This work was supported by NIDDK grants DK098143 and DK101530 from the NIH (JPA), Stanford's Spectrum Child Health Research Institute (CHRI) and SPARK (UL1 TR001085, JPA).

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The authors declare that they have no competing financial interests.

שיטות ניסיוניות ללימוד המסלולים המולקולריים השולטים צמיחת β-cell והתחדשות הן כלים חשובים לחוקרי סוכרת. בזאת, פלטפורמת הקרנה מבוססת עכברוש-איון לזהות ולאפיין לגירוי-מולקולה הקטנה של שכפול β-cell מוצג. בעוד רוב ההיבטים של פרוטוקול זה ...

סוכרת כוללת אוסף של הפרעות שיתוף הסוף-נקודה המשותפת של הומאוסטזיס הגלוקוז שבש. למרות מנגנוני פתוגניים של תת סוכרת הם ברורים, הם חולקים תוצאה של מסת β-cell ירד, כלומר, אובדן כושר אינסולין ייצור 1,2. נכון להיום, אסטרטגיות טיפול בסוכרת לסמוך על ממשל כרוני של אינסולין אקסוגני, גירוי תרופתי של ייצור אינסולין או שיפור רגישות לאינסולין, ולעתים נדיר, השתלת איי לבלב או 3,4 לבלב כולו. למרבה הצער, הצלחת יישום אסטרטגיה זו היא קצרת מועד ו / או לא מצליח לשחזר את הפונקציה מספיק של אינסולין אנדוגני. למרות התועלת של מפתחי שיטה לעורר התחדשות β-cell, אין גישה כזו. כתוצאה מכך, מטרת מחקר הסוכר עיקרית היא לפתח שיטות כדי ליצור תאי β חדשים או להרחיב המוני β-cell אנדוגני 5 </sup&gt;. למרות התחדשות β-cell ממקורות מתחדשים כמו תאי גזע עובריים היא מתקדמת, חששות בטיחות ויעילות להפוך את המרדף של אסטרטגיות חלופיות, כולל הרחבת β-תאים בוגרים, בעדיפות 6,7. חשוב לציין כי מקור השולט של תאי-β חדש in vivo הוא-תאי β קיימים ולא ובתאים מיוחדים 8,9. למרות β-תאים נראה שיש יכולת שכפולה מוגבלת, עלייה קטנה במסת β-cell (~ 30%) עשויים להיות מספיק כדי לשחזר הומאוסטזיס הגלוקוז אצל חולי סוכרת רבה. יתר על כן, בגירוי תרופתי באתרו של המוני β-cell אסטרטגיית טיפול זול וניתן להרחבה פוטנציאלי. בזאת שיטה גבוה הקרנת תוכן לזיהוי ואפיון מולקולות קטנות המעודדות צמיחת β-cell מוצגת. מגוון שיטות במבחנה ניסיונית עשוי לשמש לזיהוי מוצרי גן ו / או מולקולות that לקדם שכפול β-תא ראשוני. מאמצים מוקדמים למדידת שטף שכפול תאי β בשימוש תרבות pancreata מכרסמים עוברים או תרבויות איון מבודד ללא פגע למדוד [3 ח] התאגדות thymidine, התאגדות BrdU או גופי mitotic בתוך האוכלוסייה מוכתמת אלדהיד-thionine או אינסולין בתגובה לתנאי טיפול ספציפי 10, 11. גישות במבחנה אלה הגירסות וקרובות יש ממנו מספר מגבלות. ליקויים בולטים כוללים (1) שימוש בתאי עובר אשר, בניגוד β-תאים בוגרים, להציג קצב שכפול β-תאי בסיס גבוה והם צמיחה מוסדרת באופן מובהק 12; (2) אופי סובייקטיבי שפיטה הנסיין תלויות אירועים שכפולים β-cell; (3) את העבודה וזמן הטבע אינטנסיבי של ספירת הנסיין תלוי אירועים שכפולים β-cell מפגר תפוקה ניסיון; (4) שימוש התאגדות / כתם / מראה גרעיני לזהות שכפול אפילוts וכתם ציטופלסמית שאינה חופף לזהות β-תאים מובילים misattribution