Development of human fetal pancreas after transplantation into SCID mice

Only a small component of human fetal pancreas consists of beta cells, and yet this tissue is capable of normalizing the blood glucose levels of diabetic recipients when transplanted. The time taken to achieve this goal is several months, during which time the tissue proliferates and eventually differentiates into beta cells. The dynamics of beta cell development have not been described previously. We transplanted human fetal pancreas beneath the renal capsule of immunodeficient mice and analysed the grafts for a period of 12 weeks using antibodies against exocrine cells (lipase), endocrine cells and protodifferentiated duct cells. Exocrine cells constituted 48% of all epithelial cells in the untransplanted pancreas, with duct cells comprising 29% and endocrine cells 16% (beta cells 7%). The percentage of exocrine cells declined with time after transplantation, with only a small number undergoing apoptosis, and the duct cells increased, the values for these two cell types at 12 weeks being 20 and 57%, respectively. Both cell types appeared to proliferate equally for up to 8 weeks after transplantation, but only duct cells thereafter. Endocrine cells began to increase from 8 weeks after transplantation, representing 28% of epithelial cells (beta cells 11%) at this time. Intermediate cells, that is, cells expressing the characteristics of more than one type of mature pancreatic cell, were observed both in the ungrafted pancreas and after transplantation. The commonest intermediate cell type was duct/exocrine, with exocrine/endocrine and duct/endocrine cells also observed, suggesting active transdifferentiation from one cell type to another. We hypothesize that following the transplantation of human fetal pancreatic tissue, exocrine cells mostly transdifferentiate into duct cells and these eventually develop into endocrine cells, in particular beta cells.     

Pancreatic stem cells

Conclusions Available evidence strongly suggests that different cell types in pancreas are capable of undergoing transdifferentiation and dedifferentiation. Evidence also strongly supports the existence of stem cells in both the exocrine and endocrine pancreas. In the rat copper dificiency model, dormant stem cells present in the ductal and ductular system can be stimulated to proliferate and they have been shown to clearly differentiate into hepatocytes, a cell type not normally present in pancreas. Putative stem cells derived from the pancreas of rats after prolonged copper deficiency, can be easily grown in culture, but there is need to identify appropriate signals and defined culture conditions that can direct the in a reproducible fashion to differentiate into a specific cell type such as β-cells or liver cells. When these specific signaling molecules are identified pancreatic stem cells will be a source of an unlimited supply of self-renewable cells for transplantation. Although, the existence of pancreatic stem cells is being increasingly accepted and their multidirectional differentiation potential is recognized, their role, if any, in pancreatic cancer development remains to be clarified and investigated.     

Proliferation and differentiation of pancreatic β-cells: ultrastructural analysis of the pancreas in diabetic mice induced by selective alloxan perfusion

To clarify the mechanism of regenerative processes of pancreatic β-cells, we constructed a new diabetic model of mice and investigated their pancreatic endocrine cells by electron microscopy. Male ICR mice (8 weeks old) were partially and chemically depancreatized by perfusing alloxan (100 mg/kg body weight) via the caudal vein after clamping the cranial mesenteric artery. By this method, we could render the mice diabetic by partial reduction of β-cells localized in the splenic, gastric, and parabiliary segment. Glucose intolerance gradually ameliorated without any treatment. In the perfused segments, pancreatic β-cells showed pyknosis and the mitochondria were swollen 6h after the treatment, while non-β-cells including α-cells remained intact. At 5 days, β-cells were few and the islets became smaller in size. At 20 weeks, small islet cell clusters (ICCs) were observed budding from interlobular and intralobular ductal epithelial cells. β-cells scattering in the exocrine pancreas were also frequently observed. In the alloxan-nonperfused segment, β-cells with thin rough endoplasmic reticulum and immature secretory granules without an electron-opaque halo were observed, and the number of mitochondria increased in some β-cells at 1 day and 5 days after the treatment. At 20 weeks, β-cells that contained only mature granules were observed in hypertrophic islets. In this model, both proliferation of residual β-cells and differentiation of pancreatic endocrine cells from the ductal epithelial cells were recognized.     

