Differentiation Of Pancreatic Progenitor Cells Research Articles

Gene expression profile of hiPSC-derived cells differentiated with growth factors, forskolin and conditioned medium from human adrenocortical cell line.

Adrenocortical carcinoma (ACC) affects approx. 2 in 1,000,000 individuals in the USA, and is more common in females than males. Adrenocortical carcinoma often presents with severe symptoms, such as abdominal pain, high blood pressure, acne, hair overgrowth, and voice deepening. Research on ACC constitutes a large body of published data. There is an increased need for easy access to ACC-derived biological material. Moreover, there are limited numbers of human cell lines available. For this reason, we attempted to differentiate human induced pluripotent stem cells (hiPSCs) into adrenocortical-like cells to establish a new functional cell line. We conducted a long-term differentiation process (35 and 70 days) in the presence of growth factors (GFs), forskolin and conditioned medium collected from the human adrenal carcinoma (HAC15) cell line. Then, we analyzed the gene expression profile of the differentiated cells. The obtained cells possess features characteristic of all 3 primary germ layers. Interestingly, the differentiated cells demonstrated an extremely high level of gene expression for those involved in endocrine processes, namely glycoprotein hormones, alpha polypeptide (CGA), insulin receptor substrate 4 (IRS4), and pancreatic progenitor cell differentiation and proliferation factor-like protein (PPDPFL). The results of the study indicate that we obtained progenitors derived from endoderm with some characteristics of pancreatic-like cells. The endodermal derivative differentiation is a very challenging and complicated process; thus, the results presented in this study deserve closer consideration.

PPDPF suppresses the development of hepatocellular carcinoma through TRIM21-mediated ubiquitination of RIPK1

Pancreatic progenitor cell differentiation and proliferation factor (PPDPF) has been reported to play a role in tumorigenesis. However, its function in hepatocellular carcinoma (HCC) remains poorly understood. In this study, we report that PPDPF is significantly downregulated in HCC and the decreased PPDPF expression indicates poor prognosis. In the dimethylnitrosamine (DEN)-induced HCC mouse model, hepatocyte-specific depletion of Ppdpf promotes hepatocarcinogenesis, and reintroduction of PPDPF into liver-specific Ppdpf knockout (LKO) mice inhibits the accelerated HCC development. Mechanistic study shows that PPDPF regulates nuclear factor κB (NF-κB) signaling through modulation of RIPK1 ubiquitination. PPDPF interacts with RIPK1 and facilitates K63-linked ubiquitination of RIPK1 via recruiting the E3 ligase TRIM21, which catalyzes K63-linked ubiquitination of RIPK1 at K140. In addition, liver-specific overexpression of PPDPF activates NF-κB signaling and attenuates apoptosis and compensatory proliferation in mice, which significantly suppresses HCC development. This work identifies PPDPF as a regulator of NF-κB signaling and provides a potential therapeutic candidate for HCC.

Metformin induces mitochondrial remodeling and differentiation of pancreatic progenitor cells into beta-cells by a potential mechanism including suppression of the T1R3, PLCβ2, cytoplasmic Ca+2, and AKT.

The main goal of this study was to investigate the molecular changes in pancreatic progenitor cells subject to high glucose, aspartame, and metformin in vitro. This scope of work glucose, aspartame, and metformin were exposed to pancreatic islet derived progenitor cells (PID-PCs) for 10days. GLUT1's role in beta-cell differentiation was examined by using GLUT1 inhibitor WZB117. Insulin+ cell ratio was measured by flow cytometry; the expression of beta-cell differentiation related genes was shown by RT-PCR; mitochondrial mass, mitochondrial ROS level, cytoplasmic Ca2+, glucose uptake, and metabolite analysis were made fluorometrically and spectrophotometrically; and proteins involved in related molecular pathways were determined by western blotting. Findings showed that glucose or aspartame exposed cells had similar metabolic and gene expression profile to control PID-PCs. Furthermore, relatively few insulin+ cells in aspartame treated cells were determined. Aspartame signal is transmitted through PLCβ2, CAMKK2 and LKB1 in PID-PCs. The most obvious finding of this study is that metformin significantly increased beta-cell differentiation. The mechanism involves suppression of the sweet taste signal's molecules T1R3, PLCβ2, cytoplasmic Ca+2, and AKT in addition to the direct effect of metformin on mitochondria and AMPK, and the energy metabolism of PID-PCs is remodelled in the direction of oxidative phosphorylation. These findings are very important in terms of determining that metformin stimulates the mitochondrial remodeling and the differentiation of PID-PCs to beta-cells and thus it may contribute to the compensation step, which is the first stage of diabetes development.