של אירועים שכפולים שאינו β-cell בסמוך בטאו תאים. לאחרונה β-תאים ראשוניים בוגרים שמשו להעריך את ההשפעה של transgene יתר הביטוי כמו גם טיפול מוצר או תרכובת גן על שכפול β-cell 13-16. עם זאת, מחקרים אלה גם יש לסמוך על ספירת סובייקטיבית של אירועים שכפול, cytoplasmic staining- או שאינו ספציפי שיטות לזיהוי β-cell ו / או צעדים עתירי עבודה כי להגביל את התפוקה, למשל, ציפוי שקופית היטב פרט של תאים או איון שלם הטבעת פרפין ועיבוד 17. יש לציין, מתודולוגיה הקרנה שכפולה β-תאים אנושיים מבוסס תמונה, דומה לזו המוצגת כאן, כבר פורסמה 18; עם זאת, שימוש מוצלח של assay זה לא הודגם ושימוש איי אדם להקרנה עיקרית לא יכול להיות רחב FEAsible. אסטרטגיה חלופית לזיהוי חומרים לקידום שכפול היא להעריך אינדוקציה הצמיחה של קווי β-cell. מאמצים ראשוניים המשמשים β-cell-קווי טרנספורמציה כגון min6 תאים או INS 832/13-תאי 14,19-21. עם זאת,-שורות תאים אלה להפגין צמיחה רסן לשאת מעט דמיון β-תאים מובחנים היטב 22. כתוצאה מכך, יכולת צמיחת אינדוקציה היא מינימאלית, של רלוונטיות ברורות ולפעמים קשה לשחזר. אסטרטגיה משופרת להקרנה מבוססת שורת תאים מנצלת &quot;טרנספורמציה הפיך&quot; תאים הנמצאים עצירת גדילה בהעדר טטרציקלין (דוקסיציקלין) ביטוי אנטיגן התלוי SV40 T 23,24. עם זאת, לא ברור אם תאים אלה לחזור למצב &quot;נורמלי&quot; β-דמוי תא עם הסרת דוקסיציקלין. למרבה הצער, שימוש בתאים אלה הניב תרכובות מקדמת גדילה כללית כי לא נראה שיש שתועלת מיידית24. באופן כללי, השימוש-שורות תאים ללמוד ויסות צמיחה של תאים מסוג מוצגים פעילות שכפולה ספונטנית מינימאלית עשוי להיות תחולה מוגבלת. פלטפורמת ההקרנה שהכפולה β-cell המוצגת כאן מנצלת עכברוש העיקרי בוגרים β-תאים לשמר ברגולציה של צמיחת vivo במידת ההאפשר, תרבויות תאי איון של רכב תא מסוג מעורב כדי לאפשר זיהוי של פעילויות לקידום צמיחה מוגבלת שושלת, רבה &quot;טוב עיצוב על מנת למקסם את תפוקת ניתוח אוטומטי לחסל הטיה ולהקל תפוקה. שימוש מוצלח של פלטפורמה זו אפשר זיהוי של מספר תרכובות המקדמות שכפול β-cell 25,26. בנוסף, assay שמש במשך מחקרי יחסי מבנה-פעילות וניסויי epistasis כימיים לספק תובנות מכניסטית לתוך תקנה המולקולרי של שכפול β-cell. הפלטפורמה הציג הותאמה בהצלחה FOr lentiviral RNAi מבוסס חקירת שכפול β-cell מסלולי 25. מגבלות של assay כוללות מוגבלות מדרגיות (שימוש של תאים ראשוניים), שימוש מכרסם ולא-תאי איון אדם (אם כי assay יכול להיות מותאם עבור מחקרי איון אדם), הוצאות הקשורות הדמיה מבוססת נוגדן ושימוש איון עיקרי, השימוש איים מפוזרים (אדריכלות איון שבש) כדי להקל על רכישה ותלות תמונה אוטומטיות על הזמינות של מיקרוסקופ אוטומטית עם רכישת תמונה ויכולת ניתוח. למרות מתודולוגיה הקרנה הקלילה in vivo לזיהוי מוצרי גנים או תרכובות מעודדי התחדשות β-cell באתרו תהיה אידיאלית, פלטפורמת דוגמת אינה זמינה עדיין 27. כתוצאה מכך, הפלטפורמה תארה מתאימה חוקר מעוניין לחקור את רוב ההיבטים של שכפול β-cell.

