Diabetes mellitus is a disease that can lead to dangerously high blood sugar levels, causing numerous complications such as heart disease, glaucoma, skin disorders, kidney disease, and nerve damage. In healthy individuals, beta cells in the pancreas produce a hormone called insulin, which stimulates cells in the liver, muscles and fat to take up glucose from the blood. However, this process is disrupted in people with diabetes, who either have too few pancreatic beta cells (type 1 diabetes) or do not respond appropriately to insulin (type 2 diabetes).

All patients with type 1 diabetes, and some with type 2, must inject themselves regularly with insulin, but this does not always fully control the disease. Some type 1 patients have been successfully treated with beta cells transplanted from deceased donors, but there are not enough donor organs available for this to become routine. Thus, intensive efforts worldwide are focused on generating insulin-producing cells in the lab from human stem cells. However, the cells produced in this way can give rise to tumors.

Now, Lee et al. have shown that duct cells, which make up about 30% of the human pancreas, can be converted into cells capable of producing and secreting insulin. Ductal cells obtained from donor pancreases were first separated from the remaining tissue and grown in cell culture. Viruses were then used to introduce genes that reprogrammed the ductal cells so that they acquired the ability to make, process and store insulin, and to release it in response to glucose—hallmark features of functional beta cells.

As well as providing a potential source of cells for use in transplant or cell conversion therapies for diabetes, the ability to grow and maintain human pancreatic ductal cells in culture may make it easier to study other diseases that affect the pancreas, including pancreatitis, cystic fibrosis, and adenocarcinoma.

The pancreas is a vital organ with exocrine and endocrine cell functions, and a root of lethal human diseases including diabetes mellitus, pancreatitis, and pancreatic ductal adenocarcinoma. Exocrine acinar cells produce digestive zymogens that are delivered to the intestines by a branching network of exocrine ductal cells that secrete bicarbonate and other products. Pancreatic endocrine functions derive from clusters of epithelial cells (islets of Langerhans) called α-, β-, δ-, and PP-cells that respectively synthesize, store, and secrete the hormones Glucagon, Insulin, Somatostatin, and Pancreatic polypeptide (Benitez et al., 2012). Insulin production by islet β-cells is highly regulated: key features of mature β-cells include preproinsulin (INS) transcription, proinsulin processing by endo- and exo-peptidases and storage of the proinsulin cleavage products insulin and C-peptide in dense core vesicles. Likewise, cardinal β-cell functions regulate insulin release in response to glucose and other secretagogues, including glucose sensing and metabolism through the enzyme glucokinase, and use of ATP-dependent potassium channels (K_ATP) and voltage-gated calcium channels to induce insulin exocytosis (reviewed in Suckale and Solimena, 2010). Deficiency or malfunctioning of β-cells produces impaired glucose regulation and diabetes mellitus, a disease with autoimmune (type 1, T1DM) and pandemic forms (type 2; Ashcroft and Rorsman, 2012). Thus, replacement or regeneration of functional human β-cells is an intensely-sought goal.

Human islet transplantation can be used to replace β-cell function in T1DM (reviewed in Vardanyan et al., 2010), but a shortage of donors currently precludes broad use of human pancreatic islets for β-cell replacement. Because of their expandability and multipotency, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been explored as sources of replacement insulin-producing cells (reviewed in Hebrok, 2012). However, directing the differentiation of these developmentally ‘primitive’ cells through multiple sequential fates into β-cell-like progeny that synthesize, process, store, and secrete insulin while lacking tumorigenic potential has challenged investigators worldwide (Fujikawa et al., 2005; McKnight et al., 2010; Cheng et al., 2012). Moreover, different hESC and iPSC cell lines exhibit significant variability during development into insulin-producing cells (Nostro and Keller, 2012). Recent work demonstrated that differentiated cell types in adult organs, including the mouse pancreas, can be experimentally ‘reprogrammed’ into progeny resembling islet cells, suggesting a new strategy for β-cell replacement (Vierbuchen and Wernig, 2011). For example, adult mouse pancreatic acinar cells can be converted into insulin-producing cells in vitro and in vivo (Minami et al., 2005; Zhou et al., 2008). However, little progress has been made in reprogramming primary human epithelial cells into different cell types, including conversion of pancreatic non-β-cells toward a human β-cell fate (Vierbuchen and Wernig, 2011). Thus, systems permitting expansion and genetic modulation of human pancreatic cells could powerfully influence studies of β-cell biology and replacement.