PPDPF promotes lung adenocarcinoma progression via inhibiting apoptosis and NK cell-mediated cytotoxicity through STAT3.

Lung cancer is the most common malignancy and the leading cause of cancer death worldwide, and lung adenocarcinoma (LUAD) is the most prevalent subtype. Considering the emergence of resistance to therapies, it is urgent to develop more effective therapies to improve the prognosis. Here we reported that pancreatic progenitor cell differentiation and proliferation factor (PPDPF) deficiency inhibited LUAD development both in vitro and in vivo. Mechanistically, PPDPF induces hyperactive STAT3 by interfering STAT3-PTPN1 interaction. Activated STAT3 promoted BMPR2 transcription, which further inhibited apoptosis. Moreover, PPDPF reduced NK cell infiltration and activation to develop an immunosuppressive microenvironment, which was also mediated by STAT3. Furthermore, we identified that the expression of PPDPF was positively correlated with the malignant features of LUAD, as well as BMPR2 and p-STAT3 level in clinical samples. Therefore, our study suggests that PPDPF positively regulates BMPR2 expression and facilitates immune escape via regulating STAT3 activity, providing a potential therapy target for LUAD.

Suppression of the Toll-like receptors 3 mediated pro-inflammatory gene expressions by progenitor cell differentiation and proliferation factor in chicken DF-1 cells.

Toll-like receptors (TLRs), as a part of innate immunity, plays an important role in detecting pathogenic molecular patterns (PAMPs) which are structural components or product of pathogens and initiate host defense systems or innate immunity. Precise negative feedback regulations of TLR signaling are important in maintaining homeostasis to prevent tissue damage by uncontrolled inflammation during innate immune responses. In this study, we identified and characterized the function of the pancreatic progenitor cell differentiation and proliferation factor (PPDPF) as a negative regulator for TLR signal-mediated inflammation in chicken. Bioinformatics analysis showed that the structure of chicken PPDPF evolutionarily conserved amino acid sequences with domains, i.e., SH3 binding sites and CDC-like kinase 2 (CLK2) binding sites, suggesting that relevant signaling pathways might contribute to suppression of inflammation. Our results showed that stimulation with polyinosinic:polycytidylic acids (Poly [I:C]), a synthetic agonist for TLR3 signaling, increased the mRNA expression of PPDPF in chicken fibroblasts DF-1 but not in chicken macrophage-like cells HD11. In addition, the expression of pro-inflammatory genes stimulated by Poly(I:C) were reduced in DF-1 cells which overexpress PPDPF. Future studies warrant to reveal the molecular mechanisms responsible for the anti-inflammatory capacity of PPDPF in chicken as well as a potential target for controlling viral resistance.

Suppression of the TLR3 mediated pro-inflammatory gene expressions by PPDPF in chicken DF-1 cells

Toll-like receptors (TLRs), as a part of innate immunity, plays an important role in detecting pathogenic molecular patterns (PAMPs) which are structural components or product of pathogens and initiate host defense systems or innate immunity. Precise negative feedback regulations of TLR signaling are important in maintaining homeostasis to prevent tissue damage by uncontrolled inflammation during innate immune responses. In this study, we identified and characterized the function of the pancreatic progenitor cell differentiation and proliferation factor (PPDPF) as a negative regulator for TLR signal-mediated inflammation in chicken. Bioinformatics analysis showed that the structure of chicken PPDPF evolutionarily conserved amino acid sequences with domains, i.e., SH3 binding sites and CDC-like kinase 2 (CLK2) binding sites, suggesting that relevant signaling pathways might contribute to suppression of inflammation. Our results showed that stimulation with polyinosinic:polycytidylic acids (Poly (I:C)), a synthetic agonist for TLR3 signaling, increased the mRNA expression of PPDPF in chicken fibroblasts DF-1 but not in chicken macrophage-like cells HD11. In addition, the expression of pro-inflammatory genes stimulated by Poly(I:C) were reduced in DF-1 cells which overexpress PPDPF. Future studies warrant to reveal the molecular mechanisms responsible for the anti-inflammatory capacity of PPDPF in chicken as well as a potential target for controlling viral resistance.

