Multipotent Stem/Progenitor Cells in Human Biliary Tree Give Rise to Hepatocytes, Cholangiocytes, and Pancreatic Islets
Vincenzo Cardinale,1,2* Yunfang Wang,1* Guido Carpino,3 Cai-Bin Cui,4 Manuela Gatto,2 Massimo Rossi,5 Pasquale Bartolomeo Berloco,5 Alfredo Cantafora,2 Eliane Wauthier,1 Mark E. Furth,6 Luca Inverardi,7 Juan Dominguez-Bendala,7 Camillo Ricordi,7 David Gerber,4 Eugenio Gaudio,3*,† Domenico Alvaro,2*,† and Lola Reid1,6*,†
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. (HEPATOLOGY 2011;54:2159-2172)
he extrahepatic biliary tree contains a system of biliary glands (PBGs) are tubulo-alveolar glands found branching ducts connecting the liver to the within the duct walls.3 The glands communicate with Tintestine and plays a vital role in the passage of the bile duct lumens through channels opening bile from liver to gut with the gallbladder operating as into diverticula that occur with regularity around the an overflow compartment and a site for removal of mucosal surface. water, resulting in concentration of bile.1,2 The ventral Stem cells and progenitors have been identified and pancreas is connected to the gut by way of the hepato- isolated from livers of all donor ages.4-6 They can be pancreatic common duct, shared with the liver. Peri- culture selected with a serum-free, hormonally defined
Abbreviations: AFP, a-fetoprotein; ASMA, a-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. From the 1Department of Cell and Molecular Physiology, Biomedical Engineering, Program in Molecular Biology and Biotechnology, UNC School of Medicine, Chapel Hill, NC, USA; 2Division 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; 3Department of Health Sciences, University of Rome ‘‘Foro Italico’’, Rome, Italy; 4Department of Surgery, UNC School of Medicine, Chapel Hill, NC, USA; 5‘‘Paride Stefanini’’ Department of General Surgery and Organ Transplantation, Sapienza University of Rome, Rome, Italy; 6Wake Forest Institute of Regenerative Medicine, Winston Salem, NC, USA; and 7Diabetes Research Institute, University of Miami, Miami, FL, USA. Received May 14, 2011; Revised July 14, 2011; accepted July 20, 2011.
2159 2160 CARDINALE ET AL. HEPATOLOGY, December 2011 medium, Kubota’s medium (KM), supportive of Materials and Methods hepatic progenitors but not of mature cells7 and can be driven to adult fates by specific mixes of systemic The materials and methods can be found in the and paracrine signals8 and/or by biomatrix scaffolds.9 online Supporting Information. By contrast, numerous studies claim that there are no Results stem cells but only committed progenitors within adult 10,11 pancreas. Peribiliary Glands Are Sites for Stem/Progenitors Another source of progenitors is in the biliary tree. Within the Biliary Tree. Hematoxylin and eosin stain- It was reported recently that gallbladder epithelial cells ing (Fig. 1) of different regions of the biliary tree shows 12 can differentiate into hepatocyte-like cells and that that peribiliary glands are found throughout the biliary regeneration of extrahepatic bile ducts occurs with a bio- tree but not in the gall bladder. Quantitative assessments absorbable polymer tube within 11 weeks after surgical of the numbers of peribiliary glands and their sizes indi- 13 removal of the common bile duct in pigs. Also, it was cate that the highest numbers are in the hepato-pancreatic b shown that extrahepatic bile ducts in mice have - common duct, and also in branching points in the biliary 14 cells, with secretory granules that are immunoreactive tree such as the cystic duct and common hepatic duct at for insulin and that exhibit glucose-stimulated insulin the hilum. The percentages (within parentheses) are cal- b secretion. Histological studies indicate that the -cells culated as surface area occupied by all PBGs contained in form directly from the bile duct epithelium in late the specimen (duct wall)/total area of the specimen. embryogenesis. Connections between biliary tree, liver, Immunohistochemistry and reverse-transcription po- and pancreas have been made evident most recently by lymerase chain reaction (RT-PCR) (Fig. 2; Supporting reports that SOX17 is a molecular ‘‘toggle’’ switch driv- Fig. S2) on tissue samples (in situ) and from primary ing pancreas formation in one direction and the biliary cultures of biliary tree tissue (Figs. 2, 3) show that 15 tree in another and that SOX9-positive cells can be there are cell populations expressing classic endodermal lineage-traced genetically in intestine, liver, and pan- transcription factors (SOX17, SOX9, FOXA2, HNF6, 16 creas. Other investigations implicating the existence of PROX1, SALL4) and surface markers found on endo- common progenitors within the biliary tree for liver and dermal progenitors (CD326/EpCAM, CD56/NCAM, pancreas are summarized in a recent review (Cardinale CD133, CXCR4). The biliary tree stem/progenitors et al., submitted). Our studies corroborate and comple- expressed no or low levels of lineage markers of the ment those prior findings and clarify further that the liver (a-fetoprotein [AFP], albumin, gamma-glutamyl- biliary tree, even in adults, is replete with multipotent transpeptidase [GGT]) and endocrine pancreas (insu- cells that are stem cells, committed progenitors, or a lin, glucagon). Although not all of these markers are mixture of these and able to lineage restrict to differenti- unique to endoderm the constellation is strongly char- ated cells within liver, bile duct, and pancreas. acteristic, enabling us to hypothesize that the biliary