Pancreatic ducts constitute 30–40% of human pancreas and have been proposed as a potential source of replacement β-cells (Bouwens and Pipeleers, 1998; Bonner-Weir et al., 2004). During pancreas development, fetal endocrine cells derive from primitive ductal epithelium (reviewed by Pan and Wright, 2011; Pictet and Rutter, 1972). In addition, some studies have suggested that in adult mice, β-cells may be produced from pancreatic ductal epithelium (Inada et al., 2008; Xu et al., 2008; Rovira et al., 2010). However, recent lineage tracing evidences have not supported this view (Solar et al., 2009; Furuyama et al., 2011; Kopp et al., 2011). In humans, prior studies have suggested that adult human primary ductal cells in heterogeneous cell mixtures may harbor the potential to generate endocrine-like progeny (Bonner-Weir et al., 2000; Heremans et al., 2002; Swales et al., 2012), but interpretation in these studies was limited by the probability of islet cell contamination. Therefore, the potential for conversion of pancreatic ductal cells toward an endocrine fate remains unclear. Moreover, prior studies have revealed only limited proliferative capacity of primary human pancreatic ductal cells in culture (Rescan et al., 2005). Thus, despite their relative abundance, multiple practical issues have prevented development of human pancreatic ductal cells as a source of replacement β-cells.

Here we report that normal human adult pancreatic duct cells can be sorted, clonally expanded, and genetically converted into endocrine cells. Human insulin-producing cells (IPCs) produced from sorted duct cells exhibited hallmark features of functional neonatal β-cells including high-level preproinsulin (INS) expression, proinsulin processing and dense-core granule formation. Moreover, secretion of insulin and insulin C-peptide from IPCs is stimulated by glucose and K_ATP channel stimulants in a calcium-dependent manner. Together these studies reveal a new system for investigating human pancreatic duct cell biology, genetics, and β-cell regeneration.

Methods to regenerate lost or injured cells in diseases like diabetes mellitus are the focus of intensive investigations (McKnight et al., 2010; Benitez et al., 2012). Generation of insulin-producing cells from human stem cell lines like human ES cells (D’Amour et al., 2006; Kroon et al., 2008) is an important, and oft-cited ‘benchmark’, in efforts to achieve β-cell replacement. However, in these prior reports, progeny of human ES cells developed largely as poly-hormonal cells, most frequently expressing both glucagon and insulin. Moreover such hESC progeny failed to secrete insulin in response to glucose or other secretagogues unless transplanted as progenitors for >2 months in mice (Nostro and Keller, 2012). This transplant-based maturation strategy was complicated by tumor formation (Fujikawa et al., 2005). Thus, it has remained unknown whether human cells can develop solely in vitro to generate glucose-responsive insulin-secreting progeny without tumorigenicity. Our data indicate that in principal this can be achieved, using a small number of genes in sorted human pancreatic ductal cells that convert them toward an islet fate, including progeny that produce, store, and secrete insulin in response to glucose.

Conversion of mouse acinar cells into insulin-producing cells using adenoviral delivery of Neurog3, Pdx1, and MafA was previously reported (Zhou et al., 2008). However, it has remained unknown whether human pancreatic cells can be converted using transgenic methods toward a β-cell fate. We were unable to culture and expand primary human pancreatic acinar cells (Figure 1B and data not shown); moreover, we found that the combination of these three genes (MNP) was insufficient to reprogram primary or expanded human pancreatic ductal cells toward a β-cell fate, suggesting transgenic conversion may be restricted by species and cell type. Thus, we postulated that additional transcriptional regulators might be needed to promote human ductal conversion toward a β-cell fate. Like Neurog3, MafA, and Pdx1, the transcription factor Pax6 is expressed in both fetal and adult pancreas, and required to achieve appropriately high levels of Ins and Gcg expression in mouse islet cell development (Sander et al., 1997; Wang et al., 2009, 2010; Pan and Wright, 2011). Together with the other factors, we found that Pax6 significantly enhanced expression of β-cell markers during ductal reprogramming into β-cells, and was shown as an essential factor for this process. By systematic addition or omission of each transcription factor, we found PDX1 is not required for IPC formation. Thus, unlike mouse acinar cells (Zhou et al., 2008) and human hepatocytes (Sapir et al., 2005), human ductal cells do not require exogenous Pdx1 expression for conversion toward an endocrine fate, for reasons that remain unclear. Our findings are also consistent with recent reports that transgenic adult mouse ductal cells can generate endocrine cells in vivo (Al-Hasani et al., 2013).

