Effects of exogenous ghrelin on the tissue structure and somato-statin secretion in the pancreas of African ostrich chicks / Efectos de la grelina exógena en la estructura del tejido y la secreción de somato-estatinas en el páncreas de los polluelos de avestruz africanos

SUMMARY: This study aimed to investigate the effect of exogenous ghrelin on pancreatic growth and development in African ostrich chicks. Sixteen 40-day-old African ostrich chicks (male or female) were randomly divided into four groups and injected intravenously metatarsal vein with saline (control) or ghrelin (10, 50, and 100 μg/kg) for 6 days. Body and pancreas weight were determined, structural characteristics were observed using HE staining, somatostatin-immunopositive cells were detected using immunohistochemistry. The results were as follows: 1. The 50 and 100 μg/kg groups showed lower relative pancreas weight than the control group (P 0.05. Moreover, compared with the control, the islet cells in treatment groups were loosely arranged and showed reduced cytoplasm. In the exocrine pancreas, the volume of acinar cells in the 10, 50, and 100 μg/kg groups all decreased to varying degrees. 3. Somatostatin immunopositive cells were mainly located around the periphery of the islets and sporadically distributed in the center. The density of the somatostatin immunopositive cells in the 10, 50, and 100 μg/kg groups was higher than that in the control (P < 0.05). These findings suggest that exogenous ghrelin increases the area and number of islets and number of somatostatin immunopositive cells but reduces relative pancreas weight and effects the morphological and structural development of the pancreas, which may inhibit the pancreatic growth and development in African ostrich chicks.

RESUMEN: Este estudio tuvo como objetivo investigar el efecto de la grelina exógena sobre el crecimiento y desarrollo del páncreas en polluelos de avestruz africana. Dieciséis pollos de avestruz africana de 40 días (machos o hembras) se dividieron al azar en cuatro grupos y se inyectaron por vía intravenosa con solución salina (control) o grelina (10, 50 y 100 μg / kg) durante 6 días. determinadas, se observaron las características estructurales mediante tinción Hematoxilina-Eosina, se detectaron células inmunopositivas a somatostatina mediante inmunohistoquímica. Los resultados fueron los siguientes: ¨Los grupos de 50 y 100 μg / kg mostraron un menor peso relativo del páncreas que el grupo de control (P <0,05). El área de islotes por unidad de área del páncreas fue mayor en los grupos de 10, 50 y 100 μg / kg grupos que en el grupo de control (P <0,05). El número de islotes por unidad de área del páncreas fue menor en el grupo de 10 μg / kg que en el control (P <0,05). Además, en comparación con el control, las células de los islotes en los grupos de tratamiento estaban dispuestas de forma holgada y mostraban un citoplasma reducido. En el páncreas exocrino, el volumen de células acinares en los grupos de 10, 50 y 100 μg / kg disminuyó en diversos grados. Las células inmunopositivas de somatostatina se ubicaron principalmente alrededor de la periferia de los islotes y se distribuyeron esporádicamente en el centro. La densidad de las células inmunopositivas a la somatostatina en los grupos de 10, 50 y 100 μg / kg fue mayor que la del control (P <0,05). Estos hallazgos sugieren que la grelina exógena aumenta el área y el número de islotes y el número de células inmunopositivas a la somatostatina, pero reduce el peso relativo del páncreas, lo que puede inhibir el crecimiento y desarrollo pancreático en los polluelos de avestruz africana.

Tissue distribution of stem cell factor in adults.

Stem cell factor (SCF) is an essential cytokine during development and is necessary for gametogenesis, hematopoiesis, mast cell development, stem cell function, and melanogenesis. Here, we measure SCF concentration and distribution in adult humans and mice using gene expression analysis, tissue staining, and organ protein lysates. We demonstrate continued SCF expression in many cell types and tissues into adulthood. Tissues with high expression in adult humans included stomach, spleen, kidney, lung, and pancreas. In mice, we found high SCF expression in the esophagus, ovary, uterus, kidney, and small intestine. Future studies may correlate our findings of increased, organ-specific SCF concentrations within adult tissues with increased risk of SCF/CD117-related disease.

Pancreatic Ppy-expressing γ-cells display mixed phenotypic traits and the adaptive plasticity to engage insulin production.

