This research was supported by the S-Innovation project of the Japan Science and technology Agency (JST), the Graduate Program for Leaders in Life Innovation (GPLLI) of the University of Tokyo, and the Research Fellowship for Young Scientists of Japan Society for the Promotion of Science. We thank nac Image Technology Inc. for taking movies using a hi-speed camera.

1. חומרי הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן 10 חיץ HEPES מ&quot;מ. התאם את ה- pH 7.0 ב RT ולהוסיף NaCl 0.9%. </li><li style=";text-align:right;direction:rtl"> הכן 5% פתרון אלגינט ופתרון 10 מ&quot;מ EDTA על ידי ערבוב מלח HEPES שנאגר ערוכים 1.1. התאם את ה- pH 7.0 ב RT. </li><li style=";text-align:right;direction:rtl"> החיטוי חומרים כימיים (1.1, 1.2) במשך 20 דקות ב 121 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> להכין מאכלי 60 מ&quot;מ מצופה ג&#39;לטין שכבתי עם 1.2-2.0 פיברובלסטים עכבר עובריים × 10 6 (MEF) תאים, אשר נחשבים כתאים מזינים על ידי דגירה בתוך DMEM המכיל 10% FBS ES-מוסמך ו -10 מיקרוגרם / מיליליטר של C mitomycin 90 - 120 דקות. </li><li style=";text-align:right;direction:rtl"> לשמור על גזע pluripotent עכבר מושרה (iPS) תאים בצלחת עם התאים מזינים בנוכחות 5 מיליליטר הגבוה של גלוקוז DMEM המכיל 20% של ES-המוסמך FBS, 50 מיקרומטר של 2-mercaptoethanol, חומצת אמינו לא חיונית, ואנטיביוטיקה ( 100 / מיליליטר יחידות של פניצילין G נתרן, 100 מיקרוגרם / מיליליטר של סולפט סטרפטומיצין, ו -250 / מיליליטר ng של amphoteriCIN B). </li><li style=";text-align:right;direction:rtl"> זרע 4 × 5 תאי iPS 10 על צלחת 60 מ&quot;מ מצופה ג&#39;לטין ודגירתם על 37 מעלות צלזיוס, 5% CO 2. לשנות את מדיום התרבות בכל יום במהלך דגירה. לאחר 3-4 ימים של תרבות, trypsinize תאי דקות 1 עם 500 μl של 0.05% טריפסין המכיל EDTA 0.02% על 37 מעלות צלזיוס, 5% CO 2. אחרי זה, לנתק את התאים על ידי הקשה על צלחת התרבות עשר פעמים. </li><li style=";text-align:right;direction:rtl"> להוסיף 2.5 מיליליטר של DMEM עם 10% מהשעית תא צנטריפוגות ES-מוסמכת FBS ואנטיביוטיקה (להלן תאי iPS עכבר ניתוק לתוך תאים בודדים ידי pipetting עם micropipette 1000 μl שלוש פעמים) בשעה 160 × גרם במשך 3 דקות ולהסיר supernatant ולספור את התאים . בעקבות ספירת תאים, לזרוע תאים שנאספו על צלחת ג&#39;לטין מצופה ו דגירה למשך 30 דקות לבודד את תאי iPS מתאי MEF. לאחר הספירה של התאים (בדרך כלל 1-3 × 10 6 תאים / מנה יכולה להיות שנאספו), reseed 4 × 10 5 תאים iPS עלהצלחת. </li></ol> 2. תא Encapsulation לאלגינט Hydrogel קפסולות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאסוף את התאים על ידי אותו ההליך מתואר ב1.5 ו -1.6. בעקבות צנטריפוגה 160 × גרם במשך 3 דקות, לשטוף את התא גלולה iPS עם מי מלח HEPES שנאגר שלוש פעמים. לאחר מחדש השעיית התאים בתמיסת מלח HEPES שנאגר, לסנן את ההשעיה התא דרך מסננת 40 מיקרומטר תא כדי להסיר אגרגטים גדולים, שעלולים לסתום את החרירים. </li><li style=";text-align:right;direction:rtl"> מערבבים 2 מיליליטר של השעיה תא בתוך תמיסת מלח HEPES שנאגר ו 3 מיליליטר של 5% פתרון ALG-Na. לבסוף 5 מיליליטר של השעיה תא בתוך 3% פתרון ALG-Na מתקבל. צפיפות תאים היא desirably יותר מ 10 6 תאים / מיליליטר. </li><li style=";text-align:right;direction:rtl"> לאחר איסוף ההשעיה התא לתוך מזרק 5 מיליליטר, להתקין זרבובית שיתוף צירי (איור 1 א &#39;, ב&#39;) על המזרק ולהגדיר אותם על משאבת מזרק. זרימת גז 2 לפלוט N (נמוך מ1 ליטר / דקה) באמצעות המחט החיצונית של נחיר קואקס ולגרש את ההשעיה התא דרך הנחיר הפנימי. </li><li style=";text-align:right;direction:rtl"> לאסוף טיפות לתוך 250 מיליליטר של 0.5% פתרון CaCl 2 ולהמתין 10 - 20 דקות לgelation תוך ערבוב ב60-90 סל&quot;ד. </li></ol> 3. טיפול באלגינט קפסולות (אופציונאלי) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> דגירה כמוסות ALG-Ca ב0.05% (w / v) פתרון פולי-L ליזין (PLL) במשך 5 דקות ב 37 מעלות צלזיוס, 5% CO 2. </li><li style=";text-align:right;direction:rtl"> לאחר שטיפת הכמוסות עם מי מלח HEPES שנאגר, דגירתם בDMEM המכיל 10% FBS כדי לנטרל את המטען החשמלי על פני השטח שלהם. לנצל אותם ככמוסות מצופים (איור 1 ג). </li><li style=";text-align:right;direction:rtl"> דגירה הכמוסות במלח HEPES שנאגר המכיל 10 mM EDTA במשך 5 דקות. הכמוסות טופל EDTA מנוצלות ככמוסות חלולות (איור 1 ג). </li></ol> 4. תאי תרבות ואיסוף בקפסולות אלגינט <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> דגירה התאים במארז ב 75 סל&quot;ד עם רעד על 37 מעלות צלזיוס, 5% CO 2במשך 10 ימים. </li><li style=";text-align:right;direction:rtl"> לאסוף דגירה הכמוסות ב 10 מ&quot;מ EDTA על 37 מעלות צלזיוס, 5% CO 2 למשך 10 דקות. איסוף תאים מכמוסות אלגינט ללא טיפול PLL על ידי צנטריפוגה בז × 1,000 3 דקות. </li><li style=";text-align:right;direction:rtl"> אם מטופלים כמוסות אלגינט עם PLL, לשבור את קרום ALG-PLL ידי pipetting למעלה ולמטה כעשר פעמים עם מחט מחוברת למזרק ו צנטריפוגות זה ב 1000 - 5000 × גרם במשך 5 דקות. קוטר מחט צריך להיות נמוך יותר מגודל כמוסה ו -25 G מחטים (260 מיקרומטר) משמשות בניסוי זה (600 מיקרומטר) </li><li style=";text-align:right;direction:rtl"> לאחר צנטריפוגה, לאסוף תא גלולה לחלוטין מקרומי ALG-PLL שבור אם אתה רוצה לאסוף דגימות ה- mRNA מהתאים. קרומי ALG-PLL נותרו אולי למנוע טיהור mRNA. </li></ol>