<table><tbody><tr><td>250 g male Male Sprague Dawly Rat</td><td>Charles River</td><td>Stain # 400</td><td></td></tr><tr><td>12 cm teeth tisuue forceps</td><td>Fine Science Tools</td><td>11021-12</td><td></td></tr><tr><td>11.5 cm fine scissors</td><td>Fine Science Tools</td><td>14058-11</td><td></td></tr><tr><td>14.5 cm surgical scissors</td><td>Fine Science Tools</td><td>14001-14</td><td></td></tr><tr><td>16 cm curved forceps</td><td>Fine Science Tools</td><td>11003-16</td><td></td></tr><tr><td>12 cm curved hepostat</td><td>Fine Science Tools</td><td>13011-12</td><td></td></tr><tr><td>12 cm scalpel handle</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>Tissue sieve-30 mesh</td><td>Bellco Glass</td><td>1985-85000</td><td></td></tr><tr><td>Cizyme RI, 375,000 CDA units</td><td>VitaCyte</td><td>005-1030</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt solution (Ca&lt;sup&gt;++&lt;/sup&gt; and Mg&lt;sup&gt;++&lt;/sup&gt;)</td><td>Gibco</td><td>24020-117</td><td></td></tr><tr><td>Ketamine HCl (200 mg/20 ml)</td><td>JHP Pharmaceuticals</td><td>NDC# 42023-113-10</td><td>to make anesthetic cocktail&amp;nbsp;</td></tr><tr><td>Xylazine (5 g/50 ml)</td><td>LLOYD</td><td>NADA# 139-236</td><td>to make anesthetic cocktail&amp;nbsp;</td></tr><tr><td>Histopaque 1077</td><td>Sigma</td><td>H-1077</td><td>to make histopaque 1100</td></tr><tr><td>Histopaque 1119</td><td>Sigma</td><td>H-1119</td><td>to make histopaque 1100</td></tr><tr><td>Newborn Calf Serum 500 ml</td><td>Hyclone</td><td>SH30118.03</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt solution</td><td>Hyclone</td><td>SH30268.01</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium/Low Glucose&amp;nbsp;</td><td>Hyclone</td><td>SH30021.01</td><td></td></tr><tr><td>Functionality/Viability Solution&amp;nbsp;</td><td>Mediatech</td><td>99-768-CV</td><td></td></tr><tr><td>RPMI1640 media&amp;nbsp;</td><td>Hyclone</td><td>SH30096.01</td><td>to make conditioned medium</td></tr><tr><td>804G rat bladder carcinoma cell-line</td><td>Available upon request</td><td></td><td>to make conditioned medium</td></tr><tr><td>Fetal Bovine Serum, Qualified</td><td>Gibco</td><td>26160</td><td></td></tr><tr><td>GlutaMax-I</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>Penicillin (5,000 IU/ml/Strptomycin (5 mg/ml)&amp;nbsp;</td><td>MP Biomedicals</td><td>1670049</td><td></td></tr><tr><td>Formamide 500 ml</td><td>Fisher BioReagents</td><td>BP227-500</td><td></td></tr><tr><td>Antigen Unmasking Solution 250 ml (pH 6.0)</td><td>Vector Laboratories</td><td>H-3300</td><td>to make 0.15 M Sodium Sitrate solution</td></tr><tr><td>Dextrose, Anhydrous</td><td>EMD Chemicals</td><td>DX0145-1</td><td>to make 1 M glucose solution</td></tr><tr><td>Nomal Donkey Serum (Powder)</td><td>Jackson ImmunoResearch</td><td>017-000-121</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787-100ML</td><td></td></tr><tr><td>Mouse anti-human Ki67 antibody</td><td>BD Biosciences</td><td>556003</td><td></td></tr><tr><td>Goat anti-human PDX-1 antibody</td><td>R&amp;amp;D Systems</td><td>AF2419</td><td></td></tr><tr><td>Polyclonal Guinea Pig anti-insulin antibody</td><td>Dako</td><td>2016-08</td><td></td></tr><tr><td>Polyclonal Rabbit anti-glucagon antibody</td><td>Dako</td><td>2014-06</td><td></td></tr><tr><td>Polyclonal Rabbit anti-somatostatin antibody</td><td>Dako</td><td>2011-08</td><td></td></tr><tr><td>Polyclonal chicken anti-vimentin antibody</td><td>abcam</td><td>ab24525</td><td></td></tr><tr><td>Biotin-SP-conjugated, Donkey Anti-Mouse IgG</td><td>Jackson ImmunoResearch</td><td>715-065-150</td><td></td></tr><tr><td>StreptAvidin, Alex Flour 488 conjugated&amp;nbsp;</td><td>Invitrogen</td><td>S32354</td><td></td></tr><tr><td>Rhodamine-conjugated Donkey Anti-Goat IgG&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>705-025-147</td><td></td></tr><tr><td>Rhodamine-conjugated Donkey Anti-Guinea Pig IgG&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>706-025-148</td><td></td></tr><tr><td>Rhodamine-conjugated Donkey Anti-Rabbit IgG</td><td>Jackson ImmunoResearch</td><td>711-025-152</td><td></td></tr><tr><td>Cy 5-conjugated Donkey Anti-Guinea Pig IgG&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>706-175-148</td><td></td></tr><tr><td>Cy 5-conjugated Donkey Anti-Goat IgG</td><td>Jackson ImmunoResearch</td><td>705-175-147</td><td></td></tr><tr><td>Cy 5-conjugated Donkey Anti-Rabbit IgG</td><td>Jackson ImmunoResearch</td><td>711-175-152</td><td></td></tr><tr><td>Cy 5-conjugated Donkey Anti-Chicken IgG</td><td>Jackson ImmunoResearch</td><td>703-175-155</td><td></td></tr><tr><td>DAPI</td><td>Millipore</td><td>S7113</td><td></td></tr><tr><td>Disposable Reagent Reservoir 25 ml</td><td>Sorenson BioScience</td><td>39900</td><td></td></tr><tr><td>384 well, black/clear, tissue culture treated plate</td><td>BD Falcon</td><td>353962</td><td></td></tr><tr><td>96 well, black/clear, tissue culture treated plate</td><td>Costar</td><td>3603</td><td></td></tr><tr><td>Multi-channel pipettor</td><td>Costar</td><td>4880</td><td></td></tr><tr><td>12-channel vaccume aspirator</td><td>Drummond</td><td>3-000-096</td><td></td></tr><tr><td>Cell Scraper</td><td>Falcon</td><td>353085</td><td></td></tr><tr><td>Isotemp Water Bath Model 2223&amp;nbsp;</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>High-content screening instrument: ArrayScan VTI</td><td>Thermo Scientific</td><td></td><td></td></tr></tbody></table>