昆明小鼠胰腺导管上皮样细胞向胰岛样细胞的诱导分化

目的研究正常昆明小鼠胰腺导管上皮样细胞向胰岛样细胞分化的诱导条件。方法取正常成年昆明小鼠胰腺导管上皮进行原代培养,利用细胞贴壁时间的差异在培养24、48、72 h连续换液除去腺体细胞,在含2%胎牛血清的培养条件下除去成纤维细胞,使可以在无血清培养基中生长的胰腺导管上皮样细胞形成优势生长。在此条件下培养并传代。取第三代细胞以无血清诱导培养基促使其分化。取诱导后3、5、7、14 d的细胞进行免疫细胞化学染色鉴定。ELISA检测诱导生成的胰岛样细胞在高糖刺激下分泌胰岛素的功能。结果经过无血清诱导培养基诱导7 d后,胰腺导管上皮样细胞开始向胰岛素分泌细胞分化,胰岛素抗体染色阳性。14 d胰岛素抗体染色阳性细胞明显增多。该细胞在高糖刺激下可以分泌胰岛素。结论昆明小鼠胰腺导管上皮样细胞经过无血清培养基诱导可以向胰岛样细胞方向分化。并且该胰岛样细胞在高糖刺激下具有分泌胰岛素的功能。     

What's new in in vitro studies of exocrine pancreatic cell injury?

Exocrine pancreas in vitro models are useful for the study of pancreatic differentiation, secretion mechanisms, cell injury, and lysosomal processing of secretory product. Syrian hamster pancreas in explant organ culture undergoes a series of morphologic changes which parallel in vitro acinar cell injury, differentiation, and phenotypic alteration. Within 48 hours, the cultured acinar cells show morphologic evidence of sublethal cell injury. Autophagy and crinophagy are particularly striking. The autophagic processes can be inhibited by the addition of the protein synthesis inhibitor cycloheximide or by culture at lowered temperatures (20 degrees C). Acinar cells lethally damaged show pyknotic nuclei, high amplitude swelling, and necrosis. Approximately 25% of each explant is viable after 72 hr in culture and the viability remains constant at 25-35% for up to 60 days of culture. The morphological changes of the explants are consistent with many of the features of pancreatitis and carcinoma of the exocrine pancreas. There is an increase in the ductal elements and a decrease in acini over time in culture. This may be due to: (a) an increased replication of ductal epithelial cells concomitant with necrosis of acinar epithelial cells and/or (b) phenotypic alteration of acinar cells to ductal cells. Acinar cell necrosis and phenotypic alterations may in part be due to the activation of lysosomal degradation pathways. Processes which inhibit lysosomal activation proved protective against these alterations, while processes which promote zymogen activation were deleterious.     

An immunocytochemical study of the cytogenesis of pancreatic endocrine cells in the lizard,Anolis carolinensis

The differentiation of the pancreatic endocrine cells in the lizard Anolis carolinensis following oviposition was examined. Immediately postoviposition (PO) there was no apparent differentiation of epithelioid cells into endocrine or exocrine components. Individual subpopulations of the endocrine-like cells, which could not be identified during the early PO period on the basis of either their tinctorial properties at the light-microscopic level or their granule morphologies at the electron-microscopic level, exhibited specific hormonal localization by peroxidase-antiperoxidase complex immunocytochemistry. All four hormones searched for, insulin, glucagon, somatostatin, and pancreatic polypeptide (PP), were present in epithelioid cells shortly after oviposition. However, the immunostained secretory granules in the early PO period were smaller than those of the adult. Secretory granule morphologies that are typical of the adult were acquired at different times during development. Delta granules were observed first and were followed by alpha granules, and beta granules which appeared shortly before birth. The secretory granules of the PP-containing F cells could not be readily placed within this maturation sequence. Mosaic cells (containing more than one hormone) were not seen. Levels of immunoreactive insulin and glucagon in the pancreas increased several fold from day 10 to day 28 PO, but the attainment of adult beta-granule morphologies did not appear to be directly related to insulin itself. The results show that cytodifferentiation of the anolian endocrine pancreas occurs postoviposition and that immunocytochemical methods can be used to follow an organelle sequence during development. These findings suggest that subcellular organelles undergo structural remodeling during maturation which, at least in the case of secretory granules, may have functional significance.     

