Controlled induction of human pancreatic progenitors produces functional beta‐like cells in vitro
2015; Springer Nature; Volume: 34; Issue: 13 Linguagem: Inglês
10.15252/embj.201591058
ISSN1460-2075
AutoresHolger A. Russ, Audrey V. Parent, Jennifer J Ringler, Thomas G. Hennings, Gopika G. Nair, Mayya Shveygert, Tingxia Guo, Sapna Puri, Leena Haataja, Vincenzo Cirulli, Robert Blelloch, Gregory L. Szot, Peter Arvan, Matthias Hebrok,
Tópico(s)Diabetes Management and Research
ResumoArticle23 April 2015free access Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro Holger A Russ Holger A Russ Diabetes Center, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Audrey V Parent Audrey V Parent Diabetes Center, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Jennifer J Ringler Jennifer J Ringler Diabetes Center, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Thomas G Hennings Thomas G Hennings Diabetes Center, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Gopika G Nair Gopika G Nair Diabetes Center, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Mayya Shveygert Mayya Shveygert Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences and Department of Urology, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Tingxia Guo Tingxia Guo Diabetes Center, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Sapna Puri Sapna Puri Diabetes Center, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Leena Haataja Leena Haataja Division of Metabolism, Endocrinology & Diabetes, University of Michigan Medical School, Brehm Tower, Ann Arbor, MI, USA Search for more papers by this author Vincenzo Cirulli Vincenzo Cirulli Diabetes and Obesity Center of Excellence, Department of Medicine, Institute for Stem Cells and Regenerative Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author Robert Blelloch Robert Blelloch Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences and Department of Urology, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Greg L Szot Greg L Szot Diabetes Center, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Peter Arvan Peter Arvan Division of Metabolism, Endocrinology & Diabetes, University of Michigan Medical School, Brehm Tower, Ann Arbor, MI, USA Search for more papers by this author Matthias Hebrok Corresponding Author Matthias Hebrok Diabetes Center, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Holger A Russ Holger A Russ Diabetes Center, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Audrey V Parent Audrey V Parent Diabetes Center, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Jennifer J Ringler Jennifer J Ringler Diabetes Center, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Thomas G Hennings Thomas G Hennings Diabetes Center, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Gopika G Nair Gopika G Nair Diabetes Center, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Mayya Shveygert Mayya Shveygert Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences and Department of Urology, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Tingxia Guo Tingxia Guo Diabetes Center, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Sapna Puri Sapna Puri Diabetes Center, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Leena Haataja Leena Haataja Division of Metabolism, Endocrinology & Diabetes, University of Michigan Medical School, Brehm Tower, Ann Arbor, MI, USA Search for more papers by this author Vincenzo Cirulli Vincenzo Cirulli Diabetes and Obesity Center of Excellence, Department of Medicine, Institute for Stem Cells and Regenerative Medicine, University of Washington, Seattle, WA, USA Search for more papers by this author Robert Blelloch Robert Blelloch Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences and Department of Urology, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Greg L Szot Greg L Szot Diabetes Center, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Peter Arvan Peter Arvan Division of Metabolism, Endocrinology & Diabetes, University of Michigan Medical School, Brehm Tower, Ann Arbor, MI, USA Search for more papers by this author Matthias Hebrok Corresponding Author Matthias Hebrok Diabetes Center, University of California San Francisco, San Francisco, CA, USA Search for more papers by this author Author Information Holger A Russ1, Audrey V Parent1, Jennifer J Ringler1, Thomas G Hennings1, Gopika G Nair1, Mayya Shveygert2, Tingxia Guo1,5, Sapna Puri1, Leena Haataja3, Vincenzo Cirulli4, Robert Blelloch2, Greg L Szot1, Peter Arvan3 and Matthias Hebrok 1 1Diabetes Center, University of California San Francisco, San Francisco, CA, USA 2Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences and Department of Urology, University of California San Francisco, San Francisco, CA, USA 3Division of