Biliary tree stem cells, precursors to pancreatic committed progenitors: evidence for possible life-long pancreatic organogenesis

Abstract

Peribiliary glands (PBGs) in bile duct walls, and pancreatic duct glands (PDGs) associated with pancreatic ducts, in humans of all ages, contain a continuous, ramifying network of cells in overlapping maturational lineages. We show that proximal (PBGs)-to-distal (PDGs) maturational lineages start near the duodenum with cells expressing markers of pluripotency (NANOG, OCT4, and SOX2), proliferation (Ki67), self-replication (SALL4), and early hepato-pancreatic commitment (SOX9, SOX17, PDX1, and LGR5), transitioning to PDG cells with no expression of pluripotency or self-replication markers, maintenance of pancreatic genes (PDX1), and expression of markers of pancreatic endocrine maturation (NGN3, MUC6, and insulin). Radial-axis lineages start in PBGs near the ducts' fibromuscular layers with stem cells and end at the ducts' lumens with cells devoid of stem cell traits and positive for pancreatic endocrine genes. Biliary tree-derived cells behaved as stem cells in culture under expansion conditions, culture plastic and serum-free Kubota's Medium, proliferating for months as undifferentiated cells, whereas pancreas-derived cells underwent only approximately 8-10 divisions, then partially differentiated towards an islet fate. Biliary tree-derived cells proved precursors of pancreas' committed progenitors. Both could be driven by three-dimensional conditions, islet-derived matrix components and a serum-free, hormonally defined medium for an islet fate (HDM-P), to form spheroids with ultrastructural, electrophysiological and functional characteristics of neoislets, including glucose regulatability. Implantation of these neoislets into epididymal fat pads of immunocompromised mice, chemically rendered diabetic, resulted in secretion of human C-peptide, regulatable by glucose, and able to alleviate hyperglycemia in hosts. The biliary tree-derived stem cells and their connections to pancreatic committed progenitors constitute a biological framework for life-long pancreatic organogenesis.

Keywords: biliary tree stem cells; maturational lineages; organogenesis; pancreas; pancreatic duct glands; peribiliary glands.

Figure 1. Panel 1. The peribiliary glands and pancreatic duct glands form a continuous network withinthe biliary tree and pancreatic ducts

Panel 1. The hepato-pancreatic common duct. The biliary tree and its peribiliary glands (PBGs) are part of a continuous network connecting to the pancreatic duct and its pancreatic duct glands (PDGs). (A) The schematic drawing shows the bile duct, the main pancreatic duct and their fusion at the hepato-pancreatic ampulla (the papilla of Vater). The sectioning planes indicate the regions from which the proximal (B) and distal (C) histological sections were taken. (B) A section in the proximal plane shows the histology of the bile duct and pancreatic duct in the immediate proximity of the fusion in the hepato-pancreatic ampulla. Just before the formation of the hepato-pancreatic ampulla, the fibromuscular layer, dividing bile and pancreatic ducts, is mostly absent; PBGs and PDGs are connected and indistinguishable from each other. The dashed lines indicate the PBGs (in green) versus the PDGs (in red). Note the striking similarities in the histological appearance of bile duct versus pancreatic duct and of PBGs versus PDGs. Hypercellular foci are marked with asterisks*. (C) A section in a distal plane shows the histological appearance of PBGs and PDGs in the separated ducts, divided by a thick fibromuscular layer (FM layer). (D) A plane intermediate between B and C shows glands intermingled and crossing the FM layer (artificially shown as a green area). Note the evidence of ductal and alveolar cuboidal and columnar cells, eosinophilic acinar-like cells and mucinous cells in the PBGs and PDGs. Several hypercellular foci are present (marked with asterisks*) in the PBGs of the hepato-pancreatic common duct, suggesting intense proliferative activity at the site. (E) Higher magnification of a hypercellular focus from the boxed area in D. Cells in hypercellular foci display a high nucleus/cytoplasm ratio and are tightly packed. (F) A compartment of the PDGs can be identified by Mucin 6 (MUC6) staining. The PDG cells show PDX-1 staining. Alveolar and ductal spaces are filled with amylase. For more detailed schematics of the hepato-pancreatic common duct see Figs S1 and S2. Scale bar=250 μm

Figure 1. Panel 2. Stem Cells are present in PBGs versus committed progenitors inPDGs. See also Figs.2-3, S3 and S7

(G-J) Immunofluorescent stainings show the co-expression of SOX17, OCT4A and SOX2 in the cells of PBGs. (K-M) Immunohistochemistry also show that OCT4A, SOX2 and LGR5 are evident in the cells in PBGs. (N2P) By contrast, only rare or no cells in PDGs were found expressing OCT4A or SOX2; none were found expressing LGR5 (data not shown); and a significant proportion of the PDG cells expressed the transcription factor, NGN3, a marker of endocrine progenitor cells. Scale bar = 50 μm.