Rapid acinar to ductal transdifferentiation in cultured human exocrine pancreas.

Experiments have been performed to define conditions for the primary culture of human exocrine pancreas, as a first step towards molecular reconstruction experiments of pancreatic neoplasia. Normal human exocrine pancreas was digested using collagenase and dispase and the resulting cellular aggregates were cultured in vitro. The phenotype of the digested pancreatic cells was almost exclusively acinar (amylase-positive, keratin 19 and mucin antigens-negative), yet within 4 days of culture the cells had taken on a ductal phenotype (amylase-negative, keratin 19 and mucin antigens-positive). The kinetics of these observations exclude the possibility of overgrowth of the acinar population by a ductal sub-population, and selective adherence is excluded by examination of those cells that do not adhere, which are representative of the initiating population. We interpret these data as indicating that, under the conditions of culture, the acinar cell phenotype is not stable and can transdifferentiate to a ductal phenotype. Taken together with recent data from transgenic animals, this in vitro observation has possible implications for our view of the pathogenesis of pancreatic neoplasia.     

Expression patterns of epiplakin1 in pancreas, pancreatic cancer and regenerating pancreas

Epiplakin1 (Eppk1) is a plakin family gene with its function remains largely unknown, although the plakin genes are known to function in interconnecting cytoskeletal filaments and anchoring them at plasma membrane-associated adhesive junction. Here we analyzed the expression patterns of Eppk1 in the developing and adult pancreas in the mice. In the embryonic pancreas, Eppk1+/Pdx1+ and Eppk1+/Sox9+ pancreatic progenitor cells were observed in early pancreatic epithelium. Since Pdx1 expression overlapped with that of Sox9 at this stage, these multipotent progenitor cells are Eppk1+/Pdx1+/Sox9+ cells. Then Eppk1 expression becomes confined to Ngn3+ or Sox9+ endocrine progenitor cells, and p48+ exocrine progenitor cells, and then restricted to the duct cells and a cells at birth. In the adult pancreas, Eppk1 is expressed in centroacinar cells (CACs) and in duct cells. Eppk1 is observed in pancreatic intraepithelial neoplasia (PanIN), previously identified as pancreatic ductal adenocarcinoma (PDAC) precursor lesions. In addition, the expansion of Eppk1-positive cells occurs in a caerulein-induced acute pancreatitis, an acinar cell regeneration model. Furthermore, in the partial pancreatectomy (Px) regeneration model using mice, Eppk1 is expressed in "ducts in foci", a tubular structure transiently induced. These results suggest that Eppk1 serves as a useful marker for detecting pancreatic progenitor cells in developing and regenerating pancreas.     

Dual origin,development, and fate of bovine pancreatic islets

Endocrine cells are evident at an early stage in bovine pancreatic development when the pancreas still consists of primitive epithelial cords. At this stage, the endocrine cells are interspersed between the precursor cells destined to form the ductulo‐acinar trees of later exocrine lobules. We here demonstrate that, in bovine fetuses of crown rump length ≥ 11 cm, the endocrine cells become increasingly segregated from the developing exocrine pancreas by assembly into two units that differ in histogenesis, architecture, and fate. Small numbers of ‘perilobular giant islets’ are distinguishable from larger numbers of ‘intralobular small islets’. The two types of islets arise in parallel from the ends of the ductal tree. Aside from differences in number, location, and size, the giant and small islets differ in cellular composition (predominantly insulin‐synthesising cells vs. mixtures of endocrine cells), morphology (epithelial trabeculae with gyriform and rosette‐like appearance vs. compact circular arrangements of endocrine cells), and in their relationships to intrapancreatic ganglia and nerves. A further difference becomes apparent during the antenatal period; while the ‘interlobular small islets’ persist in the pancreata of calves and adult cattle, the perilobular giant islets are subject to regression, characterised by involution of the parenchyma, extensive haemorrhage, leukocyte infiltration (myeloid and T‐cells) and progressive fibrotic replacement. In conclusion, epithelial precursor cells of the ductolo‐acinar tree may give rise to populations of pancreatic islets with different histomorphology, cellular composition and fates. This should be taken into account when using these cells for the generation of pancreatic islets for transplantation therapy.     

