This study was supported by Natural Sciences and Engineering Research Council of Canada Discovery Grant (371697-2011, AT). The authors want to acknowledge the help of bariatric surgeons Drs S. Biron, F-S. Hould, S. Lebel, O. Lescelleur, P. Marceau as well as Christine Racine and Caroline Gagnon from the IUCPQ Tissue Bank. We thank Mr Jacques Cadorette from the IUCPQ&#8217;s audiovisual services for video shooting and editing.

הצהרת אתיקה: הפרויקט אושר על ידי ועדת האתיקה של מחקר IUCPQ לפני גיוס חולה. לצורך המאמר / הווידאו הזה, שהשגנו רקמות מ2 חולים: 1) מטופל זכר 62 שנים ישנות עם BMI של 50.7 קילוגרם / מ 2 ו- 2) מטופלת בת 35 עם BMI של 57 קילוגרם / M 2. ניתן לעשות ניסויים עם שני תאי השומן, אך היו מוגבלים לתא אחד שומן לצורך זה וידאו. היבטים טכניים של הווידאו בוצעו עם מטופל 1 וimmunofluorescence FABP4 בוצע בתאי dedifferentiated מחולה 2. עיבוד 1. לדוגמא <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> שאל מנתחים לאסוף רקמת שומן מתאי השומן תת עורי וomental בעת הניתוח בריאטרי לפרוסקופי. </li><li style=";text-align:right;direction:rtl"> במהירות להביא דוגמאות שומן למעבדה בRT ולעבד באופן מיידי. </li><li style=";text-align:right;direction:rtl"> בצע עיכול במעבדה, באווירה שאינה סטרילי.תאי סופו של דבר הועברו לחדר התרבות וטיפחו בתנאים סטריליים. כדי למנוע זיהום, להכין חיץ KRH עם מים מזוקקים וfiltrated ומעקב על ידי סינון (מסנן 0.22μM) לפני העיכול. צינורות לנקות ביסודיות עם העברה לפני אתנול במכסת מנוע תרבית תאים לבקבוק והכנת צלחת. </li><li style=";text-align:right;direction:rtl"> הנח רקמת שומן במנה משוקללת מראש ומשקל שיא. תקן את חתיכה קטנה של כל דגימת רקמה (סנטימטר פחות מ 1 2) ב 10% חיץ פורמלין ב RT לפחות 24 שעות לפני הטבעת פרפין. השתמש בדוגמה זו הגלומה לניסויי אימונוהיסטוכימיה (טכניקה לא מוצגת). </li><li style=";text-align:right;direction:rtl"> מניחים חתיכה בצינור 50 מ&quot;ל ובזק-הקפאה בחנקן נוזלי אחרת לפני האחסון ב -80 ° C למחקרים נוספים ברקמות שומן שלמות (למשל, ביטוי גנים - טכניקה לא מוצג). </li></ol> 2. Collagenase עיכול <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים את פיסת רקמת שומן שנותרה במיליליטר tu 50להיות לעיכול. </li><li style=";text-align:right;direction:rtl"> הוסף 4 מיליליטר של KRH-WB בתוספת collagenase (350 U / ml) לגרם של מדגם בצינור העיכול. </li><li style=";text-align:right;direction:rtl"> ברר ברקמת שומן במספריים. </li><li style=";text-align:right;direction:rtl"> מניחים השעיה רקמת שומן טחון שייקרה, 37 ° C, 90 מקסימום סל&quot;ד, לדגירה של 45 דקות (שעה 1 המרבית). </li></ol> 3. טיהור של תאי שומן וPreadipocytes <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> יוצקים את הפתרון השקוף עם כמה נתחי השומן באמצעות ניילון 400 מיקרומטר רשת לתוך כוס פלסטיק. </li><li style=";text-align:right;direction:rtl"> עם פינצטה, לשפשף את הכנת התא ברשת הניילון ולשטוף עם 5 מיליליטר של KRH-WB. </li><li style=";text-align:right;direction:rtl"> בעדינות להעביר את ההשעיה תא filtrated לתוך צינור 50 מיליליטר עם צינורות פלסטיק בזה ומזרק 60cc המצורף בקצה הצינור. </li><li style=";text-align:right;direction:rtl"> בואו ההשעיה עם adipocytes הבוגרת עומדת על כ 10 דקות, שמאפשרת לתאים כדי להגיע לחלק העליון של המאגר על ידי ציפה. </li><li style=";text-align:right;direction:rtl"> לאט