1242-P: Defining the Nkx6.1 Interactome in Beta Cells Reveals a Novel Interaction with Pdx1

The beta cell transcription factor Nkx6.1 plays a critical role in pancreatic progenitor cell differentiation, mature beta cell proliferation and insulin secretion. The pathways regulated by Nkx6.1 that results in these phenotypes are still being defined. Given that transcription factors generally function in complexes, we sought to determine proteins that interact with Nkx6.1 and may be harnessed to expand functional beta cell mass. INS-1 832/13 beta cells were transduced with AdCMV-Nkx6.1-BioID, AdCMV-GFP-BioID or left untreated. Biotinylated proteins were isolated and the Nkx6.1 interactome was defined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We defined 176 biotinylated proteins in cells transduced with AdCMV-Nkx6.1-BioID that were not observed with AdCMV-GFP-BioID or naturally biotinylated proteins from untreated lysates (P≤0.05, n=9). These 176 proteins are enriched in terms of nucleotide binding (P=3.6E-5), ribosomal proteins (P=9.1E-4), and transcription factors (P=4.3E-4). Among the enriched transcription factors, we validated that Pdx1, Mef2d and Nr2c2 were biotinylated in response to transduction with AdCMV-Nkx6.1 using biotin-targeted immunoprecipitation and western blot. To examine if these transcription factors are likely to interact with Nkx6.1 on the genome, we analyzed the frequency of proximal binding sites for Nkx6.1 and our candidate genes. Nr2c2 and Pdx1 binding sites in close proximity to Nkx6.1 binding sites were statistically overrepresented in the genome (P≤0.01). Given the role of Pdx1 on functional beta cell mass, we further characterized the interaction of endogenous Nkx6.1 and Pdx1 by co-immunoprecipitation (Co-IP). Our data demonstrates a significant Pdx1 binding by Nkx6.1 Co-IP, as compared to Co-IP with the IgG control (P=0.015, n=3). These data define potential Nkx6.1 binding partners in INS-1 832/13 cells, as well as a novel interaction between two essential beta cell transcription factors, Nkx6.1 and Pdx1. Disclosure C. C. Littlefield: None. N. C. Jensen: None. J. T. Hill: None. J. C. Price: None. J. S. Tessem: None. Funding Thomas J. Beatson, Jr. Foundation (2019-003)

Reduced replication fork speed promotes pancreatic endocrine differentiation and controls graft size.

Limitations in cell proliferation are important for normal function of differentiated tissues and essential for the safety of cell replacement products made from pluripotent stem cells, which have unlimited proliferative potential. To evaluate whether these limitations can be established pharmacologically, we exposed pancreatic progenitors differentiating from human pluripotent stem cells to small molecules that interfere with cell cycle progression either by inducing G1 arrest or by impairing S phase entry or S phase completion and determined growth potential, differentiation, and function of insulin-producing endocrine cells. We found that the combination of G1 arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells toward insulin-producing cells and could substitute for endocrine differentiation factors. Reduced replication fork speed during differentiation improved the stability of insulin expression, and the resulting cells protected mice from diabetes without the formation of cystic growths. The proliferative potential of grafts was proportional to the reduction of replication fork speed during pancreatic differentiation. Therefore, a compromised ability to enter and complete S phase is a functionally important property of pancreatic endocrine differentiation, can be achieved by reducing replication fork speed, and is an important determinant of cell-intrinsic limitations of growth.

