cellulose), they proliferate and differentiate to generate colonies known as embryoid bodies (EBs). These EBs consist of differentiated cells from a number of lineages including those of the hematopoietic system. Following 10 days of culture in the presence of 10% fetal calf serum, more than 40% of all EBs from three different ES cell lines, CCEG2, D3 and SQ1.2S8 contained visible erythropoietic cells (i.e. red with hemoglobin). Beta H1 (z globin) mRNA is detectable in EBs within 5 days of differentiation, whilst beta(maj)-globin RNA appears by day 6. In the presence of erythropoietin (Epo), the frequency of EBs with erythropoietic activity increases to greater than 60%; Epo also prolongs this erythropoietic activity. Interleukin-3 (IL-3) does not significantly increase the frequency of EBs that contain erythroid cells, but increases slightly the number of erythropoietic cells associated with them. In the presence of IL-3, in addition to cells of the erythroid lineage, macrophages, mast cells and in some instances neutrophils are found within differentiating EBs. The development of macrophages is significantly enhanced by the addition of IL-3 alone or in combination with IL-1 and M-CSF or GM-CSF. When well-differentiated EBs are allowed to attach onto tissue-culture plates and grown in the presence of IL-3, a long-term output of cells from the mast cell lineage is observed.(ABSTRACT TRUNCATED AT 250 WORDS)
The two peptides pancreastatin and diazepam binding inhibitor (DBI) were recently demonstrated in pancreatic islets and were shown to inhibit insulin secretion in short term experiments. In the present study we investigated long term effects of pancreastatin and DBI on the DNA synthesis, polyamine content, and insulin secretion of pancreatic beta-cells in tissue culture. For this purpose fetal rat pancreatic islets enriched in beta-cells were isolated and cultured for 3 days at different concentrations of rat pancreastatin and porcine DBI. It was found that pancreastatin dose-dependently decreased beta-cell DNA synthesis, reaching maximal inhibition at 100 nM. In parallel with this, pancreastatin also decreased insulin secretion and the islet contents of insulin and the polyamines spermidine and spermine. These effects were abolished by a high glucose concentration or addition of GH. Also, DBI evoked a dose-dependent inhibition of beta-cell DNA synthesis but affected neither the islet contents of insulin or polyamines nor insulin secretion. Like pancreastatin, DBI was ineffective in preventing the increased beta-cell DNA synthesis, insulin content, or secretion in response to high glucose or GH. It is concluded that pancreastatin and DBI inhibit beta-cell DNA synthesis and function in vitro. In the case of pancreastatin these inhibitory effects may be mediated by a decrease in islet polyamine content. It is suggested that pancreastatin and DBI may influence beta-cell replication and function in vivo in an autocrine or paracrine fashion.
Effects of a number of possible trophic factors on growth of guinea pig pancreatic duct epithelial monolayers were investigated. Isolated fragments of main and interlobular ducts were prepared and explanted onto both tissue culture plastic and thick gels of type I collagen. Monolayers growing out from explants were first cultured in a basal medium for 3 or 4 days. Next, the medium was supplemented individually with bombesin, carbachol, caerulein, epidermal growth factor (EGF), secretin, 12-O-tetradecanoylphorbol 13-acetate (TPA), or vasoactive intestinal peptide (VIP). Cells were cultured in the absence or presence of these possible trophic factors, and monolayer areas were determined morphometrically at 0, 2, and 4 days. Rate of growth was determined from increase in area over each 2-day period. Monolayers grown in basal medium alone on plastic increased to 479% of initial area over the 4-day test period; those grown on collagen increased to 523%. Explants cultured in presence of bombesin, carbachol, caerulein, secretin, TPA, and VIP on either substrate grew at rates not significantly different from those cultured in basal medium. By contrast, duct monolayers grown on plastic or collagen in presence of 10 nM EGF expanded in area to 722 and 1,070%, respectively, of their initial areas. The EC50 for this trophic effect was approximately 1 nM. These results show that EGF exerts a potent trophic effect on guinea pig pancreatic duct cells in vitro but also indicate that cell division in the pancreatic main and interlobular ducts is not regulated by caerulein and related peptide hormones that have been reported to have growth-promoting effects on exocrine pancreas in vivo.