The cellular identity of pancreatic polypeptide (Ppy)-expressing γ-cells, one of the rarest pancreatic islet cell-type, remains elusive. Within islets, glucagon and somatostatin, released respectively from α- and δ-cells, modulate the secretion of insulin by ß-cells. Dysregulation of insulin production raises blood glucose levels, leading to diabetes onset. Here, we present the genetic signature of human and mouse γ-cells. Using different approaches, we identified a set of genes and pathways defining their functional identity. We found that the γ-cell population is heterogeneous, with subsets of cells producing another hormone in addition to Ppy. These bihormonal cells share identity markers typical of the other islet cell-types. In mice, Ppy gene inactivation or conditional γ-cell ablation did not alter glycemia nor body weight. Interestingly, upon ß-cell injury induction, γ-cells exhibited gene expression changes and some of them engaged insulin production, like α- and δ-cells. In conclusion, we provide a comprehensive characterization of γ-cells and highlight their plasticity and therapeutic potential.

A new hypothetical model for pancreatic development based on change in the cell division orientation.

Although lifelong renewal and additional compensatory growth in response to demand are undeniable facts, so far, no specific stem cells have been found for pancreatic cells. According to the consensus model, the development of pancreas results from the hierarchical differentiation of pluripotent stem cells towards the appearance of the first endocrine and exocrine cells at approximately 7.5 to 8th gestation week (GW) of human embryo. However, the primitive endocrine cells arising from the embryonic phase of development do not appear to be mature or fully functional. Asymmetric localization of cellular components, such as Numb, partition protein complexes (PAR), planar cell polarity components, and certain mRNAs on the apical and basal sides of epithelial cells, causes cellular polarization. According to our model, the equal distribution of cellular components during symmetric cell division yields similar daughter cells that are associated with duct expansion. In contrast, asymmetric cell division is associated with uneven distribution of cellular components among daughter cells, resulting in different fates. Asymmetric cell division leads to duct branching and the development of acinar and stellate cells by a daughter cell, as well as the development of islet progenitor cells through partial epithelial-to-mesenchymal transition (EMT) and delamination of another daughter cell. Recently, we have developed an efficient method to obtain insulin-secreting cells from the transdifferentiation of hESC-derived ductal cells inducing a partial EMT by treatment with Wnt3A and activin A in a hypoxic environment. Similar models can be offered for other tissues and organs such as mammary glands, lungs, prostate, liver, etc. This model may open a new horizon in the field of regenerative medicine and be useful in explaining the cause of certain abnormalities, such as the occurrence of certain cysts and tumors.

The Map3k12 (Dlk)/JNK3 signaling pathway is required for pancreatic beta-cell proliferation during postnatal development.

Unveiling the key pathways underlying postnatal beta-cell proliferation can be instrumental to decipher the mechanisms of beta-cell mass plasticity to increased physiological demand of insulin during weight gain and pregnancy. Using transcriptome and global Serine Threonine Kinase activity (STK) analyses of islets from newborn (10 days old) and adult rats, we found that highly proliferative neonatal rat islet cells display a substantially elevated activity of the mitogen activated protein 3 kinase 12, also called dual leucine zipper-bearing kinase (Dlk). As a key upstream component of the c-Jun amino terminal kinase (Jnk) pathway, Dlk overexpression was associated with increased Jnk3 activity and was mainly localized in the beta-cell cytoplasm. We provide the evidence that Dlk associates with and activates Jnk3, and that this cascade stimulates the expression of Ccnd1 and Ccnd2, two essential cyclins controlling postnatal beta-cell replication. Silencing of Dlk or of Jnk3 in neonatal islet cells dramatically hampered primary beta-cell replication and the expression of the two cyclins. Moreover, the expression of Dlk, Jnk3, Ccnd1 and Ccnd2 was induced in high replicative islet beta cells from ob/ob mice during weight gain, and from pregnant female rats. In human islets from non-diabetic obese individuals, DLK expression was also cytoplasmic and the rise of the mRNA level was associated with an increase of JNK3, CCND1 and CCND2 mRNA levels, when compared to islets from lean and obese patients with diabetes. In conclusion, we find that activation of Jnk3 signalling by Dlk could be a key mechanism for adapting islet beta-cell mass during postnatal development and weight gain.

LDL cholesterol promotes the proliferation of prostate and pancreatic cancer cells by activating the STAT3 pathway.

