Multipotent stem/progenitor cells in human biliary tree give rise to hepatocytes, cholangiocytes, and pancreatic islets§


  • Vincenzo Cardinale,

    1. Department of Cell and Molecular Physiology, Biomedical Engineering, Program in Molecular Biology and Biotechnology, UNC School of Medicine, Chapel Hill, NC, USA
    2. Division of Gastroenterology, University Sapienza of Rome, Division of Gastroenterology, Department of Scienze e Biotecnologie Medico-Chirurgiche, Fondazione Eleonora Lorillard Spencer Cenci, Polo Pontino, Rome, Italy, Department of Clinical Medicine, Polo Pontino, Rome, Italy
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    • These authors contributed equally to the study.

  • Yunfang Wang,

    1. Department of Cell and Molecular Physiology, Biomedical Engineering, Program in Molecular Biology and Biotechnology, UNC School of Medicine, Chapel Hill, NC, USA
    Current affiliation:
    1. Stem Cell and Regenerative Medicine Lab, Beijing Institute of Transfusion Medicine, Beijing, PR China, 100850
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  • Guido Carpino,

    1. Department of Health Sciences, University of Rome “Foro Italico”, Rome, Italy
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  • Cai-Bin Cui,

    1. Department of Surgery, UNC School of Medicine, Chapel Hill, NC, USA
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  • Manuela Gatto,

    1. Division of Gastroenterology, University Sapienza of Rome, Division of Gastroenterology, Department of Scienze e Biotecnologie Medico-Chirurgiche, Fondazione Eleonora Lorillard Spencer Cenci, Polo Pontino, Rome, Italy, Department of Clinical Medicine, Polo Pontino, Rome, Italy
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  • Massimo Rossi,

    1. “Paride Stefanini” Department of General Surgery and Organ Transplantation, Sapienza University of Rome, Rome, Italy
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  • Pasquale Bartolomeo Berloco,

    1. “Paride Stefanini” Department of General Surgery and Organ Transplantation, Sapienza University of Rome, Rome, Italy
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  • Alfredo Cantafora,

    1. Division of Gastroenterology, University Sapienza of Rome, Division of Gastroenterology, Department of Scienze e Biotecnologie Medico-Chirurgiche, Fondazione Eleonora Lorillard Spencer Cenci, Polo Pontino, Rome, Italy, Department of Clinical Medicine, Polo Pontino, Rome, Italy
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  • Eliane Wauthier,

    1. Department of Cell and Molecular Physiology, Biomedical Engineering, Program in Molecular Biology and Biotechnology, UNC School of Medicine, Chapel Hill, NC, USA
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  • Mark E. Furth,

    1. Wake Forest Institute of Regenerative Medicine, Winston Salem, NC, USA
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  • Luca Inverardi,

    1. Diabetes Research Institute, University of Miami, Miami, FL, USA
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  • Juan Dominguez-Bendala,

    1. Diabetes Research Institute, University of Miami, Miami, FL, USA
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  • Camillo Ricordi,

    1. Diabetes Research Institute, University of Miami, Miami, FL, USA
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  • David Gerber,

    1. Department of Surgery, UNC School of Medicine, Chapel Hill, NC, USA
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  • Eugenio Gaudio,

    1. Department of Health Sciences, University of Rome “Foro Italico”, Rome, Italy
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    • Coequal senior authors.

  • Domenico Alvaro,

    Corresponding author
    1. Division of Gastroenterology, University Sapienza of Rome, Division of Gastroenterology, Department of Scienze e Biotecnologie Medico-Chirurgiche, Fondazione Eleonora Lorillard Spencer Cenci, Polo Pontino, Rome, Italy, Department of Clinical Medicine, Polo Pontino, Rome, Italy
    • Division of Gastroenterology, University Sapienza of Rome, Division of Gastroenterology, Department of Scienze e Biotecnologie Medico-Chirurgiche, Fondazione Eleonora Lorillard Spencer Cenci, Polo Pontino, Rome, Italy
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    • Coequal senior authors.

    • fax: 011 39 06 4453319

  • Lola Reid

    Corresponding author
    1. Department of Cell and Molecular Physiology, Biomedical Engineering, Program in Molecular Biology and Biotechnology, UNC School of Medicine, Chapel Hill, NC, USA
    2. Wake Forest Institute of Regenerative Medicine, Winston Salem, NC, USA
    • Department of Cell and Molecular Physiology, RM 34 Glaxo, UNC School of Medicine, Chapel Hill, NC 27599
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    • These authors contributed equally to the study.

    • fax: 919-966-6112

  • Potential conflict of interest: Dr. Reid consults, received grants from, and holds intellectuals property rights for Vesta and GigaCyte. She also received grants from and holds intellectual property rights for Vertex.