Rat insulinoma (RIN) cells, in comparison with adult islet cells, are relatively undifferentiated. They secrete low amounts of islet hormones, are unresponsive to glucose, and display pluripotency. A minority of RIN cells react with monoclonal antibodies A2B5 and 3G5 which recognize complex gangliosides on normal islet cells. In order to determine whether the expression of A2B5- or 3G5-reactive gangliosides is modulated during differentiation RIN cells were cultured with various concentrations of sodium butyrate (NaB), a known inducer of cellular differentiation. Expression of A2B5- and 3G5-reactive gangliosides was determined by indirect immunofluorescence and flow cytofluorimetry. NaB exposure resulted in a dose-dependent decrease in cell proliferation over 5 days of 1.5-, 2.9-, and 17.5-fold at 0.5, 1.0 and 3.0 mM, respectively, and a distinct change in cellular morphology. Cells exposed to NaB displayed prominent neurite-like projections. At 3 mM NaB, insulin secretion increased 7.9-fold and the percentage of cells expressing A2B5- and 3G5-reactive gangliosides increased by up to 4.4- and 5.5-fold, respectively. The expression of A2B5- or 3G5-reactive gangliosides per cell also increased, by 2.4- and 1.3-fold, respectively, at 3 mM NaB. These findings demonstrate that the expression of cell surface A2B5- and 3G5-reactive gangliosides is not static but increases with cell differentiation. NaB-treated RIN cells may serve as a model to study the role of gangliosides in the function and lineage relationships of islet cells.
The extent to which adult pancreatic beta-cells can respond in vivo to a sustained glucose stimulus by increasing their mass through either hyperplasia or hypertrophy has remained unanswered. Therefore, we studied the in vivo effect of short-term (96-h) hyperglycemia on the growth of beta-cells by infusing adult rats with 35 or 50% glucose or 0.45% saline. After 96 h of glucose infusion, the beta-cell mass, quantified by point-counting morphometrics of immunoperoxidase-stained paraffin sections, showed a 50% increase (9.57 +/- 0.87 mg, n = 5, 50% glucose infused; 9.50 +/- 1.23, n = 7, 35% glucose infused; 6.15 +/- 0.55, n = 6, 0.45% saline infused). This growth was selective for beta-cells; the non-beta-cell mass was unchanged. The mitotic index, measured by accumulated mitotic frequency after a 4-h colchicine treatment, increased fivefold in glucose-infused animals compared to saline-infused animals. This enhanced replication of beta-cells provides evidence for increase in cell number or hyperplasia. In addition, hypertrophy of the beta-cell was also quantified. Mean cell volume, determined from the mean cell cross-sectional area measured planimetrically from low-magnification electron micrographs, increased to 150% of control values after 96 h of 50% glucose infusion. Seven days after the 96-h infusion, in reversal experiments, the beta-cell mass had not returned to saline-infused levels. In addition, the non-beta-cell mass of glucose-infused animals had increased. The mitotic index of the beta-cell of glucose-infused rats was, however, significantly lower than that of the saline controls, but the mean cell volume of the beta-cells remained elevated.(ABSTRACT TRUNCATED AT 250 WORDS)
A specialized exocrine pancreatic cell can transform into a different phenotypic cell—the hepatocyte. Although in an adult animal the pancreatic and liver cells are functionally and morphologically different, both organs arise from gut endoderm, thus, sharing a common ancestry. Under proper conditions, repressed liver-specific genes in pancreatic cells are probably derepressed. The way in which such dormant genes are activated is not clear. Although some carcinogens, such as N-methyl-N-nitroso- guanidine, were shown to induce transdifferentiation, the possibility that 4-HAQO-induced genetic alterations may lead to hepatocyte conversion, when subjected to the added stress of copper depletion and repletion, can be excluded in view of the recent observation that a simple copper depletion-repletion regimen can lead to the development of pancreatic hepatocytes. The stability of a differentiated cell could be due to various molecular mechanisms such as DNA methylation, chromatin structure, DNA protein interactions, and DNA rearrangements. Modification of any of these could initiate transdifferentiation. By utilizing a variety of molecular techniques, it is possible to analyze the control mechanisms of tissue-specific gene regulation in pancreatic hepatocytes. Studies with transdifferentiated hepatocytes may be expected to yield considerable new information about the mechanism(s) of cell differentiation and the attendant transcriptional controls.