Hypercholesterolemia has been found to be closely linked with a significant increase in both cancer incidence and mortality. However, the exact correlation between serum cholesterol levels and cancer has not been completely deciphered. Here we analyzed the effect of low-density lipoprotein (LDL) cholesterol on prostate and pancreatic cancer cells. We noted that LDL induced a substantial STAT3 activation and JAK1, JAK2, Src activation in diverse prostate and pancreatic tumor cells. Moreover, LDL promoted cancer cell proliferation, migration, and invasion as well as upregulated the expression of diverse oncogenic gene products. However, deletion of LDL-activated STAT3 in LNCaP and PANC-1 cells and reduced LDL-induced cell viability. Simvastatin (SV) treatment also alleviated LDL-induced cell viability and migration ability in both the prostate and pancreatic tumor cells. These results demonstrate that LDL-induced STAT3 activation may exert a profound effect on the proliferation and survival of tumor cells.

Establishment of Pancreatic Organoids from Normal Tissue and Tumors.

Establishment of patient-derived adult stem cell-based pancreatic (tumor) organoids was first described in 2015. Since then, multiple laboratories have demonstrated the robustness of this method. We recently described the generation of a pancreatic cancer biobank containing 30 well-characterized tumor organoid models. Here, we describe the applied methods in detail. Use of tumor-selective media prevents contamination with normal cells. Generated organoids can be cryopreserved and can serve as a living biobank of pancreatic cancer for biomarker identification and drug screening. For complete information on the generation and use of this protocol, please refer to Driehuis et al. (2019).

Mouse Pancreas Stem/Progenitor Cells Get Augmented by Streptozotocin and Regenerate Diabetic Pancreas After Partial Pancreatectomy.

Existence of stem cells in adult pancreas remains contentious. Single cells suspensions obtained by collagenase and trypsin digestion separately from adult mouse pancreas and pancreatic islets were spun at 1000 rpm (250 g) to collect the cells. At this speed the stem/ progenitor cells remained buoyant and were further enriched by spinning the supernatant at 3000 rpm (1000 g). Two distinct populations of stem cells were detected including pluripotent, very small (2-6 µm) embryonic-like stem cells (VSELs) that expressed nuclear OCT-4A and pluripotent transcripts (Oct-4A, Sox2, Nanog, Stella) and slightly bigger progenitors, pancreatic stem cells (PSCs) that expressed cytoplasmic OCT-4B and PDX-1. Streptozotocin treated diabetic pancreas showed an increase in numbers of VSELs (2-6 µm, 7AAD-, LIN-CD45-SCA1+ cells) and up-regulation of transcripts specific for stem/ progenitor cells. Diabetic mice were further subjected to partial pancreatectomy to study involvement of VSELs/ PSCs during regeneration. VSELs/ PSCs were mobilized in large numbers, were observed in the lumen of blood vessels and PCNA expression suggested their proliferation. Initially, new acini assembled to regenerate the exocrine pancreas and later by Day 30, neogenesis of islets was observed in the vicinity of the blood vessels and pancreatic ducts by the differentiation of endogenous VSELs/ PSCs which may be targeted to regenerate diabetic pancreas in clinical settings.

Direct differentiation of insulin-producing cells from human urine-derived stem cells.

The loss of pancreatic ß-cells is a cause of diabetes. Therefore, replacement of pancreatic ß-cells is a logical strategy for the treatment of diabetes, and the generation of insulin-producing cells (IPCs) from stem cells has been widely investigated as an alternative source for pancreatic ß-cells. Here, we isolated stem cells from human urine and investigated their differentiation potential into IPCs. We checked the expression of surface stem cell markers and stem cell transcription factors, and found that the isolated human urine-derived stem cells (hUDSCs) expressed the stem cell markers CD44, CD90, CD105 and stage-specific embryonic antigen (SSEA)-4. In addition, these cells expressed octamer binding transcription factor (Oct)4 and vimentin. hUDSCs could differentiate into adipocytes and osteocytes, as evidenced by Oil-red O staining and Alizarin Red S-staining of differentiated cells, respectively. When we directly differentiated hUDSCs into IPCs, the differentiated cells expressed mRNA for pancreatic transcription factors such as neurogenin (Ngn)3 and pancreatic and duodenal homeobox (Pdx)1. Differentiated IPCs expressed insulin and glucagon mRNA and protein, and these IPCs also secreted insulin in response to glucose stimulation. In conclusion, we found that hUDSCs can be directly differentiated into IPCs, which secrete insulin in response to glucose.