  • Support: (UNC) Funding derived from a grant from the North Carolina Biotechnology Center (NCBC), GigaCyte Biotech (Branford, CT), Vesta Therapeutics (Bethesda, MD), and from NIH grants (AA014243, IP30-DK065933), NIDDK Grant (DK34987), and an NCI grant (CA016086); (Sapienza University) Dr. Cardinale received salary support from a scholarship from Sapienza University of Rome for the studies that he did at UNC. D. Alvaro and V. Cardinale were supported by FIRB grant no. RBAP10Z7FS_004; D. Alvaro, V. Cardinale, E. Gaudio, and G. Carpino were supported by a grant from Agenzia Regionale Del Lazio Per I Trapianti E Le Patologie Connesse; E. Gaudio was supported by MIUR grants: PRIN#2007, prot. 2007HPT7BA_001 and Federate Athenaeum funds from the University Sapienza of Rome; (Diabetes Research Institute) The studies were funded by grants from NIH, the Juvenile Diabetes Research Foundation, ADA, and the Diabetes Research Institute Foundation.

  • §

    Patent: A patent on the biliary tree stem cells was filed in November, 2009 and is jointly owned by UNC in Chapel Hill, NC, and Sapienza University in Rome, Italy.


Multipotent stem/progenitors are present in peribiliary glands of extrahepatic biliary trees from humans of all ages and in high numbers in hepato-pancreatic common duct, cystic duct, and hilum. They express endodermal transcription factors (e.g., Sox9, SOX17, FOXA2, PDX1, HES1, NGN3, PROX1) intranuclearly, stem/progenitor surface markers (EpCAM, NCAM, CD133, CXCR4), and sometimes weakly adult liver, bile duct, and pancreatic genes (albumin, cystic fibrosis transmembrane conductance regulator [CFTR], and insulin). They clonogenically expand on plastic and in serum-free medium, tailored for endodermal progenitors, remaining phenotypically stable as undifferentiated cells for months with a cell division initially every ≈36 hours and slowing to one every 2-3 days. Transfer into distinct culture conditions, each comprised of a specific mix of hormones and matrix components, yields either cords of hepatocytes (express albumin, CYP3A4, and transferrin), branching ducts of cholangiocytes (expressing anion exchanger-2-AE2 and CFTR), or regulatable C-peptide secreting neoislet-like clusters (expressing glucagon, insulin) and accompanied by changes in gene expression correlating with the adult fate. Transplantation into quiescent livers of immunocompromised mice results in functional human hepatocytes and cholangiocytes, whereas if into fat pads of streptozocin-induced diabetic mice, results in functional islets secreting glucose-regulatable human C-peptide. Conclusion: The phenotypes and availability from all age donors suggest that these stem/progenitors have considerable potential for regenerative therapies of liver, bile duct, and pancreatic diseases including diabetes. (HEPATOLOGY2011;)

The extrahepatic biliary tree contains a system of branching ducts connecting the liver to the intestine and plays a vital role in the passage of bile from liver to gut with the gallbladder operating as an overflow compartment and a site for removal of water, resulting in concentration of bile.1, 2 The ventral pancreas is connected to the gut by way of the hepato-pancreatic common duct, shared with the liver. Peribiliary glands (PBGs) are tubulo-alveolar glands found within the duct walls.3 The glands communicate with the bile duct lumens through channels opening into diverticula that occur with regularity around the mucosal surface.

Stem cells and progenitors have been identified and isolated from livers of all donor ages.4-6 They can be culture selected with a serum-free, hormonally defined medium, Kubota's medium (KM), supportive of hepatic progenitors but not of mature cells7 and can be driven to adult fates by specific mixes of systemic and paracrine signals8 and/or by biomatrix scaffolds.9 By contrast, numerous studies claim that there are no stem cells but only committed progenitors within adult pancreas.10, 11

Another source of progenitors is in the biliary tree. It was reported recently that gallbladder epithelial cells can differentiate into hepatocyte-like cells12 and that regeneration of extrahepatic bile ducts occurs with a bioabsorbable polymer tube within 11 weeks after surgical removal of the common bile duct in pigs.13 Also, it was shown that extrahepatic bile ducts in mice have β-cells,14 with secretory granules that are immunoreactive for insulin and that exhibit glucose-stimulated insulin secretion. Histological studies indicate that the β-cells form directly from the bile duct epithelium in late embryogenesis. Connections between biliary tree, liver, and pancreas have been made evident most recently by reports that SOX17 is a molecular “toggle” switch driving pancreas formation in one direction and the biliary tree in another15 and that SOX9-positive cells can be lineage-traced genetically in intestine, liver, and pancreas.16 Other investigations implicating the existence of common progenitors within the biliary tree for liver and pancreas are summarized in a recent review (Cardinale et al., submitted). Our studies corroborate and complement those prior findings and clarify further that the biliary tree, even in adults, is replete with multipotent cells that are stem cells, committed progenitors, or a mixture of these and able to lineage restrict to differentiated cells within liver, bile duct, and pancreas.


AFP, α-fetoprotein; ASMA, α-smooth muscle actin; CTFR, cystic fibrosis transmembrane conductance regulator; ES, embryonic stem cells; HDM, hormonally defined medium; hHpSCs, human hepatic stem cells; iSP, induced pluripotent stem cell; KM, Kubota's medium; MSC, mesenchymal stem cell; PBG, peribiliary gland; RT-PCR, reverse-transcription polymerase chain reaction; VEGFr, vascular endothelial cell growth factor receptor.

Materials and Methods

The materials and methods can be found in the online Supporting Information.


Peribiliary Glands Are Sites for Stem/Progenitors Within the Biliary Tree.