a high-content in vitro pancreatic islet &#946;-cell replication discovery platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

כדי להעריך β-cell או שכפול תאי α, פרוטוקול assay בארבעת צבעים נדרשים. ראשית, אובייקטים מזוהים על ידי מכתים DAPI (ערוץ 1, 386 ננומטר). לאחר מכן, β-תאי (אירוע 1) נספרים: יצירות כי שיתוף להביע PDX-1 + (ערוץ 2, 650 ננומטר) ואינסולין פרי-גרעיני (ערוץ 3, 549 ננומטר). בהמשך לכ...

Watch this Scientific Journal Video about A High-content In Vitro Pancreatic Islet Î²-cell Replication Discovery Platform at JoVE.com

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

אתגרים קריטיים לתחום מחקר הסוכר מעוניינים להבין את המנגנונים המולקולריים מווסת שכפול β-cell איון לפתח שיטות מגרה התחדשות תאי β. בזאת שיטה גבוהה ההקרנה תוכן לזהות ולהעריך את הפעילות β-cell לקידום שכפול של מולקולות קטנות מוצג.

A-תכולה גבוהה<em&gt; במבחנה</em&gt; פלטפורמת Discovery הלבלב איילט β-cell שכפול

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

a-high-content-vitro-pancreatic-islet-cell-replication-discovery

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

11.1K Views.  Stanford University School of Medicine.  אתגרים קריטיים לתחום מחקר הסוכר מעוניינים להבין את המנגנונים המולקולריים מווסת שכפול β-cell איון לפתח שיטות מגרה התחדשות תאי β. בזאת שיטה גבוהה ההקרנה תוכן לזהות ולהעריך את הפעילות β-cell לקידום שכפול של מולקולות קטנות מוצג. 