Metabolism, Endocrinology & Diabetes, University of Michigan Medical School, Brehm Tower, Ann Arbor, MI, USA 4Diabetes and Obesity Center of Excellence, Department of Medicine, Institute for Stem Cells and Regenerative Medicine, University of Washington, Seattle, WA, USA 5Present address: Fluidigm Corporation, South San Francisco, CA, USA *Corresponding author. Tel: +1 415 514 0820; E-mail: [email protected] The EMBO Journal (2015)34:1759-1772https://doi.org/10.15252/embj.201591058 See also: FM Spagnoli (July 2015) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Directed differentiation of human pluripotent stem cells into functional insulin-producing beta-like cells holds great promise for cell replacement therapy for patients suffering from diabetes. This approach also offers the unique opportunity to study otherwise inaccessible aspects of human beta cell development and function in vitro. Here, we show that current pancreatic progenitor differentiation protocols promote precocious endocrine commitment, ultimately resulting in the generation of non-functional polyhormonal cells. Omission of commonly used BMP inhibitors during pancreatic specification prevents precocious endocrine formation while treatment with retinoic acid followed by combined EGF/KGF efficiently generates both PDX1+ and subsequent PDX1+/NKX6.1+ pancreatic progenitor populations, respectively. Precise temporal activation of endocrine differentiation in PDX1+/NKX6.1+ progenitors produces glucose-responsive beta-like cells in vitro that exhibit key features of bona fide human beta cells, remain functional after short-term transplantation, and reduce blood glucose levels in diabetic mice. Thus, our simplified and scalable system accurately recapitulates key steps of human pancreas development and provides a fast and reproducible supply of functional human beta-like cells. Synopsis Focusing on developmental mechanisms, the results of this study further accelerate successful differentiation of human ESCs into functional pancreatic beta cells. Exclusion of commonly used BMP inhibitors during human embryonic stem cell to pancreatic progenitor differentiation prevents precocious endocrine induction. Sequential exposure of foregut cells to retinoic acid followed by combined EGF/KGF treatment establishes highly pure PDX1+ and PDX1+/NKX6.1+ progenitor populations, respectively. Precise temporal induction of endocrine differentiation in PDX1+/NKX6.1+ progenitors, but not in PDX1+/NKX6.1− progenitors, results in the generation of functional beta-like cells in vitro. Beta-like cells exhibit key features of bona fide human beta cells, remain functional after short-term transplantation, and reduce blood glucose levels in diabetic mice. Introduction Diabetes mellitus type 1 and 2 (T1D, T2D) are diseases characterized by autoimmune destruction or progressive dysfunction and subsequent loss of insulin-producing pancreatic beta cells, respectively. Current treatments for both types of patients with diabetes consist of regulating blood glucose levels through injections of exogenous insulin. While this approach provides reasonable management of the diseases, unwanted risks and long-term complications persist due to the inability of tightly maintaining glucose levels within a normal physiological range. Complications include life-threatening episodes of hypoglycemia, as well as long-term complications from hyperglycemia resulting in micro- and macro-angiopathy leading to cardiovascular pathologies and kidney failure, as well as neuropathy. Thus, there is a need for distinct treatments that provide superior control of glucose metabolism to minimize or ideally eliminate long-term complications. One existing approach to treating diabetes is transplantation of human cadaveric islet preparations into patients. This procedure typically results in better glycemic control, can render patients insulin independent for prolonged periods of time, and improves overall quality of life (Shapiro et al, 2000; Posselt et al, 2010; Barton et al, 2012). However, the severe shortage of cadaveric organ donors, requirement for lifelong immunosuppression, and variability between islet preparations hampers the use of islet transplantation as a readily available treatment for people with diabetes. Consequently, numerous research efforts have focused on identifying abundant alternative sources of surrogate glucose-responsive insulin-producing cells (Zhou & Melton, 2008; Tudurí & Kieffer, 2011; Efrat & Russ, 2012; Hebrok, 2012; Nostro & Keller, 2012; Bouwens et al, 2013; Pagliuca & Melton, 2013). One of the most appealing approaches is the directed differentiation into insulin-producing cells from pluripotent human embryonic stem cells (hESCs) (D'Amour et al, 2005; Chen et