Andrographolide promotes pancreatic duct cells differentiation into insulin-producing cells by targeting PDX-1

Regeneration of β-cells by differentiation of pancreatic progenitor cells has the potential to fundamentally solve the problems of the loss of β-cell function and mass during disease progression in both type 1 or 2 diabetes. Therefore, discovery of novel differentiation inducers to promote islet regeneration is of great significance. Pancreatic and duodenal homeobox1 (PDX-1) is a key transcription factor that promotes the development and maturation of pancreatic β-cells. To screen potential novel small molecules for enhancing differentiation of PNAC-1 cells, a human pancreatic ductal cell lines into insulin-producing cells (IPCs), we developed a high-throughput screening method through fusing the PDX-1 promoter region with a luciferase reporter gene. We screened and identified that andrographolide named C1037 stimulates PDX-1 expression in both mRNA and protein level and significantly promotes PANC-1 cells differentiation into IPCs as compared with that of control cells. The therapeutic effect of C037 in Streptozotocin induced diabetic mouse model through differentiation of pancreatic ductal cells into insulin positive islets was also observed. Our study provides a novel method to screen compounds regulating the differentiation of pancreatic progenitor cells having the potential of enhancing islet regeneration for diabetes therapy.

Human Fetal Bone Marrow-Derived Mesenchymal Stem Cells Promote the Proliferation and Differentiation of Pancreatic Progenitor Cells and the Engraftment Function of Islet-Like Cell Clusters

Pancreatic progenitor cells (PPCs) are the primary source for all pancreatic cells, including beta-cells, and thus the proliferation and differentiation of PPCs into islet-like cell clusters (ICCs) opens an avenue to providing transplantable islets for diabetic patients. Meanwhile, mesenchymal stem cells (MSCs) can enhance the development and function of different cell types of interest, but their role on PPCs remains unknown. We aimed to explore the mechanism-of-action whereby MSCs induce the in vitro and in vivo PPC/ICC development by means of our established co-culture system of human PPCs with human fetal bone marrow-derived MSCs. We examined the effect of MSC-conditioned medium on PPC proliferation and survival. Meanwhile, we studied the effect of MSC co-culture enhanced PPC/ICC function in vitro and in vivo co-/transplantation. Furthermore, we identified IGF1 as a critical factor responsible for the MSC effects on PPC differentiation and proliferation via IGF1-PI3K/Akt and IGF1-MEK/ERK1/2, respectively. In conclusion, our data indicate that MSCs stimulated the differentiation and proliferation of human PPCs via IGF1 signaling, and more importantly, promoted the in vivo engraftment function of ICCs. Taken together, our protocol may provide a mechanism-driven basis for the proliferation and differentiation of PPCs into clinically transplantable islets.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the differentiation of embryonic stem cells towards pancreatic lineage and pancreatic beta cell function

Animal and epidemiological studies demonstrated association of persistent exposure of TCDD, an endocrine disrupting chemical, to susceptibility of type 2 diabetes (T2D). High doses of TCDD were commonly employed in experimental animals to illustrate its diabetogenic effects. Data linking the epigenetic effects of low doses of TCDD on embryonic cells to T2D susceptibility risks is very limited. To address whether low dose exposure to TCDD would affect pancreatic development, hESCs pretreated with TCDD at concentrations similar to human exposure were differentiated towards pancreatic lineage cells, and their global DNA methylation patterns were determined. Our results showed that TCDD-treated hESCs had impaired pancreatic lineage differentiation potentials and altered global DNA methylation patterns. Four of the hypermethylated genes (PRKAG1, CAPN10, HNF-1B and MAFA) were validated by DNA bisulfite sequencing. PRKAG1, a regulator in the AMPK signaling pathway critical for insulin secretion, was selected for further functional study in the rat insulinoma cell line, INS-1E cells. TCDD treatment induced PRKAG1 hypermethylation in hESCs, and the hypermethylation was maintained after pancreatic progenitor cells differentiation. Transient Prkag1 knockdown in the INS-1E cells elevated glucose stimulated insulin secretions (GSIS), possibly through mTOR signaling pathway. The current study suggested that early embryonic exposure to TCDD might alter pancreatogenesis, increasing the risk of T2D.