Figure 2. Panel 1 (A-C). The maturational gradients in gene expression of pluripotency genes

Panel 1 (A-C) Pluripotency related transcription factors (OCT4, NANOG, SOX2) were strongly expressed in the nuclei of PBG cells. (D-F) By contrast, in PDGs, there was no expression at all of NANOG, and expression of OCT4 or SOX 2 occurred only in the cytoplasm of rare cells. The presence or absence and the subcellular localization of these transcription factors represent specific stages in lineage commitment. Scale bars= 100 μm.

Figure 2. Panel 2 (G-J)

Sections of PBGs in the hepato-pancreatic common duct were triple stained for SOX2 (green), OCT4A (red) and NANOG (blue). The merged image indicates cells co-expressing the 3 pluripotency genes (white). (K-N) Images from PDGs showing rare cells with cytoplasmic, but not nuclear, staining of SOX2 and OCT4A and lacking altogether any NANOG expression. For lower magnification images and a table with quantitation of the numbers of cells with the specific phenotypic properties, see Fig. S3. Scale bars = 100 μm

Figure 3. Panel 1 (A-H). The maturational lineage gradients in terms of endocrine traits

Panel 1 (A-H) shows a proximal (PBGs)-to distal (PDGs) gradient in expression of early-to-late pancreatic commitment markers. PDX1 and SOX 17, transcription factors of early hepato-pancreatic commitment are co-expressed in nuclei of PBG cells (A). By contrast, SOX 17 is lost, and only PDX1 is expressed in the nuclei of PDG cells. (E) MUC6 shows an affinity for a differentiated compartment of the glandular epithelium; it is found in a portion of the cells in the PBGs (B) and in all cells in PDGs (F). NGN3, a marker of endocrine committed progenitors, is not found in the nuclei of any cells of the PBGs (B) but in a large proportion of cells within PDGs (F). EpCAM is found in only a subset of the cells in the PBGs (C) but in almost all of the cells of the PDGs (G). See also Fig S6. Insulin can be observed in rare cells in PBGs (C) but is found in a large number of cells in PDGs (G). A high proliferative activity is found in PBGs, as indicated by Ki-67 staining (D), whereas Ki-67 positivity is rarely found in PDGs (H). Scale Bars = 100 μm.

Figure 3. Panel 2 (I-L)

Panel 2 (I-L) shows a radial axis maturational lineage extending from the fibromuscular layer within the duct walls to the lumens of the ducts. (I) A radial maturational lineage process begins near the fibromuscular layer and ends at the luminal surface of the both bile ducts and pancreatic ducts. (J) shows the magnified image from (I). Both show double immunofluorescence for EpCAM and Insulin. Insulin⁺ cells can be observed interspersed or in aggregates among glandular and ductal cells in the hepato-pancreatic ampulla, and undergoing commitment toward pancreatic endocrine fates. Insulin+ cells are mostly EpCAM+ (red arrows). EpCAM+/insulin- cells are also present (green arrow). Especially near the fibromuscular layer, the EpCAM- cells can be observed (white arrow) and that are negative also for insulin. (K) Immunofluorescence for Insulin in adult pancreas. Pancreatic islets are insulin positive and represent the positive control for the staining. Notably, cells of interlobular pancreatic duct are mostly insulin negative (white arrow). (L) Immunohistochemistry for EpCAM (brown) counterstained with PAS (pink) in hepato-pancreatic common duct. A radial maturational lineage can be observed also for maturation towards acinar cell. EpCAM-/PAS+ cells (pink arrows) are located at the luminal surface; EpCAM+/PAS+ cells (brown arrows) at the middle; and EpCAM-/PAS- cells (black arrow) very near the fibromuscular layer. Scale bars= 100 μm.

