We thank Lucy Brown and Alexander Spalla from the Analytical Cytometry Core at City of Hope for assistance in sorting. This work is supported in part by National Institutes of Health (NIH) grants R01DK081587 and R01DK099734 to H.T.K., and U01DK089533 to A.D.R., and by National Science Foundation grant NSF-DMR-1206121 and California Institute for Regenerative Medicine grant RB5-07398 to D.A.T. Supports from the Joseph J. Jacobs Institute for Molecular Engineering for Medicine at Caltech to D.A.T., and those from Oxnard Foundation and Ella Fitzgerald Foundation to H.T.K. are also gratefully acknowledged.
Funding: This work is supported in part by National Institutes of Health (NIH) grants R01DK081587 and R01DK099734 to H.T.K., and U01DK089533 to A.D.R., and by National Science Foundation grant NSF-DMR-1206121 and California Institute for Regenerative Medicine grant RB5-07398 to D.A.T. Supports from the Joseph J. Jacobs Institute for Molecular Engineering for Medicine at Caltech to D.A.T., and those from Oxnard Foundation and Ella Fitzgerald Foundation to H.T.K. are also gratefully acknowledged. Research reported in this publication included work performed in the Analytical Cytometry Core and Light Microscopy Digital Imaging Core supported by the National Cancer Institute of the National Institutes of Health under award number P30CA33572.
Study sponsor: The sponsor did not participate in the study design, collection, analysis, or interpretation of data.

<ol>
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The authors have nothing to disclose.

The pancreatic colony assays and single colony analyses described here were inspired by the methylcellulose-containing hematopoietic colony assays that have played major roles in deciphering the biology of hematopoietic progenitor cells in the past decades 23. In these assays (Figure 5), dissociated pancreatic cells are plated into methylcellulose-containing semi-solid media with appropriate growth factors and ECM proteins that support the formation of Ring, Dense or Endocrine/Acinar colonies ...

The pancreas is composed of three major cell lineages; acinar cells secrete digestive enzymes, ducts secrete mucin to fend off pathogens and transport digestive enzymes to the gut, and endocrine cells secrete hormones, including insulin and glucagon, that maintain glucose homeostasis. During the embryonic development of the pancreas, the early ductal cells are the source of the tri-potent progenitor cells capable of giving rise to the three lineages in the pancreata of adult animals 1,2. Because adult stem and progenitor cells, such as bone marrow stem cells, are already successfully used to treat various diseases 3, there is intense interest in finding the stem and progenitor cells in the adult pancreas. If isolation and manipulation of adult pancreatic stem and progenitor cells were possible, these cells could be used to treat diseases such as type 1 diabetes, in which the insulin-secreting cells are destroyed by autoimmunity.
Whether tri-potent progenitor cells still exist in adult pancreatic ducts after the completion of embryonic development is a question that is heavily debated in the scientific community. In this debate, and using in vivo cre-lox lineage-tracing techniques, Inada and coworkers showed that adult murine ductal cells labeled with a marker, carbonic anhydrase II, could give rise to all three pancreatic lineages 4. However, using other ductal markers, such as HNF1b 5 and Sox9 2, it was concluded that ductal cells are not the major source of beta cells in adult mice.
Several years ago, we proposed that the cause of the aforementioned debate may be due to the lack, in the field 6,7, of appropriate analytical tools that can be used to measure self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. The in vivo cre-lox lineage-tracing technique mentioned above can provide evidence for the progenitor-progeny relationship on a population level. However, this lineage tracing technique is limited in its power to discern whether single progenitor cells can self-renew and differentiate into multiple lineages. Single-cell analysis is important because if several mono-potent progenitors, each with a different lineage potential, were analyzed together, they may collectively appear to have multi-lineage differentiation abilities. In addition, stem cells are usually a minor population of an adult organ. The activities of a minor cell population could be masked by the major population. Therefore, a negative result from a population study does not necessarily indicate the absence of stem cells. Finally, cre-lox lineage tracing does not currently allow the measurement of self-renewal.
To begin addressing the technical gap in the field of pancreatic progenitor cell biology, colony 7-11 or organoid 12-15 assays using 3D culture systems were devised. Two colony assays for pancreatic progenitors were developed in our laboratory: one contains a commercial preparation of murine extracellular matrix proteins (ECM) (see Methods and Equipment Table), and the other contains laminin hydrogel, a defined artificial ECM protein 7-11. Progenitor cells are mixed in semi-solid medium containing methylcellulose. Methylcellulose is a biologically inert and viscous material prepared from wood fibers, and has been routinely used in hematopoietic colony assays 16. The methylcellulose-containing semi-solid medium restricts the movement of single progenitor cells so that they cannot re-aggregate. Yet, the medium is soft enough to allow a progenitor cell to grow and differentiate into a colony of cells in the 3D space. Following the tradition of the hematologists, a pancreatic progenitor cell that was capable of giving rise to a colony of cells was named a pancreatic colony-forming unit (PCFU). PCFUs, when grown in the murine ECM-containing colony assay, give rise to cystic colonies that are named &#34;Ring&#34; colonies 7. Upon addition of a Wnt agonist, R-spondin1, into the murine ECM-containing culture, some Ring colonies turn into &#34;Dense&#34; colonies 7. In this article, these two types of colonies grown in murine ECM culture are collectively referred to as &#34;Ring/Dense&#34; colonies. When Ring/Dense colonies are dissociated into single cell suspension and re-plated into cultures that contain laminin hydrogel, &#34;Endocrine/Acinar&#34; colonies are formed 7.
Using single colony analyses, it was found that the majority of Ring/Dense and Endocrine/Acinar colonies, either from adult (2-4 month-old) 7,11 or young (1 week-old) 9 murine pancreas, express all three lineage markers. This suggests that most of the originating PCFUs are tri-potent. In the murine ECM-containing colony assay, adult murine PCFUs robustly self-renew and expand approximately 500,000 times over 11 weeks in culture 7. Murine ECM preferentially supports the differentiation of ductal cells over endocrine and acinar lineages, whereas in the presence of laminin hydrogel, murine PCFUs are encouraged to differentiate preferentially into endocrine and acinar cells and less so to the ductal lineage 7,9,11. Importantly, Insulin+Glucagon- mono-hormonal cells are generated in the laminin hydrogel culture and secrete insulin in response to glucose stimulation in vitro 7,9, suggesting functional maturity. The tri-lineage differentiation potential 7,9 and self-renewal 11 of individual PCFUs are confirmed by single-cell micromanipulation, i.e., culturing one cell per well for colony formation. Together, these results provide evidence that there are self-renewing, tri-potent, progenitor-like cells in the postnatal murine pancreas that show activities in 3D culture.
The murine PCFU assays described in this article are derived from a prior colony assay designed for progenitor cells differentiated from murine embryonic stem cells (mESCs) 17. That protocol is documented in detail in another JoVE publication 18. The culture components and techniques required to perform the murine ECM-containing colony assay for adult PCFUs are the same as for mESC-derived progenitors 17,18. Therefore, these aspects of the assay will not be repeated here; instead the following procedures will be addressed: 1) dissociation of the adult pancreas and sorting CD133+Sox9/EGFP+ ductal cells, which enrich PCFUs from adult mice 7, 2) single-cell manipulation of the sorted cells, 3) single-colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of colonies into single-cell suspension and re-plating into murine ECM or laminin hydrogel colony assays.