Hematoxylin and eosin staining (Fig. 1) of different regions of the biliary tree shows that peribiliary glands are found throughout the biliary tree but not in the gall bladder. Quantitative assessments of the numbers of peribiliary glands and their sizes indicate that the highest numbers are in the hepato-pancreatic common duct, and also in branching points in the biliary tree such as the cystic duct and common hepatic duct at the hilum. The percentages (within parentheses) are calculated as surface area occupied by all PBGs contained in the specimen (duct wall)/total area of the specimen.

Figure 1.

Distribution and characterization of PBGs in the extrahepatic biliary tree. (A) Histological images of PGBs in various regions of the biliary tree. (B) Density of PBGs, expressed as surface occupied by PBGs acini/total area as evaluated by imaging analysis; the number and circumference were histologically analyzed in the different sites. The hepato-pancreatic ampulla showed the highest density and number of PBGs; roughly equal numbers were found in cystic duct and hilum; fewer were found in bile duct; and none in gallbladder. The percentages are calculated as surface area occupied by all PBGs contained in the specimen (duct wall)/the total area of the specimen). Magnification 10×. See also online Supporting Figs. S2-S4. Immunohistochemistry of PBGs in situ shows that PBGs are positive for CK7, CK19, NCAM, CD133, EpCAM, SOX9, SOX 17, and PDX1 but negative (or very low levels) for albumin and insulin. Magnification 40×. Online Supporting Figure showing representative RT-PCR assays show for adult hepatic hilum indicating a broad repertoire of endodermal transcription factors (SOX9, SOX17, FOXA2, PDX1, NGN3, etc.) and classic stem cell surface markers (e.g., EpCAM, NCAM, CXCR4, CD133).

Immunohistochemistry and reverse-transcription polymerase chain reaction (RT-PCR) (Fig. 2; Supporting Fig. S2) on tissue samples (in situ) and from primary cultures of biliary tree tissue (Figs. 2, 3) show that there are cell populations expressing classic endodermal transcription factors (SOX17, SOX9, FOXA2, HNF6, PROX1, SALL4) and surface markers found on endodermal progenitors (CD326/EpCAM, CD56/NCAM, CD133, CXCR4). The biliary tree stem/progenitors expressed no or low levels of lineage markers of the liver (α-fetoprotein [AFP], albumin, gamma-glutamyltranspeptidase [GGT]) and endocrine pancreas (insulin, glucagon). Although not all of these markers are unique to endoderm the constellation is strongly characteristic, enabling us to hypothesize that the biliary tree contains either a common stem cell and/or a collection of committed progenitors for liver, bile duct, and pancreas. Until these options are defined, we refer to the cells as “stem/progenitors.” There are suggestions of a maturational lineage process from peribiliary glands deep within the bile ducts and ending at the duct lumen. With progression toward the luminal surface, there is a decease or loss of stem cell or progenitor markers in parallel with acquisition of mature markers of liver (e.g., albumin) in the portion of the biliary tree close to the liver (Fig. S5).

Figure 2.

The presence of early transcription factors (A) such as SOX17 and PDX1 occurs in peribiliary gland cells both in situ and in vitro. In Supporting Fig. S5 is shown the progression in changes of the gene expression with progression toward the luminal surface. There is a decease or loss of PDX1 expression, in parallel with acquisition of mature markers of liver (e.g., albumin). The early transcription factors (e.g., PDX1 and SOX17) expressed strongly in the nucleus in the stem cell colonies cultured in KM on plastic; magnification 20× in (A) and 10× in (B). (B) RT-PCR assays indicate the expression of diverse genes in the colonies from cystic duct versus from gall bladder. The gene expressions from the two tissues are quite similar, but those from cystic duct have weak expression of both albumin and insulin, an expression pattern not found in cells from the gallbladder.

Figure 3.

Dominant types of stem/progenitor cell colonies from adult biliary trees. Most of the colonies were one of two types. (in Supporting Fig. S6 are images from a third category). (A,B) Small, round, and tight cells in type 1 colonies have a high nucleus-to-cytoplasmic ratio and have phenotypic markers closely similar to those of hHpSCs and with 100% of the cells expressing EpCAM, NCAM, and none expressing AFP. (C,D) The type 2 colonies are comprised of undulating, swirling cells with EpCAM expression at the edges but not interiors of the colonies and with high levels of expression of SOX17, PDX1, or SOX9 in the interior cells. Magnification 20×.

Cultures of Biliary Tree Tissue Results in Selection for Stem/Progenitors.

Cell suspensions prepared from different regions of the biliary tree were plated onto culture plastic and in KM, a serum-free, hormonally defined medium (HDM) designed for hepatoblasts17 and also found effective for human hepatic stem cells (hHpSCs).5 Mature epithelial cells of liver, biliary tree, and pancreas do not survive in this medium. Colonies of clonogenically expanding cells were observed in cultures from all portions of the biliary tree, but higher numbers of colonies occurred in cultures from cystic duct, hilum, and hepato-pancreatic ampulla (Figs. 2, 3, S6, S7). The colonies formed in the selection conditions were phenotypically heterogeneous, with the centers of the colonies having smaller, more undifferentiated cells, and the edges of the colonies comprised of slightly more differentiated cells, including ones qualifying to be committed progenitors.