al, 2009; Mfopou et al, 2010; Nostro et al, 2011; Van Hoof et al, 2011; Xu et al, 2011; Guo et al, 2013b; Shim et al, 2014) and, more recently, induced pluripotent stem cells (Maehr et al, 2009; Hua et al, 2013; Shang et al, 2014). Comprehensive knowledge of signaling events and temporal transcription factor (TF) expression patterns during rodent pancreas organogenesis (Hebrok, 2003; Murtaugh & Melton, 2003; Pan & Wright, 2011; Seymour & Sander, 2011) have accelerated the identification of culture conditions that allow the generation of pancreatic cell types from human pluripotent stem cells (hPSCs). Early developmental stages, including definitive endoderm, gut tube-like cells, and pancreatic progenitors can be efficiently induced in vitro. However, subsequent transitions toward hormone-expressing cells in vitro are less efficient and frequently lead to the formation of a mixed population of different pancreatic progenitors and polyhormonal endocrine cells (D'Amour et al, 2006; Nostro et al, 2011; Guo et al, 2013a). Such polyhormonal cells express insulin among other hormones, but lack expression of key beta cell transcription factors and do not secrete insulin in vitro in response to a glucose challenge—the hallmark function of bona fide beta cells (D'Amour et al, 2006; Nostro et al, 2011; Guo et al, 2013a). Nonetheless, transplantation of such heterogeneous cultures into surrogate mice results in the formation of glucose-responsive beta-like cells after several months in vivo (Kroon et al, 2008; Rezania et al, 2012; Szot et al, 2015). Sophisticated sorting experiments identified progenitor cells expressing pancreatic and duodenal homeobox 1 TF (PDX1, also known as IPF1) and homeobox protein NKX6.1 as the source for these functional beta-like cells (Kelly et al, 2011). While polyhormonal cells have been identified in human fetal pancreas, suggesting that they may reflect aspects of the normal embryonic differentiation process (De Krijger et al, 1992; Riedel et al, 2011), increasing evidence indicates that hESC-derived polyhormonal cells preferentially give rise to single-hormone-positive alpha-like cells (Rezania et al, 2011). Thus, to fully replicate human beta cell development in vitro, it is imperative to better understand and accurately recapitulate the sequence of embryonic signals required for the proper specification of beta cell precursors, rather than alpha cell precursors. During normal in vivo pancreatic organogenesis, functional beta cells are generated through a stepwise specification process starting with pancreatic progenitors, identified by the expression of Pdx1 (Herrera et al, 2002). While Pdx1+ cells can give rise to all pancreatic lineages (Herrera et al, 2002), the subsequent induction of Nkx6.1 in these cells restricts their differentiation potential to only endocrine and ductal cells (Schaffer et al, 2010). Endocrine differentiation is then initiated in Pdx1+/Nkx6.1+ progenitors by short-lived expression of the basic helix loop helix TF neurogenin 3 (Neurog3, also known as Ngn3) (Gu et al, 2002). Interestingly, the timing of Neurog3 expression has been shown to be crucial in promoting the formation of diverse endocrine islet cell types (Johansson et al, 2007). For example, precocious induction of endocrine differentiation by forced expression of Neurog3 in mice results predominantly in the generation of alpha cells (Johansson et al, 2007). Given that hESC-derived polyhormonal cells have been shown to give rise to alpha cells (Rezania et al, 2011), we hypothesized that the in vitro generation of polyhormonal endocrine cells results from premature assignment to the endocrine fate. To address this issue, we performed a detailed stepwise analysis of pancreatic progenitor generation and endocrine induction. Most current protocols efficiently establish PDX1+ progenitors by using retinoic acid in combination with molecules to inhibit bone morphogenic protein (BMP) and sonic hedgehog (SHH) signaling pathways, while simultaneously adding either fibroblast growth factor 10 or keratinocyte growth factor (KGF, also known as FGF7) (Mfopou et al, 2010; Nostro & Keller, 2012; Rezania et al, 2012; Guo et al, 2013b; Hua et al, 2013). Here, we show that the use of BMP inhibitors to specify pancreatic cells promotes the precocious induction of endocrine differentiation in PDX1+ pancreatic progenitors, which results in the formation of polyhormonal cells. Furthermore, we have identified simplified culture conditions that replicate fetal endocrine development and allow for the precise and efficient generation of PDX1+ and PDX1+/NKX6.1+ progenitor populations without precocious activation of the endocrine marker NEUROG3. Importantly, subsequent induction of endocrine differentiation in correctly