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The authors have nothing to disclose.

<html> תרבות Encapsulation ניתן להשוות עם תרבויות השעיה ישירות. תרבות השעיה היא שיטה פשוטה כדי להשיג כמויות גדולות של תאי גזע pluripotent מאשר שיטות אנקפסולציה. עם זאת, השליטה הצבירה של תאים בתרבית השעיה היא עדיין מאתגרת. בשיטת אנקפסולציה, צבירה סלולרית מוגבלת בכמוסות וניתן לשלוט ו?...

Induced pluripotent stem cells (iPS cells) are currently the source of intense research due to their role in regenerative medicine. However, huge amounts of cells are required for tissue regeneration. For instance approximately one billion pancreatic cells required for a type 1 diabetic patient1. However, conventional dish culture is only able to obtain 1 &#215; 105 cells/cm2, thus requiring 1 m2 of culture area to obtain enough stem cell-derived pancreatic cells to treat a type 1 diabetic patient. The development of a system for the mass-culture of pluripotent stem cells, such as microcarrier2 and suspension culture is therefore required for regenerative medicine. Suspension culture represents a promising method of mass culture but controlling the aggregation of cells is challenging in direct suspension cultures of human iPS cells3. Indeed, suspended cells are exposed to shear stress, which causes cell damage3 or differentiation4.
Research into hydrogel-based encapsulation has been conducted to solve problems associated with suspension culture. In hydrogel capsules, cells are protected from the flow of the medium. Previous reports have documented the use of various types of hydrogel, including agarose5, PEG6, and alginate (Alg), for cellular encapsulation. Alg-Ca hydrogel is one of the most useful hydrogels for cell encapsulation because Alg&#8722;Ca hydrogel is formed immediately after dropping alginate solution into a CaCl2 solution and is also readily digested by enzymes or chelating reagents. 
Here, we have established a stable alginate encapsulation process for iPS cells using a co-axial nozzle. By using N2 gas flow for forming droplets, it is possible to encapsulate cells into uniform capsules without the need for other reagents such as oil. In this method, the flow rate of N2 and concentration of both CaCl2 and alginate are the major operating conditions affecting the size, shape, and uniformity of capsules. This report demonstrates the optimization of these operating conditions through the use of a hi-speed camera and a microscope.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Mouse embryo fibroblast</td>
			<td>Cell Biolabs</td>
			<td>SNL 76/7</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse induced pluripotent stem cell</td>
			<td>RIKEN Bio resorce centre</td>
			<td>iPS-MEF-Ng-20D-17</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM high-glucose</td>
			<td>GIBCO</td>
			<td>11995</td>
			<td></td>
		</tr>
		<tr>
			<td>ES qualified&#160; FBS</td>
			<td>GIBCO</td>
			<td>16141079</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibacterial Antibiotics</td>
			<td>GIBCO</td>
			<td>15240</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential Amino Acid</td>
			<td>GIBCO</td>
			<td>11140</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>GIBCO</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>ESGRO Leukemia Inhibitory Factor</td>
			<td>Merck Millipore</td>
			<td>ESG1107</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>GIBCO</td>
			<td>25300</td>
			<td></td>
		</tr>
		<tr>
			<td>26G/16G needle</td>
			<td>Hoshiseido</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Syringe</td>
			<td>TERUMO</td>
			<td>SS-10ESZ</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium&#160; Chloride</td>
			<td>Wako</td>
			<td>191-01665</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>SIGMA</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Alginate</td>
			<td>Wako</td>
			<td>194-09955</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Wako</td>
			<td>039-00475</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine (Mw=15,000-30,000)</td>
			<td>SIGMA</td>
			<td>P7890</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>DOJINDO</td>
			<td>345-01865</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylinge pump</td>
			<td>AS ONE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>IX71</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica</td>
			<td>DM IRB</td>
			<td></td>
		</tr>
		<tr>
			<td>Hispeed camera</td>
			<td>nac image technology</td>
			<td>Memrecam HX-3</td>
			<td></td>
		</tr>
	</tbody>
</table>