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. *These authors contributed equally to the study. †Coequal senior authors. Current address for Yunfang Wang: Stem Cell and Regenerative Medicine Lab, Beijing Institute of Transfusion Medicine, Beijing, PR China, 100850. Address reprint requests to: Lola M. Reid, Department of Cell and Molecular Physiology, RM 34 Glaxo, UNC School of Medicine, Chapel Hill, NC 27599; [email protected]; fax: 919-966-6112 or Domenico Alvaro, 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; [email protected], fax: 011 39 06 4453319. Copyright VC 2011 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.24590 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. Additional Supporting Information may be found in the online version of this article. HEPATOLOGY, Vol. 54, No. 6, 2011 CARDINALE ET AL. 2161
Fig. 1. Distribution and characterization of PBGs in the extrahepatic biliary tree. (A) Histological images of PGBs in various regions of the bili- ary tree. (B) Density of PBGs, expressed as surface occupied by PBGs acini/total area as evaluated by imaging analysis; the number and cir- cumference 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 calcu- lated 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 repre- sentative 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). tree contains either a common stem cell and/or a col- and also found effective for human hepatic stem cells lection of committed progenitors for liver, bile duct, (hHpSCs).5 Mature epithelial cells of liver, biliary tree, and pancreas. Until these options are defined, we refer and pancreas do not survive in this medium. Colonies to the cells as ‘‘stem/progenitors.’’ There are sugges- of clonogenically expanding cells were observed in tions of a maturational lineage process from peribiliary cultures from all portions of the biliary tree, but glands deep within the bile ducts and ending at the higher numbers of colonies occurred in cultures from duct lumen. With progression toward the luminal sur- cystic duct, hilum, and hepato-pancreatic ampulla face, there is a decease or loss of stem cell or progeni- (Figs. 2, 3, S6, S7). The colonies formed in the selec- tor markers in parallel with acquisition of mature tion conditions were phenotypically heterogeneous, markers of liver (e.g., albumin) in the portion of the with the centers of the colonies having smaller, biliary tree close to the liver (Fig. S5). more undifferentiated cells, and the edges of the Cultures of Biliary Tree Tissue Results in Selection colonies comprised of slightly more differentiated for Stem/Progenitors. Cell suspensions prepared from cells, including ones qualifying to be committed different regions of the biliary tree were plated onto progenitors. culture plastic and in KM, a serum-free, hormonally There were three types of colonies identified, arbi- defined medium (HDM) designed for hepatoblasts17 trarily named types 1-3. Cells in colony type I formed 2162 CARDINALE ET AL. HEPATOLOGY, December 2011
Fig. 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. spheroids that grew slowly with divisions occurring colonies and with doubling times similar to those in every 3-4 days (Figs. 3, S6). Cells in the centers of type type 2 colonies. Cells at the colony edges expressed 2 colonies were small (7-9 lm), densely packed, uni- EpCAM and either did not express endodermal tran- form with high nucleus to cytoplasmic ratios (Figs. 2, 3, scription factors (e.g., SOX17) or these transcription S7), and phenotypically essentially identical to those of factors were perinuclear; those in the colony centers intrahepatic hHpSCs. They doubled initially every 36- expressed minimal, if any, EpCAM and yet contained 40 hours but slowed to a division every 2-3 days by 4 strong expression of transcription factors both within weeks in culture. Key features of the colony types 1 and the nuclei and/or perinuclearly. Figure 2 shows RT-PCR 2 are that 100% of the cells expressed EpCAM, NCAM, assays comparing the expression of early endodermal CXCR4, CD133 and were negative for AFP and for transcription factors (e.g., SOX17, HNF6, HES1, markers of mature cell types. Cells in type 3 colonies PDX1, NGN3, SALL4), and surface markers (e.g., consisted of flattened, swirling cells with phenotypic EpCAM, CXCR4) and mature cell markers for colonies traits distinct at the edges versus in the middle of the from cystic duct versus gall bladder. HEPATOLOGY, Vol. 54, No. 6, 2011 CARDINALE ET AL. 2163
Fig. 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 .