Formation of pancreatic duct epithelium from bone marrow during neonatal development

Recent reports suggest that bone marrow-derived cells engraft and differentiate into pancreatic tissue at very low frequency after pancreatic injury. All such studies have used adult recipients. The aim of our studies was to investigate the potential of bone marrow to contribute to the exocrine and endocrine components of the pancreas during the normal rapid growth of the organ that occurs during the neonatal period. Five to ten million bone marrow cells from adult, male, transgenic, green fluorescent protein (GFP) mice were injected into neonatal nonobese diabetic/severely compromised immunodeficient/beta2microglobulin-null mice 24 hours after birth. Two months after bone marrow transplantation, pancreas tissue was analyzed with fluorescence immunohistochemistry and fluorescence in situ hybridization (FISH). Co-staining of GFP, with anticytokeratin antibody, and with FISH for the presence of donor Y chromosome indicated that up to 40% of ducts (median 4.6%) contained epithelial cells derived from donor bone marrow. In some of these donor-derived ducts, there were clusters of large and small ducts, all comprised of GFP+ epithelium, suggesting that whole branching structures were derived from donor bone marrow. In addition, rare cells that coexpressed GFP and insulin were found within islets. Unlike pancreatic damage models, no bone marrow-derived vascular endothelial cells were found. In contrast to the neonatal recipients, bone marrow transplanted into adult mice rarely generated ductal epithelium or islet cells (p<.05 difference between adult and neonate transplants). These findings demonstrate the existence in bone marrow of pluripotent stem cells or epithelial precursors that can migrate to the pancreas and differentiate into complex organ-specific structures during the neonatal period.     

Serotonin promotes acinar dedifferentiation following pancreatitis‐induced regeneration in the adult pancreas

The exocrine pancreas exhibits a distinctive capacity for tissue regeneration and renewal following injury. This regenerative ability has important implications for a variety of disorders, including pancreatitis and pancreatic cancer, diseases associated with high morbidity and mortality. Thus, understanding its underlying mechanisms may help in developing therapeutic interventions. Serotonin has been recognized as a potent mitogen for a variety of cells and tissues. Here we investigated whether serotonin exerts a mitogenic effect in pancreatic acinar cells in three regenerative models, inflammatory tissue injury following pancreatitis, tissue loss following partial pancreatectomy, and thyroid hormone‐stimulated acinar proliferation. Genetic and pharmacological techniques were used to modulate serotonin levels in vivo. Acinar dedifferentiation and cell cycle progression during the regenerative phase were investigated over the course of 2 weeks. By comparing acinar proliferation in the different murine models of regeneration, we found that serotonin did not affect the clonal regeneration of mature acinar cells. Serotonin was, however, required for acinar dedifferentiation following inflammation‐mediated tissue injury. Specifically, lack of serotonin resulted in delayed up‐regulation of progenitor genes and delayed the formation of acinar‐to‐ductal metaplasia and defective acinar cell proliferation. We identified serotonin‐dependent acinar secretion as a key step in progenitor‐based regeneration, as it promoted acinar cell dedifferentiation and the recruitment of type 2 macrophages. Finally, we identified a regulatory Hes1–Ptfa axis in the uninjured adult pancreas, activated by zymogen secretion. Our findings indicated that serotonin plays a critical role in the regeneration of the adult pancreas following pancreatitis by promoting the dedifferentiation of acinar cells. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