specified PDX1+/NKX6.1+ progenitor cells results in the formation of glucose-responsive insulin-expressing beta-like cells in vitro within less than 3 weeks. Our study therefore details a simplified directed differentiation protocol that closely recapitulates key aspects of human endocrine development and results in the formation of large numbers of glucose-responsive beta-like cells under cell culture conditions. Results Pancreatic differentiation of hESCs using a large-scale culture system results in two distinct subsets of insulin-producing cells To generate pancreatic beta-like cells from human PSC, we established a scalable three-dimensional suspension culture system based on previously reported methods (Rezania et al, 2012; Schulz et al, 2012) (Fig 1A). To monitor the generation of live insulin-producing cells and facilitate their isolation, we employed the recently published INSGFP/W reporter cell line (Micallef et al, 2012) in which green fluorescence protein (GFP) expression is under the control of the endogenous insulin promoter. Using this differentiation protocol, GFP reporter expression was readily observed at day 13 and increased thereafter, resulting in an average of 37 ± 8% GFP+ cells between days 19 and 24 (Fig 1B–D). The validity of GFP as an accurate substitute for insulin was verified by staining with an insulin specific antibody, which revealed an even higher percentage of insulin-producing cells (up to 60%) likely due to delayed accumulation of the fluorescence marker (Fig 1E). Similar results were obtained with an antibody specific to human C-peptide, excluding antibody reactivity due to insulin uptake from culture media (Fig 1E). Co-staining for human C-peptide and glucagon (GCG), a hormone normally produced by alpha cells, showed that 4.3% and 13.2% of all cells exhibited a polyhormonal phenotype at day 13 and day 19, respectively (Fig 1F). Co-staining for C-peptide and NKX6.1 at day 20 indicated the presence of some double-positive beta-like cells (Fig 1G). Quantitative flow cytometry analysis revealed that the proportion of insulin and NKX6.1 double-positive beta-like cells increased from less than 2.5% at day 13 to approximately 12% cells at day 19 of total cells (Fig 1G). Ultrastructural analysis of differentiated cultures showed cells containing secretory vesicles with an electron-dense core surrounded by an electron-light halo (Fig 1H), a morphology reminiscent of insulin vesicles that are found in human beta cells. However, the majority of cells exhibited a mixture of secretory granules usually found in non-beta cells of human pancreas preparations (Fig 1H). Thus, differentiation experiments employing published protocols (Rezania et al, 2012; Schulz et al, 2012) result in the efficient generation of two distinct insulin-producing cell populations: INS+ cells that do not co-express the critical TF NKX6.1 and manifest as polyhormonal cells, and INS+/NKX6.1+ beta-like cells that more closely resemble human beta cells. Notably, INS+/NKX6.1+ beta-like cells are absent from cultures at earlier time points but appear and increase in number at later stages of differentiation, suggesting that they are derived from a distinct progenitor cell type. Figure 1. Pancreatic differentiation of hESCs using a large-scale culture system results in two distinct subsets of insulin-producing cells Schematic outlining the differentiation protocol employed. R, retinoic acid; C, cyclopamine; N, Noggin, E, epidermal growth factor; K, keratinocyte growth factor; T, TBP; A, ALK inhibitor. Micrograph of MEL1INS-GFP cell clusters after 17 days of differentiation demonstrating strong GFP expression (GFP expression in white). Scale bar, 200 μm. Flow cytometric analysis at day 20 of differentiation showing 41.5% of all cells expressing GFP under the control of the endogenous insulin promoter. Quantification by flow cytometry of the average percentage of GFP+ cells within differentiated cultures after 19–24 days. n = 7. Values are average ± standard deviation (SD). Flow cytometric analysis of intracellular human-specific C-peptide (C-PEP) and insulin (INS) shows comparable percentages of C-PEP− and INS+ cells. Immunofluorescence staining for C-PEP and glucagon (GCG), and flow cytometric quantification of GCG+/C-PEP+ (red gate) and GCG−/C-PEP+ (black gate) populations at days 13 and 19 of differentiation. Immunofluorescence staining for C-PEP and NKX6.1, and flow cytometric quantification of NKX6.1+/INS+ (green gate) and NKX6.1−/INS+ (red gate) populations at day 13 and 19. Immunofluorescence insets show two distinct phenotypes for C-PEP+ cells (NKX6.1+ and NKX6.1−). A robust INS/NKX6.1 double-positive population is only detected at day 19. Transmission electron microscopy of day 20 clusters. Cells contain