Integrative analysis of the inverse expression patterns in pancreas development and cancer progression.

BACKGROUND: As the malignant tumor, pancreatic cancer with a meager 5-years survival rate has been widely concerning. However, the molecular mechanisms that result in malignant transformation of pancreatic cells remain elusive. AIM: To investigate the gene expression profiles in normal or malignant transformed pancreas development. METHODS: MaSigPro and ANOVA were performed on two pancreas development datasets downloaded from the Gene Expression Omnibus database. Six pancreatic cancer datasets collected from TCGA database were used to establish differentially expressed genes related to pancreas development and pancreatic cancer. Moreover, gene clusters with highly similar interpretation patterns between pancreas development and pancreatic cancer progression were established by self-organizing map and singular value decomposition. Additionally, the hypergeometric test was performed to compare the corresponding interpretation patterns. Abnormal regions of metabolic pathway were analyzed using the Sub-pathway-GM method. RESULTS: This study established the continuously upregulated and downregulated genes at different stages in pancreas development and progression of pancreatic cancer. Through analysis of the differentially expressed genes, we established the inverse and consistent direction development-cancer pattern associations. Based on the application of the Subpathway-GM analysis, we established 17 significant metabolic sub-pathways that were closely associated with pancreatic cancer. Of note, the most significant metabolites sub-pathway was related to glycerophospholipid metabolism. CONCLUSION: The inverse and consistent direction development-cancer pattern associations were established. There was a significant correlation in the inverse patterns, but not consistent direction patterns.

Wnt Signaling Separates the Progenitor and Endocrine Compartments during Pancreas Development.

In vitro differentiation of pluripotent cells into ß cells is a promising alternative to cadaveric-islet transplantation as a cure for type 1 diabetes (T1D). During the directed differentiation of human embryonic stem cells (hESCS) by exogenous factors, numerous genes that affect the differentiation process are turned on and off autonomously. Manipulating these reactions could increase the efficiency of differentiation and provide a more complete control over the final composition of cell populations. To uncover in vitro autonomous responses, we performed single-cell RNA sequencing on hESCs as they differentiate in spherical clusters. We observed that endocrine cells and their progenitors exist beside one another in separate compartments that activate distinct genetic pathways. WNT pathway inhibition in the endocrine domain of the differentiating clusters reveals a necessary role for the WNT inhibitor APC during islet formation in vivo. Accordingly, WNT inhibition in vitro causes an increase in the proportion of differentiated endocrine cells.

Redefining the signaling pathways from pluripotency to pancreas development: In vitro ß-cell differentiation.

Pancreatic ß-cells are destroyed by the immune system, in type 1 diabetes (T1D) and are impaired by glucose insensitivity in type 2 diabetes (T2D). Islet-cells transplantation is a promising therapeutic approach based on in vitro differentiation of pluripotent stem cells (PSCs) to insulin-producing cells (IPCs). According to evolutionary stages in ß-cell development, there are several distinct checkpoints; each one has a unique characteristic, including definitive endoderm (DE), primitive gut (PG), posterior foregut (PF), pancreatic epithelium (PE), endocrine precursor (EP), and immature ß-cells up to functional ß-cells. A better understanding of the gene regulatory networks (GRN) and associated transcription factors in each specific developmental stage, guarantees the achievement of the next successful checkpoints and ensures an efficient ß-cell differentiation procedure. The new findings in signaling pathways, related to the development of the pancreas are discussed here, including Wnt, Activin/Nodal, FGF, BMP, retinoic acid (RA), sonic hedgehog (Shh), Notch, and downstream regulators, required for ß-cell commitment. We also summarized different approaches in the IPCs protocol to conceptually define a standardized system, leading to the creation of a reproducible method for ß-cell differentiation. To normalize blood glucose level in diabetic mice, the replacement therapy in the early differentiation stage, such as EP stages was associated with better outcome when compared with the fully differentiated ß-cells' graft.

The Origin of a New Progenitor Stem Cell Group in Human Development.