The findings with respect to all colony types sug- The gallbladder does not contain peribiliary glands gests that colony centers contained more primitive cells (Figs. 1, S4) but does have related cells with weaker and those at the edges were slightly more differenti- levels of stem/progenitor markers, strong evidence of ated. Cells transferred to differentiation conditions proliferative capacity (e.g., high expression of Ki67, showed loss of EpCAM and acquisition of mature Fig. S4) but less able to give rise to adult cell types markers. With the colony type 3 cells the EpCAM was other than those of the biliary tree (data not shown). lost at the edges; acquired by cells interiorly; and Both RT-PCR and immunostaining data suggest that finally, with full differentiation, loss of EpCAM alto- they might be transit amplifying cells, a hypothesis gether. Thus, EpCAM appears to be an intermediate being tested. marker of differentiation. Transcription Factors Are Located Both Intranu- Cells in all three colony types were consistently neg- clearly and Perinuclearly. The various transcription ative by immunohistochemistry for mesenchymal factors (e.g., SOX17, SOX9, PDX1) were found pre- markers such as desmin, a-smooth muscle actin dominantly intranuclearly both in situ and in cultured (ASMA), markers of endothelia (e.g., CD31 and vas- cells (Figs. 2-4, S7). Within each peribiliary gland cular endothelial cell growth factor receptor [VEGFr]), there was heterogeneous expression of transcription and hemopoietic markers (e.g., CD45, CD34) (data factors and of cytoplasmic and membrane-associated not shown). stem cell markers, with some cells positive and others 2164 CARDINALE ET AL. HEPATOLOGY, December 2011
Fig. 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 SOX17þ and PDX1þ cells within PBGs is 10%-15% with SOX17þ cells constituting 11.2% 6 3.76% (standard deviation [SD]) and PDX1þ cells constituting the 16.6% 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 lm thick and the cell diameters, in vitro and in vivo,is 7-9 lm. 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. negative. Although most of these markers were shared multiple transcription factors relevant to liver and pan- by cell populations from all biliary tree sites examined, creas (e.g., HNF6, NGN3) in some peribiliary glands there were distinctions in the relative expression of one is a unique feature that is distinctive from findings versus another marker and in whether key transcrip- with respect to embryonic stem (ES) cells lineage tion factors (e.g., SOX 17, PDX1) were located within restricted to liver or pancreas and in which specific the nucleus (Figs. 2, 4) or perinuclearly (Figs. 3, S7). genes turn on (and then off) in stages. Marker analyses A perinuclear localization occurred in some cells completed to date of biliary tree stem/progenitors indi- in situ and in cells at the edges of the type 3 colonies. cate they are stages between definitive endoderm and The transition from intranuclear to perinuclear loca- determined stem cells and mostly at lineage stage 4 in tion is interpreted as sequestration and/or turnover of the development of the endocrine pancreas or of the transcription factors accompanying differentiation liver from ES cells.18 events. Alternatively, the perinuclear localization could Expansion Potential of the Biliary Tree Stem/ indicate that the factors are in an inactive storage form Progenitor Cells. Cultures of the biliary tree tissue that can be activated by translocation to the nucleus on plastic and in serum-free KM resulted in selection under appropriate regenerative demands. for colonies of cells that divided initially every 36- Interestingly, there were also cells coexpressing mul- 40 hours, thereafter slowing to a division every 2-3 tiple transcription factors such as SOX17 and PDX1 days, with proliferation continuing for months and (Fig. 4). The percentage of SOX17þ cells is 11.2% 6 associated with stable maintenance of the undifferenti- 3.8% and the PDX1þ cell is the 16.6% 6 3.4% and ated cell phenotype (Table S2). Figure S8 shows a rep- the percentage of the cells coexpressing SOX17 and resentative colony maintained for more than 8 weeks PDX1 was variable but ranged from 10%-15%. This on culture plastic and in KM. Cells in the colony cen- coexpression in some cells and, similarly, expression of ters (regions a and b) had an average cell diameter of HEPATOLOGY, Vol. 54, No. 6, 2011 CARDINALE ET AL. 2165