Circular RNA circ-FOXM1 facilitates cell progression as ceRNA to target PPDPF and MACC1 by sponging miR-1304-5p in non-small cell lung cancer

Circular RNAs (circRNAs) have recently been considered as key regulators in carcinogenesis. Nevertheless, the study of circRNAs involved in non-small cell lung cancer (NSCLC) remains largely unclear. In this study, we aim to explore the clinical significance, regulatory effect and mechanism of circ-FOXM1 in NSCLC. The results indicated that circ-FOXM1 overexpression was observed in NSCLC tissues and closely associated with lymph node invasion, higher TNM stage, and unfavorable prognosis. Furthermore, knockdown and ectopic expression of circ-FOXM1 significantly inhibited and promoted the growth, migration and invasion of NSCLC cells, respectively. Mechanistically, miR-1304-5p was discovered as a direct target of circ-FOXM1. Circ-FOXM1 upregulated the level of pancreatic progenitor cell differentiation and proliferation factor (PPDPF) and metastasis-associated in colon cancer 1 (MACC1) by sponging miR-1304-5p, thereby increasing the proliferation and invasion of NSCLC cells. Taken together, this work revealed that circ-FOXM1/miR-1304-5p/PPDPF/MACC1 signaling was essential for the development and progression of NSCLC.

CRISPR-Cas9 Gene Editing Restores Beta-Cell Differentiation and Function in Patient-Specific iPSCs

Genetic knockout studies in mice concluded that the differentiation of endocrine lineages in the pancreas and intestine requires the transcription factor neurogenin3 (NGN3). However, case reports of patients with NGN3 mutations show that most patients do not develop diabetes until later in childhood, suggesting that NGN3 may not be required for human beta cell differentiation in vivo. We have identified a patient with a previously undescribed NGN3 loss-of-function mutation and generated induced pluripotent stem cells from patient fibroblasts to analyze the role of NGN3 in human beta cell differentiation. We used established protocols to differentiate patient-specific iPSCs (PS-iPSCs) into pancreatic progenitor cells and beta-like cells in parallel with the H1 hESC line. The PS- iPSCs displayed significantly lower pancreatic progenitor cell differentiation with only 10.7% of the double positive PDX1+/NKX6.1+ cell population present, whereas the control H1s hESCs produce 60% double positive cells. After further differentiation into beta-like cells, H1 cells produced multiple endocrine cell types, while the PS-iPSC line was not able to make significant numbers of endocrine cells. To assess if the loss of NGN3 was the primary cause for the loss of pancreatic progenitor cells and beta-like cell differentiation, we used CRISPR-cas9 gene editing to correct the patient’s mutation in the PS-iPSCs (cPS-iPSCs). Correcting the patient’s mutation restored the PDX1+/NKX6.1+ cell population in the pancreatic progenitor stage. Beta-like cells differentiated from cPS- iPSCs were capable of glucose responsive insulin secretion at the end of the differentiation protocol. In conclusion, our data suggests that NGN3 may have a previously unidentified role important for the differentiation of pancreatic progenitor cells and that there may be a relationship between NKX6.1 and NGN3 that is critical for pancreatic development. Disclosure K. Millette: None. K.R. Vogt: None. A. Salas: None. P. Pitukcheewanont: None. J. Austin: None. M. Martin: None. S. Georgia: None.

MRI tracking of autologous pancreatic progenitor-derived insulin-producing cells in monkeys

Insulin-producing cells (IPCs) derived from a patient’s own stem cells offer great potential for autologous transplantation in diabetic patients. However, the limited survival of engrafted cells remains a bottleneck in the application of this strategy. The present study aimed to investigate whether nanoparticle-based magnetic resonance (MR) tracking can be used to detect the loss of grafted stem cell-derived IPCs in a sensitive and timely manner in a diabetic monkey model. Pancreatic progenitor cells (PPCs) were isolated from diabetic monkeys and labeled with superparamagnetic iron oxide nanoparticles (SPIONs). The SPION-labeled cells presented as hypointense signals on MR imaging (MRI). The labeling procedure did not affect the viability or IPC differentiation of PPCs. Importantly, the total area of the hypointense signal caused by SPION-labeled IPCs on liver MRI decreased before the decline in C-peptide levels after autotransplantation. Histological analysis revealed no detectable immune response to the grafts and many surviving insulin- and Prussian blue-positive cell clusters on liver sections at one year post-transplantation. Collectively, this study demonstrates that SPIO nanoparticles can be used to label stem cells for noninvasive, sensitive, longitudinal monitoring of stem cell-derived IPCs in large animal models using a conventional MR imager.

Production of pancreatic progenitor cells from human induced pluripotent stem cells using a three-dimensional suspension bioreactor system.