We initially attempted to induce spontaneous differentiation of pancreatic ductal cells using systematic variations of culture conditions, but these efforts proved unsuccessful (J Lee, unpublished results). During pancreas development, Neurog3 level surges in a subset of pancreatic progenitors located in primitive ducts, inducing development of endocrine cell fates (Zhou et al., 2007; Miyatsuka et al., 2009). Therefore, based on this model, we attempted to mimic induction of Neurog3 in human ductal cells using adenoviral overexpression of Neurog3. We found that Neurog3 was necessary and sufficient for reprogramming human ductal cells, and that the level of ectopic Neurog3 mRNA expressed in ductal cells correlated well with the extent of endocrine reprogramming, including expression of islet hormones (Figure 3G). These findings are reminiscent of studies by Gu et al. showing that reduced Neurog3 gene dosage in mice leads to respecification of pancreatic endocrine progenitors into ductal and acinar cells (Wang et al., 2010). Thus, Neurog3 functions may be evolutionarily conserved in allocating cells toward an exocrine or endocrine fate (whether in development or experimental cell conversion) in a dosage-dependent manner. Consistent with prior work revealing that Neurog3 attenuates islet cell proliferation (Miyatsuka et al., 2011), we did not observe multiple rounds of cell division, an important prerequisite for some de-differentiation events (Hanna et al., 2009), during Neurog3-dependent cell conversion. Also, we observed Neurog3 induction alone can rapidly upregulate endocrine molecular signatures in cultured human ductal cells. Thus endocrine cell conversion described here may involve direct conversion of human ductal cells into endocrine cells, rather than de-differentiation, but additional studies are required to assess this possibility. Our findings, albeit with enforced transcription factor expression in adult cells, indicate that Neurog3 expression is sufficient to induce latent endocrine programs in human adult ductal cells, a capacity not yet clearly demonstrated, to our knowledge.

We demonstrated robust expansion of purified human ductal cells in 3-dimensional culture. The cells were clonally expanded and serially passaged up to seven generations over 3 months, achieving an increase in cell number calculated to be up to 3,200-fold. By contrast, in prior studies, the maximum duration of sustained culture achieved with primary human pancreatic ductal cells was 5 weeks (Trautmann et al., 1993; Bonner-Weir et al., 2000; Rescan et al., 2005; Hao et al., 2006; Yatoh et al., 2007; Hoesli et al., 2012). Moreover, cultured cells in spheres maintained cardinal features of primary pancreatic ducts such as apical-basal polarity and KRT19 expression up to seven generations (Figure 2—figure supplement 1D,E). Thus, features of our culture system may be useful for studying the genetics and biology of human ductal cells.