The state of differentiation of various neoplastic cell lines is inversely correlated with the rate of cellular growth. To
delineate the changes in hormone gene expression associated with an induced decrease in the growth rate of rat insulinoma
cells, we studied the effects of sodium butyrate on the expression of the genes encoding insulin, glucagon, and angiotensinogen.
Sodium butyrate inhibited cellular proliferation and decreased levels of c-myc mRNA. Concomitantly, steady-state levels of
mRNAs encoding insulin and glucagon increased by 10- and 8.5-fold, respectively, as a result of a specific increase in the
transcription of both genes. Sodium butyrate also inhibited angiotensinogen gene expression, which was ectopic in the insulinoma
cells. These observations suggest that sodium butyrate induces a pattern of events leading to the differentiation of the rat
insulinoma cells.
Evidence is provided for the impermeability of the human placenta for bovine insulin at term. Women in labor received glucose infusions with and without insulin added; the dosage was 1 g glucose and 0.33 units insulin per min. The insulin in cord blood was nearly always elevated after maternal infusions compared with controls, but no difference in fetal insulin concentrations were observed whether insulin was added to mother's infusion or not. Even an 18-fold increase of the maternal insulin level did not affect the insulin level in cord blood. The additional glucose transferred to the fetus by maternal infusion stimulates the fetal insulin secretion with a delay of at least 22 min.
In embryonic mice, the catecholamine biosynthetic enzyme tyrosine hydroxylase [L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] can be visualized immunocytochemically in a population of cells in epithelial cords of the developing pancreas. These embryonic catecholamine cells, first seen by day 11, are large and vacuolated and have a folded nuclear membrane. One day later, at day 12, glucagon is first detected immunocytochemically in pancreatic cells similar in location and morphology to the embryonic catecholamine cells. By use of a method for detecting both antigens in the same cell, both the hydroxylase and glucagon can be visualized between day 12 and day 14 in 10-40% of stained cells. From day 14, the number of cells stained for hydroxylase decreases; they cannot be detected after day 18. In contrast, the cells containing glucagon increase during development and persist throughout life. Endocrine cells of the embryonic pancreas also contain dopa decarboxylase but not dopamine-beta-hydroxylase or phenylethanolamine-N-methyl transferase. In adult mice, small cells containing tyrosine hydroxylase but differing in location and morphology from the embryonic catecholaminergic cells are seen in pancreatic islets. The adult catecholaminergic cells never store glucagon. We suggest that adult glucagon (A)-containing cells arise from transformation in situ of cells that transiently express a catecholaminergic (probably dopaminergic) phenotype. These results suggest that one class of peptidergic cells may arise from transformation of an aminergic precursor.