alginate encapsulation of pluripotent stem cells using a co-axial nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

בפרוטוקול 2.5, פתרון אלגינט גורש יוצר צורה כדורית מייד לאחר הגירוש (איור 2 א - ח). אם ההשעיה הוא גורש עם קצב זרימה N 2 נמוך יותר מאשר 1 ליטר / דקה, גודל הטיפות הוא אחיד (איור ט 2). עם זאת, אם את זרימת N 2 היא גבוהה יותר מאשר 1 ליטר / דקה, הטיפה מתפרקת <stro...

Watch this Scientific Journal Video about Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle at JoVE.com

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Encapsulation אלגינט של תאי גזע pluripotent שימוש נחיר Co-הצירית

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

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Developmental Biology

12.5K Views.  The University of Tokyo. We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Video: Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

דור של גזע תאים המושרה pluripotent מקפוא באפי מעילים באמצעות Episomal פלסמידים ללא שילוב

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

דור של תאי גזע האינטגרציה נטול האדם המושרה pluripotent שימוש קרטינוציטים נגזר שיער

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Differentiation of Atrial Cardiomyocytes from Pluripotent Stem Cells Using the BMP Antagonist Grem2

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed phenotypes. Researchers interested in pursuing studies involving specific myocyte subtypes require a more directed differentiation approach. By treating mouse embryonic stem (ES) cells with Grem2, a secreted BMP antagonist that is necessary for atrial chamber formation in vivo, a large number of cardiac cells with an atrial phenotype can be generated. Use of the engineered Myh6-DSRed-Nuc pluripotent stem cell line allows for identification, selection, and purification of cardiomyocytes. In this protocol embryoid bodies are generated from Myh6-DSRed-Nuc cells using the hanging drop method and kept in suspension until differentiation day 4 (d4). At d4 cells are treated with Grem2 and plated onto gelatin coated plates. Between d8-d10 large contracting areas are observed in the cultures and continue to expand and mature through d14. Molecular, histological and electrophysiogical analyses indicate cells in Grem2-treated cells acquire atrial-like characteristics providing an in vitro model to study the biology of atrial cardiomyocytes and their response to various pharmacological agents.

Generating cardiomyocytes from pluripotent stem cells in vitro allows access to large amounts of cardiac tissue in vitro for basic science and clinical applications. This protocol uses the atrializing factor Grem2 to both increase the numbers of cardiomyocytes obtained and to generate cardiomyocytes with an atrial phenotype.

דיפרנציאציה של פרפור cardiomyocytes מתאי גזע פלוריפוטנטיים שימוש Grem2 אנטגוניסט BMP

Protocols for generating populations of cardiomyocytes from pluripotent stem cells have been developed, but these generally yield cells of mixed ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

ייצור בקנה מידה גדולה של cardiomyocytes מתאי גזע פלוריפוטנטיים אדם באמצעות פרוטוקול בידול מולקולה קטן המבוססת לשחזור ביותר

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

את ייצור תאי גזע פלוריפוטנטיים מתאי מי שפירים עכבר באמצעות מערכת transposon

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

דור של תאי גזע מושרים אינטגרציה נטולה מן אדם הדם ההיקפי mononuclear תאי שימוש וקטורי Episomal

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

אופקי הר שלם: עיבוד רומן פרוטוקול הדמיה על רקמה עבה, תלת מימדי חתכים של העור

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

אינטגרציה חופשית של גזירה אנושית של תאים גזעיים Pluripotent באמצעות Laminin 521 מטריקס

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

שימוש האדם המושרה תאים דמויי Hepatocyte נגזר תאי גזע Pluripotent לגילוי סמים

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

מודל אנושי העכבר Chimeric הכבד היפרכולסטרולמיה משפחתית באמצעות Hepatocytes נגזר תאי גזע Pluripotent המושרה

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...