The observation of two precursor groups of the early stem cells (Groups I and II) leads to the realization that a first amount of fetal stem cells (Group I) migrate from the AMG (Aortal-Mesonephric-Gonadal)-region into the aorta and its branching vessels. A second group (Group II) gains quite a new significance during human development. This group presents a specific developmental step which is found only in the human. This continuation of the early development along a different way indicates a general alteration of the stem cell biology. This changed process in the stem cell scene dominates the further development of the human stem cells. It remains unclear where this phylogenetic step first appears. By far not all advanced mammals show this second group of stem cells and their axonal migration. Essentially only primates seem to be involved in this special development.

Desarrollo y cambios con la edad en el tubo digestivo, hígado y páncreas / Digestive tract, liver and pancreas: development and changes with age

An adequate functioning of the digestive tract, liver and pancreas is fundamental to providing the organism with the necessary conditions for its development and maintaining its digestive and systemic homeostasis. Life expectancy has increased, it is estimated that adults over 65 years old by 2050, will represent 25% of the local population. The morphological and functional changes associated with aging in the digestive system, liver and pancreas are modest except for those that occur in the microbiota. Recently it has been possible to establish the contribution of the microbiota to life expectancy and establish a link between gastrointestinal microbiota, inflammation associated with aging (inflammaging) and survival. This represents a shift in the paradigm of our understanding physiology, chronic diseases, neoplasms and for the development of new therapies.

Un adecuado funcionamiento del tubo digestivo, hígado y páncreas es fundamental para poder brindar al organismo las condiciones necesarias para su desarrollo y mantener su homeostasis digestiva y sistémica. La expectativa de vida se ha incrementado, estimándose a nivel nacional que para el año 2050 los adultos mayores de 65 años representarán el 25% de la población. Los cambios morfológicos y funcionales asociados al envejecimiento en el aparato digestivo, hígado y páncreas son modestos a excepción, de los que se producen en la microbiota. Recientemente se ha podido establecer la contribución de la microbiota a la esperanza de vida y establecer un nexo entre microbiota gastrointestinal, inflamación asociada al envejecimiento y sobrevida. Esto representa un cambio en el paradigma sobre cómo comprendemos la fisiología, las patologías crónicas, neoplásicas y en el desarrollo de nuevas terapias.

Statistical theory of branching morphogenesis.

Branching morphogenesis remains a subject of abiding interest. Although much is known about the gene regulatory programs and signaling pathways that operate at the cellular scale, it has remained unclear how the macroscopic features of branched organs, including their size, network topology and spatial patterning, are encoded. Lately, it has been proposed that, these features can be explained quantitatively in several organs within a single unifying framework. Based on large-scale organ reconstructions and cell lineage tracing, it has been argued that morphogenesis follows from the collective dynamics of sublineage-restricted self-renewing progenitor cells, localized at ductal tips, that act cooperatively to drive a serial process of ductal elongation and stochastic tip bifurcation. By correlating differentiation or cell cycle exit with proximity to maturing ducts, this dynamic results in the specification of a complex network of defined density and statistical organization. These results suggest that, for several mammalian tissues, branched epithelial structures develop as a self-organized process, reliant upon a strikingly simple, but generic, set of local rules, without recourse to a rigid and deterministic sequence of genetically programmed events. Here, we review the basis of these findings and discuss their implications.

Growth of Epithelial Organoids in a Defined Hydrogel.

Epithelial organoids are simplified models of organs grown in vitro from embryonic and adult stem cells. They are widely used to study organ development and disease, and enable drug screening in patient-derived primary tissues. Current protocols, however, rely on animal- and tumor-derived basement membrane extract (BME) as a 3D scaffold, which limits possible applications in regenerative medicine. This prompted us to study how organoids interact with their matrix, and to develop a well-defined hydrogel that supports organoid generation and growth. It is found that soft fibrin matrices provide suitable physical support, and that naturally occurring Arg-Gly-Asp (RGD) adhesion domains on the scaffold, as well as supplementation with laminin-111, are key parameters required for robust organoid formation and expansion. The possibility to functionalize fibrin via factor XIII-mediated anchoring also allows to covalently link fluorescent nanoparticles to the matrix for 3D traction force microscopy. These measurements suggest that the morphogenesis of budding intestinal organoids results from internal pressure combined with higher cell contractility in the regions containing differentiated cells compared to the regions containing stem cells. Since the fibrin/laminin matrix supports long-term expansion of all tested murine and human epithelial organoids, this hydrogel can be widely used as a defined equivalent to BME.