Islet replacement is a promising strategy for the treatment of patients with type 1 diabetes and patients who have undergone total pancreatectomy. Recent progress in cellular reprogramming technology may allow the transplantation of a patient's own pancreatic cells. Although many studies have reported the differentiation of pancreatic progenitor cells from mouse and human pluripotent stem cells (PSCs), obtaining sufficient cell numbers for clinical applications remains problematic. Here, we describe the mass production of human pancreatic progenitor cells from human induced (i)PSCs using a three-dimensional suspension bioreactor system. Bioreactor culture of cells with stage-specific provision of growth factors and small compounds for 17 days produced approximately 1.6 × 108 cells/100 ml vessel in a single batch. About 95% of cells expressed pancreatic and duodenal homeobox factor 1, and 22% co-expressed the transcription factor NKX6.1. Furthermore, culture of pancreatic progenitor cells for an additional 2 weeks yielded mature pancreatic cells, including C-peptide-, glucagon- and trypsin-expressing cell populations. Moreover, differentiated β-cells secreted insulin in response to increased glucose in vitro. These findings suggest that a three-dimensional suspension culture system can generate human pancreatic progenitor cells from human iPSCs. Further optimization of culture conditions should provide sufficient functional islet cells for transplantation therapy. Copyright © 2017 John Wiley & Sons, Ltd.

Stress-induced adaptive islet cell identity changes.

The different forms of diabetes mellitus differ in their pathogenesis but, ultimately, they are all characterized by progressive islet β-cell loss. Restoring the β-cell mass is therefore a major goal for future therapeutic approaches. The number of β-cells found at birth is determined by proliferation and differentiation of pancreatic progenitor cells, and it has been considered to remain mostly unchanged throughout adult life. Recent studies in mice have revealed an unexpected plasticity in islet endocrine cells in response to stress; under certain conditions, islet non-β-cells have the potential to reprogram into insulin producers, thus contributing to restore the β-cell mass. Here, we discuss the latest findings on pancreas and islet cell plasticity upon physiological, pathological and experimental conditions of stress. Understanding the mechanisms involved in cell reprogramming in these models will allow the development of new strategies for the treatment of diabetes, by exploiting the intrinsic regeneration capacity of the pancreas.

NADPH Oxidase-Dependent Reactive Oxygen Species Stimulate β-Cell Regeneration Through Differentiation of Endocrine Progenitors in Murine Pancreas.

Reactive oxygen species (ROS) act as second messengers for redox modification of transcription factors essential for differentiation. The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a major source of ROS, has been shown to regulate differentiation of various progenitor cells, while its role in pancreatic endocrine cell differentiation is unclear. This study was aimed at this knowledge gap. Our results showed that ROS levels were dynamically changed during pancreas development concomitant with endocrine cell differentiation induced by modest exogenous ROS in rudiment cultures. NOX4, but not NOX2, the member of NADPH oxidase, was expressed persistently in endocrine lineage and showed high activity in critical pancreas development phase. Inhibition of NADPH oxidase activity impeded the differentiation of endocrine progenitors in vitro, and exogenous ROS reversed this effect. Studies performed in streptozotocin (STZ)-injected neonatal rats showed that diphenyleneiodonium (DPI) obstructed β-cell regeneration through the suppression of neurogenin 3 (NGN3) expression, but not Ki67-labeling β-cells, indicating that ROS stimulation promoted differentiation beyond proliferation of β-cells. Inhibition of NADPH oxidase also reduced expression of SRY (sex-determining region Y)-box 9 (SOX9), a transcriptional regulator of Ngn3, in endocrine precursor cells, both in vivo and in vitro. Overexpression of SOX9 attenuated the reduction of NGN3 induced by suppression of NADPH oxidase. This is the first study to demonstrate NADPH oxidase, especially NOX4-dependent ROS that promotes pancreatic progenitor cell differentiation into endocrine cells both in vitro and in vivo, probably through the regulation of SOX9. We provide evidence that NADPH oxidase-dependent ROS-mediated signaling is necessary for endocrine cell differentiation, which provides a potential strategy for efficient generation of insulin-producing cells in clinical application.