A population of cells containing the enzymes tyrosine hydroxylase (TH) and dopa-decarboxylase (L-AADC) but not dopamine-B-hydroxylase (DBH) nor phenylethanolamine-N-methyltransferase (PNMT) can be detected with immunocytochemical techniques in the pancreas of mouse embryos at the 11th day of development (E 11). The presence of TH in embryonal pancreas is transient: TH is not observed after E 15. By use of a method for simultaneously detecting two antigens in the same section both TH and glucagon were visualized in the same cell on E 12. Double labelled cells comprised 10% of all stained cells. At E 14.5, some of the cells stained for TH also contained insulin. However, at the time somatostatin appeared no embryonal cells containing TH remained. We conclude that two cell types of the APUD series, i.e., the glucagon and insulin cells of pancreas, arise from transformation, in situ, of cells that transiently express a dopaminergic phenotype. These results suggest that peptide-containing cells in skin, brain and gut are linked by a common embryonic origin. They also raise the prospect that other peptidergic cells of the APUD series may have aminergic precursors.
Due to its small size and complex cellular composition, the islet of Langerhans appeared as a rather intractable tissue for studies in cell-to-cell interactions. The available data summarized here suggest, on the contrary, that the complex yet consistent pattern of cell-to-cell relationships in the islet, together with the knowledge of the role of each islet hormone upon homologous and heterologous islet cells, may precisely contribute to the elaboration of a satisfactory model of interactions accounting for the adaptative secretory response needed to ensure glucose homeostasis. We emphasize the critical role of technical improvements in the progress of cell research. If one compares the islet of Langerhans at the beginning of this century, a microcolony of pale cells, with the two types of islets of Langerhans known today and with present knowledge of the intracellular and intercellular organization of their cells, one may feel happy about the past and even the present. However, our techniques, instrumentation, and skills again require further adaptation and refinement if we wish to understand more of the intricate mechanisms that govern the integrated function of this fascinating microorgan.
1. The effects of amino acids on cytosolic free calcium concentration ([Ca2+]i) were measured, using fura-2 fluorescence imaging, in mouse pancreatic islets of Langerhans. 2. Slow [Ca2+]i oscillations appeared when isolated islets were incubated with a solution containing a mixture of amino acids and glucose at concentrations found in the plasma of fed animals. 3. In the presence of 11 mM glucose, alanine (5 mM) and arginine (10 mM) induced a transient rise in [Ca2+]i followed by an oscillatory pattern, while leucine (3 mM) and isoleucine (10 mM) triggered the appearance of slow [Ca2+]i oscillations. 4. Also in the presence of glucose (11 mM), tolbutamide (10 microM) increased the duration of the glucose-induced [Ca2+]i oscillations. While tolbutamide (10 microM) did not modify the leucine-induced slow oscillatory pattern, addition of diazoxide (10 microM) resulted in the gradual appearance of [Ca2+]i oscillations which resembled the glucose-induced fast oscillations. 5. Like stimulatory glucose concentrations (11 mM), glyceraldehyde (10 mM) induced fast oscillations of [Ca2+]i. 6. Fluoroacetate (2 mM) transformed leucine-induced slow [Ca2]i oscillations into fast [Ca2+]i oscillations. Iodoacetate (1 mM) completely inhibited any oscillatory pattern. 7. It is suggested that mitochondrially generated signals, derived from amino acid oxidative metabolism, acting in conjunction with glucose-signalled messengers, are very effective at closing ATP-dependent K+ channels (KATP+). 8. We propose that metabolic regulation of KATP+ channels is one of the mechanisms underlying the modulation of the oscillatory [Ca2+]i response to nutrient secretagogues.
The Msx genes of vertebrates comprise a small family of chromosomally unlinked homeobox-containing genes related to the Drosophila gene muscle-segment homeobox (msh). Despite their ancient pedigree, the Msx genes are expressed in a range of vertebrate-specific tissues, including neural crest, cranial sensory placodes, bone and teeth. They are active in numerous systems, which have been used as models to study pattern formation and tissue interaction, and are, therefore, attracting a growing interest among developmental biologists. But beyond their presumed role as transcription factors, we do not know what their functions are in the cell or the embryo. Here, I review recent evidence that is beginning to address this problem and might eventually increase our understanding of how the vertebrate embryo has evolved.