There were three types of colonies identified, arbitrarily named types 1-3. Cells in colony type I formed spheroids that grew slowly with divisions occurring every 3-4 days (Figs. 3, S6). Cells in the centers of type 2 colonies were small (7-9 μm), densely packed, uniform with high nucleus to cytoplasmic ratios (Figs. 2, 3, S7), and phenotypically essentially identical to those of intrahepatic hHpSCs. They doubled initially every 36-40 hours but slowed to a division every 2-3 days by 4 weeks in culture. Key features of the colony types 1 and 2 are that 100% of the cells expressed EpCAM, NCAM, CXCR4, CD133 and were negative for AFP and for markers of mature cell types. Cells in type 3 colonies consisted of flattened, swirling cells with phenotypic traits distinct at the edges versus in the middle of the colonies and with doubling times similar to those in type 2 colonies. Cells at the colony edges expressed EpCAM and either did not express endodermal transcription factors (e.g., SOX17) or these transcription factors were perinuclear; those in the colony centers expressed minimal, if any, EpCAM and yet contained strong expression of transcription factors both within the nuclei and/or perinuclearly. Figure 2 shows RT-PCR assays comparing the expression of early endodermal transcription factors (e.g., SOX17, HNF6, HES1, PDX1, NGN3, SALL4), and surface markers (e.g., EpCAM, CXCR4) and mature cell markers for colonies from cystic duct versus gall bladder.

The findings with respect to all colony types suggests that colony centers contained more primitive cells and those at the edges were slightly more differentiated. Cells transferred to differentiation conditions showed loss of EpCAM and acquisition of mature markers. With the colony type 3 cells the EpCAM was lost at the edges; acquired by cells interiorly; and finally, with full differentiation, loss of EpCAM altogether. Thus, EpCAM appears to be an intermediate marker of differentiation.

Cells in all three colony types were consistently negative by immunohistochemistry for mesenchymal markers such as desmin, α-smooth muscle actin (ASMA), markers of endothelia (e.g., CD31 and vascular endothelial cell growth factor receptor [VEGFr]), and hemopoietic markers (e.g., CD45, CD34) (data not shown).

The gallbladder does not contain peribiliary glands (Figs. 1, S4) but does have related cells with weaker levels of stem/progenitor markers, strong evidence of proliferative capacity (e.g., high expression of Ki67, Fig. S4) but less able to give rise to adult cell types other than those of the biliary tree (data not shown). Both RT-PCR and immunostaining data suggest that they might be transit amplifying cells, a hypothesis being tested.

Transcription Factors Are Located Both Intranuclearly and Perinuclearly.

The various transcription factors (e.g., SOX17, SOX9, PDX1) were found predominantly intranuclearly both in situ and in cultured cells (Figs. 2-4, S7). Within each peribiliary gland there was heterogeneous expression of transcription factors and of cytoplasmic and membrane-associated stem cell markers, with some cells positive and others negative. Although most of these markers were shared by cell populations from all biliary tree sites examined, there were distinctions in the relative expression of one versus another marker and in whether key transcription factors (e.g., SOX 17, PDX1) were located within the nucleus (Figs. 2, 4) or perinuclearly (Figs. 3, S7). A perinuclear localization occurred in some cells in situ and in cells at the edges of the type 3 colonies. The transition from intranuclear to perinuclear location is interpreted as sequestration and/or turnover of transcription factors accompanying differentiation events. Alternatively, the perinuclear localization could indicate that the factors are in an inactive storage form that can be activated by translocation to the nucleus under appropriate regenerative demands.

Figure 4.

In situ double immunofluorescence staining for SOX17 and PDX1 indicating coexpression in some cells (images represent PBGs within hepato-pancreatic ampulla). The percentage of SOX17+ and PDX1+ cells within PBGs is ≈10%-15% with SOX17+ cells constituting 11.2% ± 3.76% (standard deviation [SD]) and PDX1+ cells constituting the 16.6% ± 3.43% (SD). The heterogeneity in cellular subpopulations within the PBGs was not due to an artifact of sectioning, because the sections used were 3-5 μm thick and the cell diameters, in vitro and in vivo, is 7-9 μm. The analysis and the images were made with a wide field fluorescence microscope and not with a confocal microscope. Therefore, most of the nucleus is displayed in a single section with the signal representing the amount of antigen present in most of the nucleus.

Interestingly, there were also cells coexpressing multiple transcription factors such as SOX17 and PDX1 (Fig. 4). The percentage of SOX17+ cells is 11.2% ± 3.8% and the PDX1+ cell is the 16.6% ± 3.4% and the percentage of the cells coexpressing SOX17 and PDX1 was variable but ranged from 10%-15%. This coexpression in some cells and, similarly, expression of multiple transcription factors relevant to liver and pancreas (e.g., HNF6, NGN3) in some peribiliary glands is a unique feature that is distinctive from findings with respect to embryonic stem (ES) cells lineage restricted to liver or pancreas and in which specific genes turn on (and then off) in stages. Marker analyses completed to date of biliary tree stem/progenitors indicate they are stages between definitive endoderm and determined stem cells and mostly at lineage stage 4 in the development of the endocrine pancreas or of the liver from ES cells.18

Expansion Potential of the Biliary Tree Stem/Progenitor Cells.