Figure 4. Comparison of biliary tree cells versus pancreatic cells under expansion culture conditions

The expansion conditions are culture plastic and serum-free Kubota’s Medium. Both biliary tree tissue and fetal and adult pancreas yield colonies initiated by a small number of cells. Biliary tree stem cell (hBTSC) colonies (A-C) divide initially about every 36-40 hours, and then slow to a division every 2-3 days, continuing to expand indefinitely for months and yielding large colonies, each containing over 500,000 cells by ∼8 weeks of culture. They vary in morphology from flattened, monolayer colonies to ones that are slightly 3-dimensional. Two types of colonies have been observed: those in which the colonies are EpCAM negative (B) but give rise to EpCAM⁺ cells at the edges, and those in which every cell, from the outset, expresses EpCAM (C). As noted in the phase image in A, the type I colonies are connected to the type 2 suggesting a precursor-descendent relationship. This was confirmed by the fact that all cells become EpCAM⁺ if inducers of differentiation are added. This indicates that the type I cells are giving rise to the type 2 cells. The cells from fetal pancreas yield colonies that behave as committed progenitors (D-K). If plated on culture plastic and in Kubota’s Medium (D, E, H), the cells are EpCAM⁺ from the outset and form colonies that initially look similar to those of hBTSCs, but they are essentially negative for C-peptide (H). Yet even when on culture plastic and in serum-free Kubota’s medium, they go through only 8-10 divisions and then begin to aggregate (F and G). They also become NGN3+ (Fig S5) that is accompanied by partial endocrine differentiation with expression of other endocrine markers such as C-peptide (I), glucagon (J) and somatostatin (K). Magnification: A-E (10X); F-K (20X). Scale Bars = 100 μm.

Figure 5. Formation of neoislet-like spheroids paralleled by endocrine differentiation

(A-B) Formation of neoislet-like spheroids occurs within a few days if cells are cultured in serum-free, hormonally defined medium designed to drive the cells towards a pancreatic islet fate(HDM-P) and embedded into hydrogels consisting of 40% hyaluronans and 60% type IV collagen/laminin mixture (1:1 ratio). (C) Hematoxylin/eosin staining of a neoislet demonstrates cord-like structures. Immunohistochemistry of cells at intermediate and late stages indicate that cells become positive for mature islet markers, including C-peptide (C-pep), glucagon (GCG), and somatostatin (SST) (D-G). (H) RT-PCR analyses corroborate the immunohistochemistry in a survey of genes expressed when cells are in Kubota’s Medium versus in HDM-P. See also online supplement Figs S4 and S5. Scale bars are as labeled on the different images.

Figure 6. Functional assays for the differentiated pancreatic progenitor cells

(A-D) Transmission electron microscopy (TEM) and immune-electron microscopy of pancreatic progenitor cells that derive from differentiation of cells to spheroids in culture. (A) The progenitor cells are tightly compacted (similar to findings with hBTSCs) and have a high nucleus to cytoplasmic ratio. (B-D) After differentiation, the cell interactions become looser, and the cells grow in size. The differentiated cells contain secretory granules, large amount of mitochondria and well-arranged endoplasmic reticulum generated in the cytoplasm. The granules proved positive for insulin in differentiated neoislets; the white arrows indicate gold particles bound to insulin. (E). Stimulated C-peptide secretion assay shows that undifferentiated progenitor cells (Undiff) secrete negligible amounts of human C-peptide, whereas differentiated neoislets released significantly higher amounts of C-peptide (significance levels equal to 0.01 to 0.001). The C-peptide released is regulatable as observed after incubation with Low (5.5 mM) versus High (16.7 mM) glucose concentrations or 100 μM TOL. (F) Electrophysiological recordings show electrical responsiveness in differentiated neoislet cells (Diff), but not in the undifferentiated (Undiff) pancreatic progenitor cells. Scale bars= 1 μm

Figure 7. Transplantation of neoislets can alleviate hyperglycemia in diabetic mice

(A-B) Representative body weight and non-fasting glucose levels in transplanted versus control mice indicate improvement in transplanted mice. The body weight decreased, while the blood glucose level increased gradually in all of the control mice. In the control group, 4 out of 7 mice died at or near post- operative day (POD) 60, when their blood glucose levels were above 600mg/dl for more than 20 days. The rest of the mice survived longer than 120 days by using the long-term insulin treatments. Body weight increased steadily, while the blood glucose level decreased and then persisted at ∼150-280mg/dl in the transplanted mice. In the transplanted mice, the blood glucose levels increased only after the epididymal grafts were explanted (see arrows). (C) Serum C-peptide was detected in transplanted mice at low levels on POD 37 and at higher levels on POD 60. On POD 60, the levels were also responsive to glucose administration (2g/kg weight) via IP. (D) A macroscopic view of the explanted epididymal fat pads two months after transplantation. Note the residual grey sutures remaining at the top of each of the testes. Hematoxylin and Eosin staining of the explanted epididymal fat pads show that the transplanted cells either fused into the fat pads loosely or grew as a solid mass (E-F). The graft area is well-vascularized. Immunofluorescence staining shows high levels of expression of human C-peptide (arrows) in the transplanted sites (G). Magnification, E-G (10X); H (20X). Scale bars are as labeled on the images.