both secretory vesicles with electron-dense cores surrounded by electron-light halos (green box), akin to bona fide beta cell vesicles, as well as other granules similar to those found in non-beta pancreatic cells (red boxes). Download figure Download PowerPoint Defining the temporal activities of individual signaling factors to efficiently generate PDX1+ and PDX1+/NKX6.1+ pancreas progenitor populations while preventing precocious induction of endocrine differentiation To characterize the type of progenitors present in differentiating cultures at the point of endocrine induction, we performed a detailed time-course analysis for the expression of pancreatic markers PDX1, NKX6.1, NEUROG3, GCG, and INS (Supplementary Fig S1). High expression of the progenitor marker PDX1 was efficiently induced and maintained starting 1 day after the combined addition of retinoic acid (R), the SHH inhibitor cyclopamine (C), and the BMP inhibitor Noggin (N) to the culture media (referred to as RCN, day 6, Supplementary Fig S1A and B). Subsequent treatment with epidermal growth factor (EGF), KGF, and N (EKN) resulted in the robust generation of PDX1+/NKX6.1+ double-positive cells, reaching 67% of the total population at day 11 (Supplementary Fig S1A and B). Immunofluorescence analysis revealed that the RCN cocktail of factors widely used to generate pancreatic endoderm also induces precocious expression of NEUROG3 in PDX1+ pancreatic progenitors. Indeed, the expression of NEUROG3 can be detected as early as day 6, when NKX6.1 protein is absent from all cells (Supplementary Fig S1A and B). Consequentially, insulin-expressing cells that are first detected 4 days after NEUROG3 induction (starting at day 10) do not co-express NKX6.1 and are mostly polyhormonal (Fig 1F and G, and Supplementary Fig S1C). In contrast, INS/NKX6.1 double-positive beta-like cells can be readily detected only at later time points (day 19, Fig 1G), suggesting that these cells differentiate from PDX1/NKX6.1 double-positive progenitor cells. We thus hypothesized that robust generation of PDX1+/NKX6.1+ progenitor cells prior to induction of NEUROG3 would allow efficient generation of beta-like cells in vitro. To determine which of the factors used between days 6 and 8 in the original protocol (R, C, and N) are responsible for the induction of PDX1, NKX6.1, and NEUROG3, we incubated spheres with each of the factors alone or in different combinations over days 6–8 (Fig 2A). Basal media with B27 but lacking any additional factors served as the control condition. At the end of day 8, each of these six conditions was further subdivided into three different treatment groups: Media composition remained the same as during days 6–8 (group 1) or changed either to EK (group 2) or to EKN (group 3), resulting in 18 individual experimental conditions (Fig 2A). Spheres cultured under each condition were analyzed at day 9.5 by flow cytometry to quantify the expression of PDX1 and NKX6.1, and by conventional immunofluorescence analysis for NKX6.1 and NEUROG3 expression. As shown in Fig 2B, spheres within group 1 that had been exposed to retinoic acid during days 6–8, either alone or in combination with other factors (conditions 4, 5, and 6), exhibited highly efficient generation of PDX1+ progenitors (> 88%), while the addition of C or N alone (conditions 2 and 3) did not result in enhanced generation of PDX1+ cells over basal media alone. NKX6.1 was induced only weakly in all group 1 conditions, with the exception of RC (condition 5), which produced 45% PDX1/NKX6.1 double-positive cells. NKX6.1 expression was also strongly induced when cells were exposed to retinoic acid alone or in combination with other factors followed by treatment with EK (group 2) or EKN (group 3) (Fig 2B and C, conditions 10–12 and 16–18). Endocrine differentiation, marked by NEUROG3 expression, was noted only when spheres had been exposed to N, either between days 5 and 9.5 (Fig 2C, conditions 3, 6, 9, and 12) or starting at the end of day 8 (Fig 2C, group 3, conditions 13–18). Very few NEUROG3+ cells were detected in all other conditions (Fig 2C, conditions 1, 2, 4, 5, 7, 8, 10, and 11). qPCR analysis at day 8 of NEUROG3 and its downstream target NKX2.2 mRNA transcripts revealed significantly lower levels of these endocrine markers with R treatment when compared to the commonly employed RCN condition (Supplementary Fig S1D). Notably, the addition of vitamin C, recently shown to reduce endocrine differentiation in hESCs (Rezania et al, 2014), did not significantly lower NEUROG3 or NKX2.2 transcripts in our suspension culture system during RCN or R treatment (Supplementary Fig S1D). Taken together, these results indicate that R followed by EK treatment leads to highly efficient generation of