Cultures of the biliary tree tissue on plastic and in serum-free KM resulted in selection for colonies of cells that divided initially every 36-40 hours, thereafter slowing to a division every ≈2-3 days, with proliferation continuing for months and associated with stable maintenance of the undifferentiated cell phenotype (Table S2). Figure S8 shows a representative colony maintained for more than 8 weeks on culture plastic and in KM. Cells in the colony centers (regions a and b) had an average cell diameter of ≈6-7 μm, whereas those at the colony edges were larger (≈11-12 μm) (regions c-e). We sampled sufficient regions to enable us to have an estimate of ≈400,000-500,000 cells for the entire colony by Metamorph analyses; this was an underestimate given the dense aggregates of cells in some regions of the colony. Similar analyses were done on other colonies at 4-8 weeks of culture (Table S3) and indicated that the cells went through divisions every ≈3 days such that by 2 months they had gone through ≈18-20 divisions.

Biliary Tree Stem Cell/Progenitors Lineage Restrict Either to Hepatocytes, Bile Ducts, or Pancreatic Islets Depending on the Microenvironment.

The biliary tree stem/progenitors were maintained for 4-8 weeks in an undifferentiated state in culture on plastic and in KM resulting in 100% of the cells of colony types 1 and 2 and ≈20%-30% of those in colony type 3 being positive for EpCAM. At the timepoint of transfer to culture conditions other than KM and plastic, the cells in all colony types contained cells strongly expressing markers of stem cells (e.g., CXCR4, SOX9, SOX17, PDX1, CD133) and negligible levels of expression of genes indicative of mature cells (e.g., albumin, secretin receptor, insulin). The potential adult fates of biliary tree stem/progenitors were realized by passaging equal numbers of them from cultures in KM into one of three distinct differentiation conditions tailored either for liver, bile duct, or pancreatic islets. Each condition was comprised of a serum-free HDM tailored for the adult tissue of interest: HDM-L (hepatocytes), HDM-C (cholangiocytes), and HDM-P (pancreatic islets). For the 2D cultures the cells were plated onto culture plastic and in just HDM; for the 3D cultures the specific HDM was used in combination with embedding the cells into a mixture of extracellular matrix components also tailored for the desired adult cell type. Passaging the cells again onto plastic and in KM resulted in self-replication, conditions used as the stem cell (SC) controls.

Lineage Restriction Yielding Hepatocytes.

Cells with hepatocyte markers did not occur in the SC control conditions. The numbers of cells coexpressing CK18 and albumin increased to 36.7% ± 10.4% in 2D (monolayer) cultures in HDM-L (Fig. S9A-C), and present mostly at the periphery of the colony, whereas colony centers consisted primarily of undifferentiated cells (negative for albumin and positive for EpCAM; data not shown).

In the HDM-L and embedded into matrix in 3D, cords of cuboidal-shaped cells with ultrastructural and functional features of hepatocytes were observed (Figs. 5, 6, S10) accompanied by significant increases in hepatocyte-specific gene expressions that included early (e.g., HNFα4, AFP, CK8 and 18, and albumin), intermediate or zone 2 (e.g., transferrin, tyrosine aminotransferase [TAT]), and late or zone 3 genes (e.g., P450 3A4) (Fig. 7).

Figure 5.

Differentiation toward a pancreatic islet fate in cells on plastic and in HDM-P. When the biliary tree stem cells were cultured as monolayers in HDM-P the cultures changed within a week to have cell aggregation and condensation at the edges of the colonies, resulting in the formation of islet-like structures containing C-peptide (B,C) insulin (D), and PDX-1 (not shown). (A) Undifferentiated cells (EpCAM+ cells) were found within the colony centers. Magnification 20×. The number of islet-like structures/colony increased from 1 ± 0.7 to 3.8 ± 1.3 in the cultures in HDM-P. (E) The levels of human C-peptide in ng/μg of protein at low (basal) glucose concentration (5.5 mM) for a 2-hour incubation in cultures in KM was 4.5 ± 2.25 versus 12.3 ± 1.9 ng/μg of protein in the cultures in HDM-P. The data are expressed as means ± standard error; n = 4; *P < 0.05. Glucose stimulated C-peptide secretion (F) was observed in the HDM-P with the levels of human C-peptide in the medium at 1.10 ± 0.32 μg/L under low (basal) glucose concentration (5.5 mM) to 1.92 ± 0.43 μg/L in high glucose concentration (22 mM); n = 7; *P < 0.01.

Figure 6.

Adult fates of biliary tree stem cells when given a specific HDM and embedded in mixtures of extracellular matrix components. (A) Macroscopic images of branching and ramifying bile duct formation lined by layers of epithelial cells within 7-9 days in cultures of biliary tree stem cells under 3D conditions in MKM-C show the differentiation toward cholangiocytes. Magnifications are 10× and 20× for (A,B), respectively. (C,D) After 9 days of restriction under MKM-L conditions the differentiation toward hepatocytes, cords of cuboidal cells formed and were interspersed with bile canaliculi. Magnification 20× (see also Fig. S10 for ultrastructural images of the cells under the stem cell control condition versus this 3D differentiation condition). (E-G) After 7-14 days of differentiation toward a pancreatic islet fate, islet-like structures are observed budding from the surface of the ball of cells and expressed PDX1 (pink) and C-peptide (blue/green). The nuclei are blue from DAPI staining. (E) Higher magnification images (20×) show two of the islet-like structures that formed at the edges of the hydrogels at day 5 and day 7 in the 3D differentiation conditions. The aggregates became positive for dithizone staining by day 7, indicating expression of insulin.