<table><tbody><tr><td>Murine ECM proteins (Matrigel)</td><td>Becton Dickson (Franklin Lakes, NJ, USA)</td><td>354230</td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Laminin Hydrogel</td><td>Provided by David Tirrell (Pasadena, CA USA)</td><td></td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Methylcellulose</td><td>Shinetsu Chemical (Tokyo, Japan)</td><td></td><td>1,500 centipoise (dynamic viscosity unit equal to 15 g/cm/sec) (high viscosity)</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline</td><td>Mediatech (Manassas, VA, USA)</td><td>21-031-CV</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Gibco (Grand Island, NY, USA)</td><td>15070-063</td><td></td></tr><tr><td>50 ml Flacon Conical vial</td><td>Corning Inc. (Corning, NY, USA)</td><td>352070</td><td></td></tr><tr><td>100 mm x 20 mm Suspension culture dish</td><td>Corning Inc. (Corning, NY, USA)</td><td>430591</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma (St. Louis, MO, USA)</td><td>A8412</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco (Grand Island, NY, USA)</td><td>15070-063</td><td></td></tr><tr><td>DNase1</td><td>Calbiochem (Darmstadt, Germany)</td><td>260913</td><td></td></tr><tr><td>Collagenase B</td><td>Roche (CH-4070, Basel, Schweiz, Switzerland)</td><td>11088831001</td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Anti-mouse CD16/32</td><td>Biolegend (San Diego, CA, USA)</td><td>101310</td><td>low endotoxin, azide free</td></tr><tr><td>PE-Cy7 Rat IgG2a &amp;kappa; Isotype Control</td><td>Biolegend (San Diego, CA, USA)</td><td>400522</td><td></td></tr><tr><td>Rat IgG1 &amp;kappa; Isotype Control</td><td>eBioscience (San Diego, CA, USA)</td><td>13-4301-82</td><td></td></tr><tr><td>Anti-CD133-Biotin</td><td>eBioscience (San Diego, CA, USA)</td><td>13-1331-82</td><td></td></tr><tr><td>Anti-CD71-PE-Cy7</td><td>Biolegend (San Diego, CA, USA)</td><td>113812</td><td></td></tr><tr><td>Streptavidin-Allophycocyanin</td><td>Biolegend (San Diego, CA, USA)</td><td>405207</td><td></td></tr><tr><td>4&#039;,6-Diamidino-2-phenylindole</td><td>Invitrogen (Waltham, MA,&amp;nbsp; USA)</td><td>3571</td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Anti-mucin 1</td><td>Thermo Fisher Scientific (Waltham, MA USA)</td><td>HM-1630-P1</td><td></td></tr><tr><td>Dylight 649 Goat anti-Armenian Hamster</td><td>Jackson Immuno (West Grove, PA, USA)</td><td>127-495-160</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium: Nutrient Mixture F-12</td><td>Mediatech(Manassas, VA, USA)</td><td>10-092-CV</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Tissure Culture Biologicals (Long Beach, CA, USA)</td><td>101</td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Tris Ethylenediaminetetraacetic acid</td><td>TEKnova (Hollister, CA, USA)</td><td>T0221</td><td></td></tr><tr><td>Rneasy Micro Kit</td><td>Qiagen (Venlo, Netherlands)</td><td>74004</td><td></td></tr><tr><td>QuantiTec Reverse Transcription Kit</td><td>Qiagen (Venlo, Netherlands)</td><td>205310</td><td></td></tr><tr><td>CellsDirect One-Step qRT-PCR Kit</td><td>Ambion/Invitrogen(Grand Island, NY, USA)</td><td>11753-100</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Santa Cruz Bio (Santa Cruz, CA, USA)</td><td>SC-281692</td><td></td></tr><tr><td>Goat serum</td><td>Jackson Immuno (West Grove, PA, USA)</td><td>005-000-121</td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Donkey serum</td><td>Jackson Immuno (West Grove, PA, USA)</td><td>0017-000-121</td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Triton X-100</td><td>Sigma (St. Louis, MO, USA)</td><td>T9284</td><td></td></tr><tr><td>Trypsin</td><td>Sigma (St. Louis, MO, USA)</td><td>T-4799</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Invitrogen (Waltham, MA,&amp;nbsp; USA)</td><td>15575-020</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Life Technologies (Waltham, MA, USA)</td><td>25200-056</td><td></td></tr><tr><td>Sterile Water</td><td>Gibco (Grand Island, NY, USA)</td><td>15230-147</td><td>Molecular biology grade</td></tr><tr><td>Pasteur Pipette</td><td>Fisher Scientific (Pittsburgh, PA , USA)</td><td>13-678-8B</td><td></td></tr><tr><td>40 &amp;mu;m Filter Mesh</td><td>Fisher Scientific (Pittsburgh, PA , USA)</td><td>08-771-1</td><td></td></tr><tr><td>70 &amp;mu;m filter mesh</td><td>Fisher Scientific (Pittsburgh, PA , USA)</td><td>08-771-2</td><td></td></tr><tr><td>TC Plate 96 Well Suspension</td><td>Sarstedt</td><td>83.3924 (Previously 83.1835)</td><td></td></tr><tr><td>1 cc Syringe</td><td>Becton Dickson (Franklin Lakes, NJ, USA)</td><td>309659</td><td></td></tr><tr><td>10 cc Syringe</td><td>Becton Dickson (Franklin Lakes, NJ, USA)</td><td>301604</td><td></td></tr><tr><td>48.48 Dyanmic Array Chip</td><td>Fluidigm (San Francisco, CA, USA)</td><td>BMK-M-48.48</td><td></td></tr><tr><td>Fluidigm GE 48.48 Dynamic Array Sample &amp;amp; Assay Loading Reagent Kit</td><td>Fluidigm (San Francisco, CA, USA)</td><td>85000800</td><td></td></tr><tr><td>TaqMan Universal PCR Master Mix</td><td>Applied Biosystems (Grand Island, NY, USA)</td><td>4304437</td><td></td></tr><tr><td>Polyethylene glycol sorbitan monolaurate</td><td>Sigma (St. Louis, MO, USA)</td><td>P7949</td><td></td></tr><tr><td>Glass Bottom Dish</td><td>MatTek (Ashland, MA, USA)</td><td>P35G-1.5-14-C</td><td>35 mm Petri dish with glass bottom</td></tr><tr><td>Mouth Piece/Rubber Tubing</td><td>Renova Life Inc. (College Park, MD, USA)</td><td>MP-SET</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma (St. Louis, MO, USA)</td><td>N0636</td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Vascular Endothelial Growth Factor</td><td>R&amp;amp;D Systems (Minneapolis, MN, USA)</td><td>293-VE</td><td>Stock kept at -80 &amp;#176;C</td></tr><tr><td>Activin B</td><td>R&amp;amp;D Systems (Minneapolis, MN, USA)</td><td>659-AB</td><td>Stock kept at -80 &amp;#176;C</td></tr><tr><td>Extendin 4</td><td>Sigma (St. Louis, MO, USA)</td><td>E7144</td><td>Stock kept at -20 &amp;#176;C</td></tr><tr><td>Rspondin-1</td><td>R&amp;amp;D Systems (Minneapolis, MN, USA)</td><td>3474-RS</td><td>Stock kept at -80 &amp;#176;C</td></tr><tr><td>Falcon 5 ml Polystyrene Round-Bottom Tube</td><td>Corning Inc. (Corning, NY, USA)</td><td>352054</td><td></td></tr><tr><td>PrecisionGlide Needle 18 G x1 1/2</td><td>Becton Dickson (Franklin Lakes, NJ, USA)</td><td>305196</td><td></td></tr><tr><td>PrecisionGlide Needle 16 G x 1 1/2</td><td>Becton Dickson (Franklin Lakes, NJ, USA)</td><td>305198</td><td></td></tr><tr><td>Costar Ultra-Low Attachment Surface 24 well flat bottom plate&amp;nbsp;</td><td>Corning Inc. (Corning, NY, USA)</td><td>3473</td><td></td></tr><tr><td>Costar 96 Black Well Plate</td><td>Corning Inc. (Corning, NY, USA)</td><td>3603</td><td>Flat, clear bottom with lid.&amp;nbsp; Black polystyrene TC-treated microplates</td></tr><tr><td>Zeiss LSM510 META NLO Axiovert 200M Inverted Microscope</td><td>Carl Zeiss AG (Oberkochen, Germany)</td><td></td><td></td></tr><tr><td>Biomark HD&amp;nbsp;</td><td>Fluidigm (San Francisco, CA, USA)</td><td></td><td></td></tr><tr><td>Aria Special Order Research Product Cell Sorter</td><td>Becton Dickson (Franklin Lakes, NJ, USA)</td><td></td><td></td></tr><tr><td>PBS/BSA</td><td></td><td></td><td>1x PBS&lt;br/&gt;
0.1% (wt/vol) BSA&lt;br/&gt;
PenStrep (49 Units/ml Penicillin, 49 &amp;mu;g/ml Streptomycin)</td></tr><tr><td>PBS/BSA/Dnase1</td><td></td><td></td><td>1x PBS&lt;br/&gt;
0.1% (wt/vol) BSA&lt;br/&gt;
PenStrep (49 Units/ml Penicillin, 49 &amp;mu;g/ml Streptomycin)&lt;br/&gt;
Dnase 1 (2,400 Units/ml per pancreas)</td></tr></tbody></table>