Prior studies have reported that duct-containing fractions from human adult pancreas can form insulin-producing cells in vitro (Bonner-Weir et al., 2000; Hao et al., 2006; Heremans et al., 2002; Noguchi et al., 2006; Koblas et al., 2008; Swales et al., 2012) or after xeno-transplantion in mice (Yatoh et al., 2007). However, the possibility of endocrine cell contamination in the initial ductal fraction or feeder/stromal cells used for co-culture was raised by the detection of mRNAs encoding islet cell hormones and other endocrine markers in these and other studies (Heremans et al., 2002; Gao et al., 2005). Therefore, it remained elusive whether human pancreatic ducts retained the potential to produce islet endocrine cells in adult. In this report, we used FACS to fractionate CD133⁺ ductal cells and used molecular and immunocytological studies to demonstrate complete elimination of cells expressing markers of differentiated endocrine cells (including islet hormones). Therefore, subsequent conversion of these cells into functional endocrine cells provided unequivocal evidence that endocrine cell-free human adult CD133⁺ ductal cell fraction can be converted into islet endocrine cells. Centroacinar cells are located at the junction of acini and tip of intercalated ducts (Cleveland et al., 2012) and their properties remain poorly understood. These cells express CD133 (Immervoll et al., 2008), raising the possibility that our fractionated CD133⁺ cells also include centroacinar cells. Based on their relative paucity in the pancreas, it is unlikely that centroacinar cells are the exclusive source of spheres within this CD133⁺ fraction, as more than 11% of CD133⁺ cells were capable of generating spheres (Figure 1B). However, because of difficulties performing lineage-tracing experiments with human samples, we cannot exclude the possibility that centroacinar cells may also contribute to the conversion into endocrine cell lineages.

While expression of Pax6 along with Neurog3, Pdx1 and MafA significantly enhanced expression of INS and other β-cell marker genes in converted ductal cells, this transcription factor combination alone was not sufficient to generate mature IPCs. We found that extending the culture period for 2 weeks after viral infection led to maturation of several hallmark β-cell functions, including expression of key β-cell factors, significant increases of INS mRNA and protein levels, proinsulin processing, dense-core granule formation, and Insulin secretion in response to glucose or other depolarizing stimuli. We tested four distinct culture media with or without serum for this extended culture, and all media permitted maturation of these β-cell properties in IPCs (Figure 5—figure supplement 1F and see ‘Materials and methods’), indicating that the duration of culture is a key variable for promoting β-cell maturation in vitro. After maturation, the spheres contained an average of 7% total insulin compared to human islet controls, and 7–11% of cells comprising these spheres produced insulin C-peptide. Thus, we calculate that each reprogrammed Insulin⁺ cell produced between 49 and 77% of insulin levels observed in native β-cell controls, a comparable level to the IPCs derived from human ES cells (D’Amour et al., 2006).

Is the capacity of human ductal cells to be converted toward endocrine islet fates unique? A prior study by Sapir et al. (2005) suggests that human hepatocytes may be induced to express insulin. However, the conversion toward an insulin-producing fate was comparatively poor; resulting cells produced about 10,000-fold lower insulin mRNA level than that of human islets, about 3–4 orders of magnitude lower than from conversion of pancreatic duct spheres. In addition, characteristic dense core vesicles in converted hepatocytes were not observed, indicating insufficient conversion towards β-cells. Here, we also assessed the endocrine potential of primary human dermal fibroblasts, cells successfully ‘reprogrammed’ toward many non-fibroblast fates, including induced pluripotent stem cells (Takahashi et al., 2007), but detected no clear evidence of conversion toward an endocrine or β-cell fate (Figure 5—figure supplement 4, see ‘Materials and methods’ for details). Thus, conversion of human adult duct spheres into cells that produce and secrete insulin is singularly robust. Moreover, unlike prior studies of human ES cells that have high variability among ES cell lines used (D’Amour et al., 2006; Kroon et al., 2008), we demonstrated conversion toward insulin⁺ fates by ductal cells from multiple unrelated donors, another feature of the robustness of our methods.

Expression of factors produced from viral transgenes persisted in Insulin⁺ cells for at least 5 months, evidenced by the GFP expression in transplanted insulin-producing cells (Figure 5—figure Supplement 2A and 3). The transgenes delivered by adenovirus do not generally persist in dividing cells (Zhou et al., 2008). We speculate that cell cycle arrest in Insulin⁺ cells may be induced by Neurog3 (Miyatsuka et al., 2011), thereby preventing dilution of viral transgene-encoded factors. Thus, further studies are needed to investigate how persistent expression of conversion factors like Neurog3 affects maintenance and maturation of endocrine phenotypes in converted cells. Survival of transplanted insulin-secreting cells produced from ductal cells was poor, and reduced yields following transplantation of ductal cells precluded physiological studies in mouse models of diabetes. Promoting survival of transplanted insulin-secreting cells is a general problem for transplant-based islet replacement approaches. Thus, studies of factors that enhance survival of Insulin⁺ ductal cell progeny are an important current focus.