לאט לשאוב החיץ בחלק התחתון של הצינור באמצעות syrin 60ccיניקת ge. </li><li style=";text-align:right;direction:rtl"> להוסיף 20 מיליליטר של KRH-WB לשטוף. חזור מצעד 3.4 ל -2 שוטף נוסף. </li><li style=";text-align:right;direction:rtl"> לאסוף את החיץ להביא את ההשעיה adipocyte לנפח סופי של 5 או 10 מיליליטר, בהתאם לכמות תאים. להמשיך עם שלבים בסעיף 5. </li><li style=";text-align:right;direction:rtl"> לשחזר את חלק סטרומה וכלי דם למאגר שנאסף עם מזרק 60cc ידי צנטריפוגה (3,000 סל&quot;ד, RT, 5 דקות) לתרבית תאים עיקריות נוספת אם תרצה בכך (טכניקה לא מוצגת). </li></ol> ספירת תאים 4. למבוגרים adipocyte <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> טען 10 μl של ההשעיה adipocyte מזועזעת בעדינות בתא ספירה (haemocytometer). לבצע ספירת תאים ארבע פעמים. </li><li style=";text-align:right;direction:rtl"> לחשב מספר התאים בוגרים מבודדים. </li></ol> 5. מבוגרים adipocyte Dedifferentiation לT-25 Flask <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מלא בקבוק תרבות 2 רקמה 25 סנטימטרים עד ¾ מההיקף עם / F12-20% בסרום עגל DMEM. </li><li style=";text-align:right;direction:rtl"> , על פי ספירת תאים לשפוך 500,000 תאים בוגרים לתוך הבקבוק. </li&gt; <li style=";text-align:right;direction:rtl"> למלא את הבקבוק באמצעות צינור 50 מ&quot;ל עם מדיום לחלוטין ולהסיר בועות רבות ככל האפשר. </li><li style=";text-align:right;direction:rtl"> לדפוק את כובע unvented על הבקבוק. </li><li style=";text-align:right;direction:rtl"> נקה את הבקבוק עם אתנול לפני דגירה, כדי למנוע זיהום. </li><li style=";text-align:right;direction:rtl"> דגירה הבקבוק הפוך במשך שבוע מבלי לגעת בו כדי למנוע תנועה בתרבות שעלולה לשבש את הדבקות סלולרית. </li><li style=";text-align:right;direction:rtl"> לפני היפוך הבקבוק ב 7 ימים של תרבות ההפוכה, בעדינות לתפעל את הבקבוק ולהסיר את כל המדיום בבקבוק על ידי שאיפה, הימנעות תנועות פתאומיות. </li><li style=";text-align:right;direction:rtl"> להוסיף 12 מיליליטר של סרום עגל% DMEM-F12-20 ולטפח תאים עם טכניקות סטנדרטיים. כובע מסונן, פרק ניתן להוסיף לבקבוק. </li></ol> 6. למבוגרים adipocyte Dedifferentiation לתוך צלחת 6-גם <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> מניחים coverslip על החלק התחתון של כל טוב של 6-גם צלחת </li><li style=";text-align:right;direction:rtl"> הוסף &quot;תותב פלסטיק ½ על גבי כל coverslip. </li><li style=";text-align:right;direction:rtl"> מלא בארות עם 8 מיליליטר של 20% בינוניים עגל סרום-DMEM. </li><li style=";text-align:right;direction:rtl"> שים coverslip על כל תותב פלסטיק. </li><li style=";text-align:right;direction:rtl"> הכנס קצה pipet בין השקופיות והצינור להזרמת תאים שמתחת לסליידר (50,000 תאים לכל טוב). </li><li style=";text-align:right;direction:rtl"> דגירה צלחות בתרבית תאי חממה סטנדרטית על 37 מעלות צלזיוס עם 5% CO 2 במשך שבוע. </li><li style=";text-align:right;direction:rtl"> coverslip ההפוך עם תאים מצורפים לכל תרבות היטב המכיל 2 מיליליטר של תקשורת בתוספת 20% בסרום עגל ולהמשיך. </li><li style=";text-align:right;direction:rtl"> השתמש coverslip עם התאים עוברים dedifferentiation למספר מטרות כוללים immunofluorescence (טכניקה לא מוצגת). </li></ol>

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The authors declare no conflict of interest.