in vitro colony assays for characterizing tri-potent progenitor cells isolated from the adult murine pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

Adult pancreatic progenitor cells can be enriched by fluorescence-activated cell sorting (Figure 1). The Sox9/EGFP transgenic mouse line used here was first generated as a result of the GENSAT Brain Atlas Project 19, and the EGFP reporter is under the control of a bacterial artificial chromosome containing ~75 kb upstream and ~150 kb downstream sequences of Sox9 20. In these mice, EGFP labels pancreatic ducts efficiently and specifically 22</sup...

Watch this Scientific Journal Video about In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas at JoVE.com

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

in-vitro-colony-assays-for-characterizing-tri-potent-progenitor-cells

Research

JoVE Journal

Developmental Biology

9.4K Views.  Beckman Research Institute of City of Hope.  In vitro testen van kolonies op zelfvernieuwing en differentiatie van progenitorcellen geïsoleerd uit volwassen muizen pancreas gedetecteerd worden gepland. In deze testen pancreatische voorlopercellen leiden tot cel kolonies in 3-dimensionale ruimte-methylcellulose bevattende halfvaste medium. Protocollen voor het manipuleren van enkelvoudige cellen en karakterisering van individuele kolonies worden beschreven. 

Video: In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic &#946;-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive &#946;-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression.

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding ...

Genetics

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Medicine

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling,&#160;however,&#160;with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell&#39;s physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to ...

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 &#181;m diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.

The differentiation of stem cells into islet cells provides an alternative solution to conventional diabetes treatment and disease modeling. We describe a detailed stem cell culture protocol that combines a commercial differentiation kit with a previously validated method to aid in producing insulin-secreting, stem cell-derived islets in a dish.

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Biology

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications 1. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages 2, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A 3 in combination with several growth factors 4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine 8. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation 9. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation 10,11.
The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.

The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...

Bioengineering

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

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