Novel Insights into Adult and Cancer Stem Cell Biology.

Adult tissues are thought to harbor two populations of "dormant" and "actively dividing" stem cells. Quiescent stem cells undergo rare asymmetric cell divisions (ACDs) through which they self-renew and give rise to tissue-committed "progenitors" of distinct fate and "progenitors" in turn undergo symmetric cell divisions (SCDs) and clonal expansion. However, quiescent stem cells have not been demonstrated in adult tissues such as skin, testis, liver, and brain. After surgical removal of part of liver and pancreas-adult differentiated cells divide and regenerate and a possible role of stem cells remains doubtful. Long-term repopulating hematopoietic stem cells are quiescent in nature but ACD has not been convincingly demonstrated even among them. Attempts by various groups to identify a common stemness program that ensures self-renewal among different kinds of stem cells have also remained futile. Uncontrolled self-renewal and compromised differentiation of stem cells possibly initiate leukemia/cancer, but the identity of leukemic stem cells and whether cancer stem cells arise by epithelial-mesenchymal transition (EMT) in solid tumors are all open-ended questions that need greater clarity. Acceptance of the presence of very small embryonic-like stem cells (VSELs) in adult tissues could clarify several of these existing dilemmas in the field. Data are compiled showing that VSELs undergo ACD in the hematopoietic system, testis, ovary, uterus, and pancreas, whereas tissue-committed progenitors undergo SCD and clonal expansion. VSELs possess similar overlapping stemness program as in embryonic stem cells, embryonic carcinoma cells, embryonic germ cells, induced pluripotent stem cells, and primordial germ cells. VSELs and leukemic and cancer cells express overlapping embryonic markers. Uncontrolled proliferation of VSELs and compromised differentiation possibly initiate leukemia. Process of EMT and initiation of solid tumor from VSELs (located among the epithelial cells) are indeed two distinct and parallel events. To conclude, VSELs provide explanation to several confounding aspects of adult stem cell biology.

Pancreas regeneration.

The pancreas is made from two distinct components: the exocrine pancreas, a reservoir of digestive enzymes, and the endocrine islets, the source of the vital metabolic hormone insulin. Human islets possess limited regenerative ability; loss of islet ß-cells in diseases such as type 1 diabetes requires therapeutic intervention. The leading strategy for restoration of ß-cell mass is through the generation and transplantation of new ß-cells derived from human pluripotent stem cells. Other approaches include stimulating endogenous ß-cell proliferation, reprogramming non-ß-cells to ß-like cells, and harvesting islets from genetically engineered animals. Together these approaches form a rich pipeline of therapeutic development for pancreatic regeneration.

Role of the Wilms' tumor suppressor gene Wt1 in pancreatic development.

The Wilms tumor suppressor gene (Wt1) encodes a transcription factor involved in the development of a number of organs, but the role played by Wt1 in pancreatic development is unknown. The pancreas contains a population of pancreatic stellate cells (PSC) very important for pancreatic physiology. We described elsewhere that hepatic stellate cells originate from the WT1-expressing liver mesothelium. Thus, we checked if the origin of PSCs was similar. WT1 expression is restricted to the pancreatic mesothelium. Between embryonic day (E) 10.5 and E15.5, this mesothelium gives rise to mesenchymal cells that contribute to a major part of the PSC and other cell types including endothelial cells. Most WT1 systemic mutants show abnormal localization of the dorsal pancreas within the mesentery and intestinal malrotation by E14.0. Embryos with conditional deletion of WT1 between E9.5 and E12.5 showed normal dorsal pancreatic bud and intestine, but the number of acini in the ventral bud was reduced approximately 30% by E16.5. Proliferation of acinar cells was reduced in WT1 systemic mutants, but pancreatic differentiation was not impaired. Thus, mesothelial-derived cells constitute an important subpopulation of pancreatic mesodermal cells. WT1 expression is not essential for pancreas development, although it influences intestinal rotation and correct localization of the dorsal pancreas within the mesogastrium. Developmental Dynamics 247:924-933, 2018. © 2018 Wiley Periodicals, Inc.