Figure 7.

Proof of multipotentiality as assessed by gene expression. Quantitative RT-PCR assays were done on biliary tree stem/progenitors maintained under self-replication conditions (SC control) versus in one of three, distinct serum-free, hormonally defined medium (MKM-L, MKM-C, or MKM-P) and either on culture plastic or used in combination with embedding the cells into a specific combination of extracellular matrix components. The mRNA relative expression levels were calculated by normalization of ΔΔCt values against the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The expression levels of a given gene under self-replication conditions are indicated as 1.0 and the levels under the other conditions are the fold difference. Below are summarized the conditions assayed; the number in parentheses is the number of times the experiment was done. 2D (monolayer) cultures on tissue culture plastic and in (A) KM (13×); (B) HDM-L: albumin assays (9×); (C) HDM-P: insulin assays (13×); (D) HDM-C: secretin receptor (6×). 3D cultures of cells embedded in mixtures of extracellular matrix components and in (E) HDM-C (3×); (F) HDM-L (3×); or (G) HDM-P (3×). The genes assayed were GGT1: gamma glutamyl transpeptidase-1; AE2: anion exchanger 2; CFTR: cystic fibrosis transmembrane conductance regulator; HNF4a: hepatocyte nuclear factor 4A; AFP: alpha-fetoprotein; ALB: albumin; TF: transferrin; TAT: tyrosine aminotransferase; CYP3A4: cytochrome P450 3A4; pdx1: Pancreatic and duodenal homeobox 1; ISL-1: ISL LIM homeobox 1; NGN3: neurogenin 3; INS: insulin; GCG: glucagon.

Lineage Restriction to Bile Ducts Lined with Cholangiocytes.

The presence of cells expressing markers of cholangiocytes (CK7, secretin receptor [SR], and CFTR) occurred minimally in the SC control conditions with an average of 3.2% ± 2.6% positive cells found in each colony. In cells on plastic and in HDM-C, clusters of cells coexpressing CK7, SR, and CFTR were observed concentrated at the periphery of the colonies and their numbers increased to 49.2% ± 11.1% of the cells/colony (Fig. S9). The colony centers remained primarily as undifferentiated cells (negative for SR and CFTR and positive for EpCAM; data not shown). Quantitative RT-PCR of the cultures in the SC controls versus those in HDM-C indicated that SR messenger RNA (mRNA) more than doubled (P < 0.05). The effect was even more profound in the cells embedded into matrix and given HDM-C, resulting in ramifying and branching ducts formation, lined by cells with a phenotype of mature cholangiocytes (Fig. 8C). Quantitative RT-PCR indicated significantly higher levels of expression of cholangiocyte-specific genes for GGT, CFTR, and AE-2 (Fig. 7).

Figure 8.

Adult fates of the stem/progenitor cells as indicated by transplantation in vivo in immunocompromised mouse. (A,B) In the liver sections prepared from the hosts, around 6.52% ± 2.5% of the total area was occupied by transplanted cells which were positive for human HepPar-1 (arrows). Magnification 20×. (B) Double immunofluorescence revealed the colocalization of human HepPar-1 and human albumin. Magnification 20×. (C) Immunohistochemistry of liver sections for human specific CK7 (arrows) indicative of cholangiocytes shows on average 12.7% ± 5.5% of all bile duct cells are derived from transplanted stem cells. It was found that ≈14.92% ± 5.9% cells in the large bile ducts and ≈5.02% ± 1.95% of the cells in small bile ducts were positive for human CK7. No immunoreactions were observed in controls (n = 6). Magnification 40×. (D) The epididymal fat pads (EFP) of male Balb/C Rag2−/−/Il2rg−/− mice were injected with 200-400 preinduced, biliary tree stem/progenitor derived cell aggregates in the 3D differentiation conditions. Glucose tolerance tests performed at postoperative days 68 and 91 showed significant blood levels of human C-peptide in experimental mice and these levels were regulatable by glucose.

Lineage Restriction to Functional Pancreatic Islets.

Formation of islet-like structures increased significantly both in cultures on plastic and in HDM-P and when embedded into matrix and given HDM-P. The monolayers in HDM-P produced dense balls of aggregated cells budding from the edges of the colonies and containing cells expressing C-peptide, PDX1, and insulin. Four to five such aggregate-structures appeared on average in cells on plastic and in HDM-P; secreted human C-peptide could be detected especially in cultures stimulated with high glucose levels (Fig. 5). In cultures embedded into matrix and given HDM-P, the islet-like clusters occurred at the edges of the hydrogels (pale blue structures) and were positive for dithizone staining, indicating cells with zinc condensed in insulin granules in the cytoplasm (Fig. 6). Immunohistochemistry indicated that these neoislet-like structures were positive for PDX1, human C-peptide, and insulin as well as for glucagon and somatostatin (data not shown). The quantitative RT-PCR assays (Fig. 7) of these cultures were the most dramatic in the elevation of expression of genes specific for pancreatic endocrine cells.