PDX1+/NKX6.1+ progenitors (90%) and that the formation of bona fide NEUROG3-positive endocrine precursors is induced by treatment with N (Fig 2A–C, condition 10, green gates). Thus, by defining the temporal activities of individual signaling factors alone and in combination, we can induce transcription factor expression patterns characteristic of different human embryonic pancreatic progenitor cells types (PDX1+ and PDX1+/NKX6.1+ progenitors) without precocious induction of endocrine differentiation. Figure 2. Defining the temporal activities of individual signaling factors to efficiently generate PDX1+ and PDX1+/NKX6.1+ pancreatic progenitor populations while preventing precocious induction of endocrine differentiation A–C. Pancreatic progenitor marker expression at day 9.5 after treatment with conventional differentiation factors alone or in different combinations. Treatments consisted of combinations of cyclopamine (C), Noggin (N), and retinoic acid (R) during days 6–8 followed by subdivision of each condition into three treatment groups during day 9–9.5. Group 1: continuation of day 6–8 treatment; Group 2: treatment with EGF and KGF (EK); Group 3: treatment with EGF, KGF, and Noggin (EKN). The condition selected for further studies (10) is marked with a green box. Data shown are representatives of results obtained in two independent experiments. (A) Table detailing 18 different culture conditions that were evaluated. (B) Quantification of PDX1 (orange gate) and NKX6.1 (blue gate) protein-expressing cells in individual conditions after 9.5 days of differentiation. (C) NKX6.1 and NEUROG3 protein expression assessed by whole-mount staining of differentiated clusters at 9.5 days. Note robust NEUROG3 expression in all clusters exposed to N (conditions 3, 6, 9, and 12–18). Download figure Download PowerPoint Recapitulating human pancreas organogenesis to generate endocrine progenitors This improved and simplified differentiation protocol provides the basis for subsequent efficient formation of insulin-producing cells in suspension (Fig 3A). Endocrine differentiation in PDX1/NKX6.1 double-positive cells was induced by exposure to a cocktail of factors consisting of TBP (T), ALK inhibitor (A), N, and K, (TANK) which have previously been shown to activate NEUROG3 expression while maintaining high expression of PDX1 and NKX6.1 (Nostro et al, 2011; Rezania et al, 2012) (Fig 3A and B). Importantly, while NEUROG3 protein was undetectable before TANK treatment (Fig 3C, day 9), cells exhibiting nuclear accumulation of NEUROG3 protein appeared as early as 1 day following TANK treatment (Fig 3C, day 10). Thus, the expression of the pro-endocrine factor NEUROG3 is rapidly induced through TANK treatment once PDX1+/NKX6.1+ progenitors are specified (Fig 3B, day 9). In contrast to the near-uniform generation of PDX1+ and PDX1+/NKX6.1+ progenitor populations following appropriate stimulation, endocrine differentiation appears to be confined to a smaller population of cells. This observation can be explained by the very short half-life of the NEUROG3 protein (Roark et al, 2012), which allows only transient detection of this marker in cells undergoing endocrine different-iation. However, NEUROG3+ cells continued to be present when clusters were exposed to the endocrine differentiation cocktail for 5 days (Fig 3C, day 14), indicating that endocrine cells were being generated throughout this period. To further characterize the progenitors present in our cultures at the initiation of endocrine differentiation, we analyzed the expression of NKX2.2, a downstream target of NEUROG3. NKX2.2 has recently been reported to have distinct expression patterns during pancreatic organogenesis in mouse and human (Jennings et al, 2013). While Nkx2.2 is readily detectable in mouse pancreatic progenitor cells before Neurog3 expression, NKX2.2 protein is only observed after endocrine commitment during human pancreas development. Similarly, we detected NKX2.2 protein expression only after endocrine differentiation is initiated at day 10, but not before in either PDX1+ or PDX1+/NKX6.1+ progenitors (Fig 3C, data not shown). Of note, some NKX2.2+ cells at day 10 co-express NEUROG3, and increasing numbers of NKX2.2+/NEUROG3− cells are found at later time points (Fig 3C). These data suggest that NKX2.2 could serve as a lineage tracer for human cells that have undergone endocrine differentiation induced by transient NEUROG3 expression. In summary, we have established a novel differentiation strategy that faithfully recapitulates human pancreas organogenesis and allows for the precise control over the generation of PDX1+ and PDX1+/NKX6.1+ progenitors. Figure 3. Recapitulating human pancreas organogenesis to generate endocrine proge
Referência(s)