新生昆明小鼠胰腺干细胞体外分离、培养及自然分化的潜能**

背景：关于胰腺干细胞在胰腺组织中的分布情况,以及如何有效的将其分离、体外优化培养,目前仍有一定的困难。 目的：从新生昆明小鼠胰腺组织中分离出胰腺干细胞,在体外条件下培养观察其形态学和生物学特征,并进行初步鉴定。 方法：取新生SPF级昆明小鼠的胰腺组织,使用Ⅴ型胶原酶消化,采用Percoll不连续密度梯度离心,使胰腺组织内、外分泌部细胞分离,分布在3个不同的密度界面;收集各界面的细胞,以无血清、添加碱性成纤维细胞生长因子和表皮细胞生长因子的DMEM培养基培养。 结果与结论：通过细胞形态学和细胞生长特性的观察,结合双硫腙染色证实：采用Percoll不连续密度梯度离心,第一、二密度界面的细胞来源于胰腺内分泌部;第三密度界面细胞来源于胰腺外分泌部。分别从胰腺内、外分泌部获取胰腺组织细胞,在体外培养观察发现均存在一类大、圆、单个核的细胞,呈集落样附壁生长,具有较活跃的分裂增殖能力,碱性磷酸酶染色阳性,表达胰腺干细胞的特异性标志巢蛋白,即为胰腺干细胞;随着体外培养时间的延长,分别表达PDX-1和CK-19,呈现向胰腺内分泌部细胞和外分泌部细胞分化的趋势。     

Pancreatic development and stem cell-based regenerative medicine

After the gut endoderm is formed, subsequent permissive induction signals from the notochord allows the pancreas to emerge and growout from a specific site of the embryonic gut epithelium. The first pancreas-specific gene, pdx1, is expressed around this stage. Interaction between pancreatic epithelium and the surrounding mesenchyme allows the pancreas bud to further grow and differentiate to form ductal, exocrine or endocrine lineages. Several lines of evidence from gene knockout mice and cell lineage studies suggest that a common pancreas stem cell first gives rise to exocrine and endocrine progenitor cells. The endocrine progenitor then give rise to four different endocrine cells: alpha, beta, delta, and PP cells, although it is not known how and when these cells arise. We have focused our studies on the understanding of endodermal induction and organogenesis of the pancreas. We specifically aimed at the isolation of pancreatic stem cells and the development of functional pancreatic endocrine beta cells in culture. For this aim, we used ES cells as a model system. Here, I review the literature on the development and regeneration of the pancreas. Some recent results on growing pancreatic cells from ES (embryonic stem) cells.     

Transplantation of human fetal pancreas: fresh vs. cultured fetal islets or ICCS

The paucity of human adult islets available for transplantation in IDDM makes the use of human fetal pancreas a potential alternative. Fetal pancreatic endocrine cells grow and differentiate over time when fresh explants or cultured islet-like cell clusters (ICCs) are transplanted under the kidney capsule in athymic nude mice. We have recently developed a procedure to isolate fetal islets, which differ from ICCs in their β-cell content. This study was undertaken to compare the maturation and growth of grafts from purified fetal islets, containing mostly β-cells, to grafts of mostly undifferentiated endocrine cell precursors, cultured as ICCs, and fresh, uncultured tissue. Total insulin content was highest in the fresh tissue pre-transplant while insulin levels fell precipitously during culture as either fetal islets or ICCs. Although 500 fetal islets contained more insulin than 500 ICCS before transplantation, the insulin content of the resulting grafts was the same 3 months post-transplantation. The degree of stimulation following glucose challenge was comparable, as was the histological appearance. However 70 mg of fresh tissue was needed to generate the fetal islets while only 30 mg was needed for the ICCs. Grafts of 30 mg fresh tissue also had similar total insulin contents and stimulation following glucose challenge, but, when normalized to DNA there was a significantly higher concentration of insulin in the grafts from ICCs or fetal islets. Moreover there were distinct morphological differences, with fibrous and ductal elements prominent in the grafts from fresh tissue, which were also much larger and more diffuse, with cystic elements evident macroscopically. Quantitative immunohistochemical analysis showed that grafts from cultured tissue were 48.3±5% positive for immunoreactive insulin compared with grafts from fresh tissue which were only 13.3±1.4% positive for insulin. In conclusion cultured ICCs, a heterogeneous mixture of hormone-containing and undifferentiated endocrine cells, are a preferable source for transplantation than either purified fetal islets or uncultured tissue.     