Transplantation of Biliary Tree Stem/Progenitor Cells In Vivo Results in Distinct Fates Depending on Site and Microenvironment.

To explore whether biliary tree stem cells have the ability to differentiate into mature liver cell type in vivo, we transplanted isolated biliary tree stem/progenitors directly into the livers of normal SCID mice (n = 3) and checked for their fate(s) 30 days later. The mice were not subjected to any injury process, meaning that the engraftment occurred under quiescent liver conditions. Human cells were found around injection sites and some were dispersed into the liver sinusoids of periportal and intralobular parenchyma. We estimate that an average of 6.52% ± 2.5% of the total area of the hepatic lobes was occupied by mature human hepatocytes positive for human HepPar-1 and albumin (Fig. 8). Moreover, the bile ducts were lined with a high percentage (average 12.7% ± 5.5%) of mature human cholangiocytes, positive for human CK7 (Fig. 8), meaning that human cholangiocytes coexisted with human hepatocytes within areas of liver parenchyma in the hosts. Human cells were not observed in sham-transplanted animals, and no tumors formed in any of the transplanted animals.

To assess ability to form functional pancreatic endocrine cells in vivo, biliary tree stem/progenitors lineage restricted partially toward a pancreatic islet fate were transplanted in Rag2−/−/Il2rg−/− mice that were subsequently rendered diabetic by treatment with streptozocin (STZ). The mice not subjected to STZ maintained normal glucose levels throughout the experiments. The sham controls given STZ became hyperglycemic and within 2 weeks had glucose levels at > 750 mg/dL. These controls maintained high levels of hyperglycemia for the duration of the experiments and some of them died at around 100 days. By contrast, the glucose levels in STZ-treated mice and transplanted with preinduced neoislet clusters remained high (>750 mg/dL) for ≈2 months and then declined steadily. By day 102 the glucose levels were less than half that of the controls. All of these mice survived, and there was no tumor formation in any of them. Significant levels of human C-peptide were detected at postoperative days 68 and 91 in the serum of hosts transplanted but not control or sham control mice (P < 0.001). The human C-peptide levels in vivo were regulatable by glucose challenge (Fig. 8).


Peribiliary glands are stem cell niches of the biliary tree and compare with and are related to intrahepatic stem cell niches in ductal plates of fetal and neonatal livers and canals of Hering in pediatric and adult livers.4, 5, 19, 20 They start at the level of intrahepatic septal bile ducts, implicating these as additional intrahepatic stem cell niches, corroborating the findings of Theise et al.19 These multipotent stem cells, located in peribiliary glands deep within the bile duct walls, express markers for endodermal stem cells and can migrate to appropriate sites and differentiate into various adult cells, contributing to the renewal/repair of biliary epithelium and also of liver and pancreas. Given that cells and the differentiation phenomena are found in biliary tree tissue from fetal, pediatric, adult, and geriatric donors, facets of organogenesis of liver, biliary tree, and pancreas appear to be ongoing throughout life.

The gallbladder does not contain peribiliary glands, but it does have related cells that possibly represent facultative progenitors. This proposal parallels the intestinal model in which proliferation of stem cells within Lieberkuhn's crypts is followed by cell migration and differentiation along the crypt-villus axis and is critical for development of the intestinal architecture.21

SOX17 is important for endodermal progenitors switching between biliary tree and pancreas,15 is associated with hedgehog proteins known also as important for liver versus pancreas differentiation, and is associated with primary cilia.22 We assume this is relevant to the SOX17 evident in the biliary tree stem/progenitors, but its relevance is not yet fully understood.

Cultures of the biliary tree stem/progenitors were obtained readily in KM, a serum-free, defined medium developed for rodent hepatoblasts and subsequently found effective for hepatic stem cells.5 In these conditions the biliary tree stem/progenitors remained phenotypically stable and underwent a division initially every 36-40 hours, but slowed to a division every ≈3 days by 4 weeks, and at all times assayed thereafter had a proliferative rate comparable to that found for hHpSCs.5 Under all conditions and with all the types of stem/progenitors identified, the edges of the colonies contained cells that were larger and more differentiated than those in the colony interiors mimicking the formation of liver tissue from ductal plates4 and that of islets from the edges of pancreatic ducts during organogenesis.11

Biliary tree stem/progenitors are logical precursors for mature cells of liver, bile duct, and pancreas given known events in organogenesis and in studies on pathologies of these tissues.6 They overcome many, if not all, of the ongoing controversies about whether or not there are stem cells in adult tissues for pancreas.11 The inability to identify true stem cells in adult pancreas10, 11, 23, 24 has fueled attempts to lineage restrict ES cells, induced pluripotent stem (iPS) cells, amniotic fluid-derived stem cells (AFSCs), or mesenchymal stem cells (MSCs), often with genetic manipulation to direct these cells to an endodermal fate; to find ways to reprogram adult pancreatic acinar cells; or to elicit proliferation of existing pancreatic islet beta cells.11, 25, 26 The process of driving these various stem cell populations to a mature endodermal tissue fate is inefficient, requiring up to 4-6 weeks in culture, and yielding adult cells with muted functions or with overexpression of or absence of expression of some genes. In the cases of the MSCs, the resulting adult cells are phenotypic hybrids of mesenchymal cells and the desired adult cell type.27 Moreover, for reasons unknown, the phenotype of the adult cells generated is distinct with every preparation and source. Fully mature hepatocytes or glucose-regulated β-cells from these precursors occur only with in vivo maturation of transplanted cells after several months. Moreover, any therapy based on ES or iPS cells may be limited by the potential of teratoma formation, usually ascribed to residual undifferentiated stem cells.28, 29