An insulin signaling feedback loop regulates pancreas progenitor cell differentiation during islet development and regeneration

As one of the key nutrient sensors, insulin signaling plays an important role in integrating environmental energy cues with organism growth. In adult organisms, relative insufficiency of insulin signaling induces compensatory expansion of insulin-secreting pancreatic beta (β) cells. However, little is known about how insulin signaling feedback might influence neogenesis of β cells during embryonic development. Using genetic approaches and a unique cell transplantation system in developing zebrafish, we have uncovered a novel role for insulin signaling in the negative regulation of pancreatic progenitor cell differentiation. Blocking insulin signaling in the pancreatic progenitors hastened the expression of the essential β cell genes insulin and pdx1, and promoted β cell fate at the expense of alpha cell fate. In addition, loss of insulin signaling promoted β cell regeneration and destabilization of alpha cell character. These data indicate that insulin signaling constitutes a tunable mechanism for β cell compensatory plasticity during early development. Moreover, using a novel blastomere-to-larva transplantation strategy, we found that loss of insulin signaling in endoderm-committed blastomeres drove their differentiation into β cells. Furthermore, the extent of this differentiation was dependent on the function of the β cell mass in the host. Altogether, our results indicate that modulation of insulin signaling will be crucial for the development of β cell restoration therapies for diabetics; further clarification of the mechanisms of insulin signaling in β cell progenitors will reveal therapeutic targets for both in vivo and in vitro β cell generation.

CXCR4 positive cell-derived Pdx1-high/Shh-low cells originated from embryonic stem cells improve the repair of pancreatic injury in mice.

Treatments for pancreatic injuries have been significantly improved recently, but full recovery of pancreatic function remains difficult. Embryonic stem cells have great potentialities for self-renewal and multiple differentiations. In this study, we explored an approach to induce the differentiation of pancreatic progenitor cells from embryonic stem cells in vitro. Male mouse embryonic stem cells were cultured by the hanging-drop method to form embryoid bodies. The definitive endoderm marked by CXCR4 in embryoid bodies was sorted by magnetic activated cell sorting and subsequently administrated with b-FGF, exendin-4, and cyclopamine to induce the differentiation of putative pancreatic progenitor cells, which was monitored by Pdx1, and Shh expressions. The putative pancreatic progenitor cells were transplanted into female BALB/c mice with pancreatitis induced by L-Arginine. Male donor cells were located by detecting sex-determining region of Y-chromosome DNA. Definitive endoderm cells (CXCR4(+) cells) were sorted from 5-day embryoid bodies. After 3-day administration with b-FGF, exendin-4, and cyclopamine, Pdx1-high/Shh-low cells were differentiated from CXCR4(+) cells. These cells developed into more amylase-secreted cells in vitro and could specifically reside in the damaged pancreas acinar area in mice with acute pancreatitis to enhance the regeneration. The putative pancreatic progenitor cells (Pdx1-high/Shh-low cells) derived from mouse embryonic stem cells through the administration of b-FGF, exendin-4, and cyclopamine on the CXCR4(+) cells in vitro could improve the regeneration of injured pancreatic acini in vivo.

Human Fetal Liver Stromal Cell Co-Culture Enhances the Differentiation of Pancreatic Progenitor Cells into Islet-Like Cell Clusters

Recent advance in directed differentiation of pancreatic stem cells offers potential to the development of replacement therapy for diabetic patients. However, the existing differentiation protocols are complex, time-consuming, and costly; thus there is a need for alternative protocols. Given the common developmental origins of liver and pancreas, we sought to develop a novel protocol, devoid of growth factors, by using liver stromal cells (LSCs) derived from human fetal liver. We examined the effects of the LSCs on the differentiation of pancreatic progenitor cells (PPCs) into islet-like cell clusters (ICCs). PPCs and LSCs isolated from 1st to 2nd trimester human fetal tissues underwent co-cultures; differentiation and functionality of ICCs were determined by examining expression of critical markers and secretion of insulin. Co-culture with 2nd but not 1st trimester LSCs enhanced ICC differentiation and functionality without the use of exogenous differentiation 'cocktails'. Differential expression profiles of growth factors from 1st versus 2nd trimester fetal liver were compared. Many morphogenic factors were expressed by LSCs, while insulin-like growth factor 1 (IGF1) was identified as one of the key molecules responsible for the ICC differentiation. This is the first report showing that an LSC-induced microenvironment can enhance ICC differentiation and functionality. Further modifications of the stroma microenvironment may offer an alternative, efficient and cost-effective approach to providing islets for transplantation.