In conclusion, our study provides unique evidence that primary human cells can generate progeny that produce, store and secrete insulin in response to glucose or depolarizing agents, the hallmark features of pancreatic β-cells. We also show that human pancreatic exocrine cells, like in mice (Zhou et al., 2008), can be converted by transgenes toward an endocrine islet-like cell fate. We speculate that gene-based strategies like those described here may be combined with other methods, including culture modulation by growth factors and small molecules (Warren et al., 2010), to optimize endocrine differentiation or conversion of diverse cellular sources to advance cell replacement for diabetes. We speculate that our cell culture system may also serve as the foundation to investigate the genetics and pathogenesis of diverse human diseases rooted in pancreatic ductal cells, including pancreatitis, cystic fibrosis, and adenocarcinoma.

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Author details

1. Jonghyeob Lee

   Department of Developmental Biology, Stanford University School of Medicine, Stanford, United States

   Contribution

   JL, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Takuya Sugiyama

   Department of Developmental Biology, Stanford University School of Medicine, Stanford, United States

   Contribution

   TS, Contributed initial conception and execution of pancreatic sphere culture

   Contributed equally with

   Yinghua Liu

   Competing interests

   The authors declare that no competing interests exist.

3. Yinghua Liu

   Department of Developmental Biology, Stanford University School of Medicine, Stanford, United States

   Contribution

   YL, Contributed cell sorting and ductal sphere culture

   Contributed equally with

   Takuya Sugiyama

   Competing interests

   The authors declare that no competing interests exist.

4. Jing Wang

   Department of Developmental Biology, Stanford University School of Medicine, Stanford, United States

   Contribution

   JW, Renal subcapsular transplantation and human tissue assessment

   Competing interests

   The authors declare that no competing interests exist.

5. Xueying Gu

   Department of Developmental Biology, Stanford University School of Medicine, Stanford, United States

   Contribution

   XG, Contributed immunohistochemical and morphometric studies

   Competing interests

   The authors declare that no competing interests exist.

6. Ji Lei

   Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, United States

   Contribution

   JL, Procurement of cadaveric human pancreas and islet-depleted cell preparation

   Competing interests

   The authors declare that no competing interests exist.

7. James F Markmann

   Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, United States

   Contribution

   JFM, Procurement of cadaveric human pancreas and islet-depleted cell preparation

   Competing interests

   The authors declare that no competing interests exist.

8. Satsuki Miyazaki

   Division of Stem Cell Regulation Research, Osaka University Graduate School of Medicine, Osaka, Japan

   Contribution

   SM, Provision of Ad-MafA and Ad-Neurog3-IRES-eGFP

   Competing interests

   The authors declare that no competing interests exist.

9. Jun-ichi Miyazaki

   Division of Stem Cell Regulation Research, Osaka University Graduate School of Medicine, Osaka, Japan

   Contribution

   J-iM, Provision of Ad-MafA and Ad-Neurog3-IRES-eGFP

   Competing interests

   The authors declare that no competing interests exist.

10. Gregory L Szot

    UCSF Transplantation Surgery, University of California, San Francisco, San Francisco, United States

    Contribution

    GLS, Procurement of cadaveric human pancreas and islet-depleted cell preparation

    Competing interests

    The authors declare that no competing interests exist.

11. Rita Bottino

    Department of Pediatrics, Division of Immunogenetics, Children’s Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, United States

    Contribution

    RB, Procurement of cadaveric human pancreas and islet-depleted cell preparation

    Competing interests

    The authors declare that no competing interests exist.

12. Seung K Kim

    1. Department of Developmental Biology, Stanford University School of Medicine, Stanford, United States
    2. Department of Medicine, Oncology Division, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, United States

    Contribution

    SKK, Conception and design, Analysis and interpretation of data, Drafting or revising the article

    For correspondence

    seungkim@stanford.edu

    Competing interests

    The authors declare that no competing interests exist.

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