Our findings that there are endodermal stem/progenitors in the biliary trees of all donor ages; that they clonogenically expand in vitro under wholly defined conditions; and that they readily and efficiently lineage restrict to liver, biliary tree, or pancreatic adult fates in culture with sets of wholly definable microenvironmental cues or in vivo suggest that they will become preferred choices for clinical programs and most experimental studies. The speed at which the differentiation occurs, even in culture, is an indication that these biliary tree stem/progenitors have progressed through most of the developmental stages for a liver or pancreatic fate. Indeed, their phenotypic characteristics in situ and in vitro indicate that they are mostly at stage 4 of the 5 stages during the progress of human ES cell stepwise-differentiation toward an islet fate.18 The other factor in the speed of the response is that we utilized a 3D microenvironment comprised of both a serum-free HDM and a specific mixture of matrix molecules; both the soluble and matrix signals were tailored for different adult cell types. The microenvironment drives the cells rapidly to a specific adult fate that can be easily identified by morphology and functions.

Further differentiation occurs with or without selective pressure in vivo. That for lineage restriction toward the intrahepatic parenchymal cell fates resulted in rapid differentiation, because the in vivo environment is toward integration of the cells within the liver plates followed by differentiation and with minimal adverse variables. By contrast, that for pancreatic islets in hosts with STZ-induced diabetes involved selective pressure for differentiation in combination with adverse effects of the toxic environment induced by hyperglycemia. Blood vessels can become narrowed or blocked by a buildup of fat and cholesterol due to abnormal glucose metabolism and poor nutrient conditions in hyperglycemic environment. Transplanted precursors for pancreatic islets can take months to fully mature given this mix of positive and negative effects resulting from hyperglycemia, as has been shown by others.28 Thus, the speed of differentiation achieved in vitro versus in vivo is distinct for each adult fate being generated and dependent on the balance of positive and adverse effects of the in vivo microenvironment.

We interpret the survival and rescue of the transplanted mice from hyperglycemia as due to the production of human insulin. One cannot exclude the possibility that some degree of spontaneous regeneration of the mouse pancreas might have occurred, resulting in the regeneration of endogenous islets releasing murine insulin, but even if this occurred it was insufficient to rescue the sham controls. The controls subjected to STZ maintained glucose levels above 750 mg/dL throughout the experiments, and some of them died. One cannot consider the option of formation of hybrids between host pancreatic islet cells and the biliary tree-derived islet-like clusters, because transplantation was done into the fat pads. The strongest evidence is that the dramatic reduction in hyperglycemia in the experimental mouse correlated with increasing levels of human insulin/C-peptide in their blood, and that the levels were regulatable by glucose injected into the mice. The extent of response observed seems logical given that low numbers of partially differentiated neoislet-like clusters were implanted; given the poor nutrient environment for the grafts due to the high blood glucose levels; and given that mouse cells are far less sensitive to human insulin than mouse insulin. We assume that a normoglycemic state would be achieved within the time frame we used if higher numbers of neoislets were transplanted.

Our observations have pathophysiologic implications given that peribiliary glands, as stem cell niches, might play a role in injury repair as sites of origin of inflammatory (primitive sclerosing cholangitis) or neoplastic (cholangiocarcinoma) diseases, paralleling the findings of Thayer and associates30 for pancreatic duct glands and the extensive studies by Bonner-Weir et al.11 on candidate progenitors in pancreatic ducts. It is of interest that peribiliary glands have the highest density at the hepato-pancreatic ampulla and common hepatic duct at the hilum, sites at which cholangiocarcinomas typically occur.31 The common embryologic origin of intestine and biliary tree opens new perspectives on certain pathologies such as ulcerative colitis and sclerosing cholangitis or in the similarities between colorectal adenocarcinoma and cholangiocarcinoma.32

Comparisons of progenitor stem/populations in biliary tree versus pancreas could provide explanations for the known distinctions in regenerative capacity of liver versus pancreas and could reveal if, as we suspect, organogenesis of liver and pancreas is ongoing throughout life. These speculations are to be addressed with future studies. Biliary tree tissue is available from fetal, neonatal, pediatric, and adult organs, including surgical materials (e.g., from cholecystectomy), tissue routinely discarded from donor livers (gallbladder, cystic ducts, periampular region), or pancreata (periampular region, bile duct) rejected for use in transplantation and made available for research. Thus, the extrahepatic biliary tree is an ideal and available source of stem/progenitor cells useful for regenerative medicine programs for liver, bile duct, and pancreas, including for treatment of diabetes.