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.     

Temporal restriction of pancreatic branching competence during embryogenesis is mirrored in differentiating embryonic stem cells

To develop methods for the generation of insulin-producing β-cells for the treatment of diabetes, we have used GFP-tagged embryonic stem cells (ESCs) to elucidate the process of pancreas development. Using the reporter Pdx1(GFP/w) ESC line, we have previously described a serum-free differentiation protocol in which Pdx1-GFP(+) cells formed GFP bright (GFP(br)) epithelial buds that resembled those present in the developing mouse pancreas. In this study we extend these findings to demonstrate that these cells can undergo a process of branching morphogenesis, similar to that seen during pancreatic development of the mid-gestation embryo. These partially disaggregated embryoid bodies containing GFP(br) buds initially form epithelial ring-like structures when cultured in Matrigel. After several days in culture, these rings undergo a process of proliferation and form a ramified network of epithelial branches. Comparative analysis of explanted dissociated pancreatic buds from E13.5 Pdx1(GFP/w) embryos and ESC-derived GFP(br) buds reveal a similar process of proliferation and branching, with both embryonic Pdx1(GFP/w) branching pancreatic epithelium and ESC-derived GFP(br) branching organoids expressing markers representing epithelial (EpCAM and E-Cadherin), ductal (Mucin1), exocrine (Amylase and Carboxypeptidase 1A), and endocrine cell types (Glucagon and Somatostatin). ESC-derived branching structures also expressed a suite of genes indicative of ongoing pancreatic differentiation, paralleling gene expression within similar structures derived from the E13.5 fetal pancreas. In summary, differentiating mouse ESCs can generate pancreatic material that has significant similarity to the fetal pancreatic anlagen, providing an in vitro platform for investigating the cellular and molecular mechanisms underpinning pancreatic development.     

The epigenetic regulators Bmi1 and Ring1B are differentially regulated in pancreatitis and pancreatic ductal adenocarcinoma

Chronic pancreatitis and pancreatic ductal adenocarcinoma (PDAC) are associated with major changes in cell differentiation. These changes may be at the basis of the increased risk for PDAC among patients with chronic pancreatitis. Polycomb proteins are epigenetic silencers expressed in adult stem cells; up‐regulation of Polycomb proteins has been reported to occur in a variety of solid tumours such as colon and breast cancer. We hypothesized that Polycomb might play a role in preneoplastic states in the pancreas and in tumour development/progression. To test these ideas, we determined the expression of PRC1 complex proteins (Bmi1 and Ring1b) during pancreatic development and in pancreatic tissue from mouse models of disease: acute and chronic pancreatic injury, duct ligation, and in K‐Ras^G12V conditional knock‐in and caerulein‐treated K‐Ras^G12V mice. The study was extended to human pancreatic tissue samples. To obtain mechanistic insights, Bmi1 expression in cells undergoing in vitro exocrine cell metaplasia and the effects of Bmi1 depletion in an acinar cancer cell line were studied. We found that Bmi1 and Ring1B are expressed in pancreatic exocrine precursor cells during early development and in ductal and islet cells—but not acinar cells—in the adult pancreas. Bmi1 expression was induced in acinar cells during acute injury, in acinar–ductal metaplastic lesions, as well as in pancreatic intraepithelial neoplasia (PanIN) and PDAC. In contrast, Ring1B expression was only significantly and persistently up‐regulated in high‐grade PanINs and in PDAC. Bmi1 knockdown in cultured acinar tumour cells led to changes in the expression of various digestive enzymes. Our results suggest that Bmi1 and Ring1B are modulated in pancreatic diseases and could contribute differently to tumour development. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.