Video: A High-content In Vitro Pancreatic Islet β-cell Replication Discovery Platform

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.

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.

מבחני מושבת Vitro לאפיון ובתאי Tri-עצמה מבודדת מלבלב Murine למבוגרים

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. ...

Developmental Biology

Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, respectively. Functional beta cell mass is determined by the anatomical beta cell mass as well as the ability of the beta cells to respond to a nutrient load. A loss of functional beta cell mass is central to both major forms of diabetes 1-3. Whereas the declining functional beta cell mass results from an autoimmune attack in type 1 diabetes, in type 2 diabetes, this decrement develops from both an inability of beta cells to secrete insulin appropriately and the destruction of beta cells from a cadre of mechanisms. Thus, efforts to restore functional beta cell mass are paramount to the better treatment of and potential cures for diabetes.
Efforts are underway to identify molecular pathways that can be exploited to stimulate the replication and enhance the function of beta cells. Ideally, therapeutic targets would improve both beta cell growth and function. Perhaps more important though is to identify whether a strategy that stimulates beta cell growth comes at the cost of impairing beta cell function (such as with some oncogenes) and vice versa.
By systematically suppressing or overexpressing the expression of target genes in isolated rat islets, one can identify potential therapeutic targets for increasing functional beta cell mass 4-6. Adenoviral vectors can be employed to efficiently overexpress or knockdown proteins in isolated rat islets 4,7-15. Here, we present a method to manipulate gene expression utilizing adenoviral transduction and assess islet replication and beta cell function in isolated rat islets (Figure 1). This method has been used previously to identify novel targets that modulate beta cell replication or function 5,6,8,9,16,17.

This protocol allows one to identify factors that modulate functional beta cell mass to find potential therapeutic targets for the treatment of diabetes. The protocol consists of a streamlined method to assess islet replication and beta cell function in isolated rat islets following manipulation of gene expression with adenoviruses.

מכרסם איי הערכת שכפול של התא פונקציה בטא Adenovirally-transduced מבודדים

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, ...

Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis. It is becoming increasingly clear that interactions between transmembrane proteins on the surfaces of adjacent beta cells are important determinants of beta-cell function.
Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of mRNA and protein expression, potentially affecting beta-cell health and/or function in ways that could confound analyses of the effects of specific interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and may prevent effects due to interactions between proteins within the same cell membrane to be distinguished from the effects of transcellular interactions.
Here a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is presented. This method is applicable to beta-cell lines, such as INS-1 cells, and to dissociated primary beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers identified proteins important for driving synapse formation. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell functional maturation might be driven by similar transcellular interactions. We developed a system where beta cells are cultured on a layer of HEK293 cells expressing a protein of interest. In this model, the beta-cell cytoplasm is untouched while extracellular protein-protein interactions are manipulated. Although we focus here primarily on studies of glucose-stimulated insulin secretion, other processes can be analyzed; for example, changes in gene expression as determined by immunoblotting or qPCR.

Transcellular protein interactions are important determinants of pancreatic beta-cell function. Detailed here is a method&#x2014;adapted from a coculture model of synaptogenesis&#x2014;for investigating how specific transmembrane proteins influence insulin secretion. Transfected HEK293 cells express proteins of interest; beta cells do not need to be transfected or otherwise directly perturbed.

ניתוח Coculture של אינטראקציות חלבון תאיות המשפיעות על הפרשת אינסולין על ידי תאים בטא בלבלב

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Transcriptomic תא בודד ניתוחים של תאים בלבלב האנדוקרינית העכבר

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Sustained Administration of &#946;-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic &#946;-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to &#946;-cells in culture and in transplanted tissue models. These technologies allow the study of candidate &#946;-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating &#946;-cell proliferation in a culture system is critical before moving forward to in vivo models. Herein, we describe a method to co-culture intact mouse islets with biodegradable compound of interest (COI)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for the purpose of assessing the effects of sustained in situ release of mitogenic factors on &#946;-cell proliferation. This technique describes in detail how to generate PLGA microspheres containing a desired cargo using commercially available reagents. While the described technique uses recombinant human Connective tissue growth factor (rhCTGF) as an example, a wide variety of COI could readily be used. Additionally, this method utilizes 96-well plates to minimize the amount of reagents necessary to assess &#946;-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of &#946;-cell proliferation. This method is suitable for determining the efficacy of candidate &#946;-cell mitogens.

מנהל מתמשך של mitogens β-cell כדי איי עכבר Intact<em&gt; Ex Vivo</em&gt; שימוש פוליפוני מתכלה (לקטית-שיתוף גליקולית וחומצה) מיקרוסכמות

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the ...

Bioengineering

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.

התמיינות של תאי גזע פלוריפוטנטיים אנושיים למבשרי תאי בטא בלבלב במערכת תרבית דו-ממדית

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.

התמיינות של תאי גזע פלוריפוטנטיים אנושיים לצברי איים מייצרי אינסולין

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

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

שיטה לבידוד עכבר הלבלב איילט וקביעת תאית cAMP

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Biology

Pancreatic Islet Embedding for Paraffin Sections

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture. For in vivo islet studies, paraffin-embedded pancreas sectioning is a powerful technique to assess cell-specific outcomes in the native pancreatic environment. Studying post-culture islets by paraffin sectioning would offer several advantages: detection of multiple outcomes on the same islets (potentially even the exact-same islets, using serial sections), cell-type-specific measurements, and maintaining native islet cell-cell and cell-substratum interactions both during experimental exposure and for analysis. However, existing techniques for embedding isolated islets post-culture are inefficient, time consuming, prone to loss of material, and generally produce sections with inadequate islet numbers to be useful for quantifying outcomes. Clinical pathology laboratory cell block preparation facilities are inaccessible and impractical for basic research laboratories. We have developed an improved, simplified bench-top method that generates sections with robust yield and distribution of islets. Fixed islets are resuspended in warm histological agarose gel and pipetted into a flat disc on a standard glass slide, such that the islets are distributed in a plane. After standard dehydration and embedding, multiple (10+) 4 - 5 &#181;m sections can be cut from the same islet block. Using this method, histological&#160;and immunofluorescent analyses can be performed on mouse, rat, and human islets. This is an effective, inexpensive, time-saving approach to assess cell-type-specific, intact-architecture outcomes from cultured islets.

Ex vivo pancreatic islet studies are important for diabetes research. Existing techniques to study cultured islets in their native 3-dimensional architecture are time consuming, inefficient, and infrequently used. This work describes a new, simple, and efficient method for generating high-quality paraffin sections of whole cultured islets.

הלבלב איון הטבעה למקטעי פרפין

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a ...

אירועי איתות תוך-תאי והפרשת הורמונים בפסאודואיסטים אנושיים

Human Pseudoislet System for Synchronous Assessment of Fluorescent Biosensor Dynamics and Hormone Secretory Profiles

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, have a central role in the control of glucose homeostasis through the secretion of insulin by beta cells, which lowers blood glucose, and glucagon by alpha cells, which raises blood glucose. Intracellular signaling pathways, including those mediated by cAMP, are key for regulated alpha and beta cell hormone secretion. The 3D islet structure, while essential for coordinated islet function, presents experimental challenges for mechanistic studies of the intracellular signaling pathways in primary human islet cells. To overcome these challenges and limitations, this protocol describes an integrated live-cell imaging and microfluidic platform using primary human pseudoislets generated from donors without diabetes that resemble native islets in their morphology, composition, and function. These pseudoislets are size-controlled through the dispersion and reaggregation process of primary human islet cells. In the dispersed state, islet cell gene expression can be manipulated; for example, biosensors such as the genetically encoded cAMP biosensor, cADDis, can be introduced. Once formed, pseudoislets expressing a genetically encoded biosensor, in combination with confocal microscopy and a microperifusion platform, allow for the synchronous assessment of fluorescent biosensor dynamics and alpha and beta cell hormone secretory profiles to provide more insight into cellular processes and function.

Author Spotlight: Investiting Islet Abnormalities and Function with a Pseudoislet Protocol 

This protocol describes a method for the synchronous acquisition and co-registration of intracellular signaling events and the secretion of insulin and glucagon by primary human pseudoislets using the adenoviral delivery of a cyclic adenosine monophosphate (cAMP) biosensor, a cAMP difference detector in situ (cADDis), and a microperifusion system.

מערכת פסאודואיסלט אנושית להערכה סינכרונית של דינמיקה ביו-חיישנית פלואורסצנטית ופרופילי הפרשת הורמונים

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, ...

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