Dedifferentiation של תאי שומן בוגרים עם טכניקת תרבות התקרה היא גישה חדשה להשגת תאי גזע שומן ממדגם קטן של רקמת שומן מקומית. בהתבסס על הניסיון שלנו ושל אחרים 2, גרם אחד של רקמה מספיקה לצלחת בקבוק 2 25 סנטימטר ולהשיג אוכלוסייה של תאי DFAT שהומוגניות הודגמה על ידי Poloni ומשת?...

In vitro dedifferentiation of mature adipocytes is achieved through a technique called ceiling culture1. Because of their natural tendency to float in aqueous solutions, isolated mature adipocytes adhere to the surface of an inverted flask fully filled with culture medium. Over a few days, cells modify their spherical morphology and become fibroblast-like cells. The resulting cells, called dedifferentiated fat (DFAT) cells, are multipotent2. Research articles on adipocyte dedifferentiation, especially on human cells, are limited. However, they have already provided interesting information regarding multipotency, cell phenotype and replicative capacity of DFAT cells2. Mature adipocytes originating from various fat compartments have been successfully dedifferentiated including those originating from human visceral and subcutaneous adipose tissues2-4. In addition to these depots, Kishimoto and collaborators sampled adipose tissue from the buccal fat pads and dedifferentiated adipocytes into DFAT cells5. Matsumoto and collaborators successfully generated subcutaneous DFAT cells from patients covering a wide range of ages, and the majority of cells had a high proliferative rate and less than 6% of senescence even after 10 passages in culture2.
DFAT cells have been successfully re-differentiated into several lineages, including adipogenic, osteogenic, chondrogenic and neurogenic lineages2,3,6. These cells express several embryonic stem cell markers such as Nanog and the four identified pluripotent factors Oct4, c-myc, Klf4 and Sox23. They also express markers specific to each of the three germ layers7. In addition, DFAT cells are similar to Bone Marrow-derived Mesenchymal Stem Cells (BM-derived MSC) based on their epigenetic signature3. Exploiting the stem cell capacity of DFAT cells, many groups have investigated their potential to treat or improve various diseases8,9. Improvements of pathologic conditions, such as infracted cardiac tissue, spinal cord injury and urethral sphincter dysfunction, have been observed with DFAT cell injections in rat models of disease10-12. 
In addition to the stem cell properties of DFAT cells, they may represent a new cellular model for adipocyte physiology studies. The 3T3-L1 cell line is often used for this purpose as these cells differentiate into adherent, lipid-storing adipocytes under adipogenic stimulation13. However, these cells originate from mouse embryo tissue13. Also, depot-specificity cannot be investigated with this model and it may not fully reflect human adipocyte physiology14. Other laboratories work with isolated adipose cells from murine fat depots, but fat distribution is not dimorphic in mice and anatomical configuration of the rodent's abdominal cavity prevents from extrapolating directly to humans15. In order to study adipocytes in the context of the physiopathology of human obesity, consideration of body fat distribution and fat depot-specific differences has become essential16. Some limitations of primary preadipocyte cultures, including cell quantities obtained from adipose tissue biopsy samples and their senescence after a few passages in culture, created the need for alternate models. Perrini and collaborators investigated depot-specificity in gene expression of DFAT cells originating from visceral and subcutaneous fat and compared them to adipose-derived stem cells (ASC) from the same fat depot. They demonstrated that differences in gene expression and function where mainly found between depots than between cell types, suggesting that DFAT cells are physiologically close to ASC from the same depot. DFAT cells may represent an interesting alternative to available models for studies on fat distribution in the pathophysiology of human obesity. Moreover, ceiling culture is a promising method to obtain adult stem cells for tissue engineering purposes. 
Here, we describe collagenase digestion, a widely-used technique to isolate mature adipocytes from the subcutaneous and/or visceral fat samples17, and the subsequent steps to perform ceiling culture and dedifferentiate these cells into multipotent, fibroblast-like cells.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Bovine serum albumine</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine</td>
			<td>Sigma</td>
			<td>A4036</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma</td>
			<td>A0278</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td></td>
			<td></td>
			<td>Any brand can be used</td>
		</tr>
		<tr>
			<td>Type I collagenase</td>
			<td>Worthington Biochemical Corp</td>
			<td>LS-004196</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12, HEPES, no phenol red</td>
			<td>Gibco-Life Technologies</td>
			<td>11039-021</td>
			<td>Add to medium : 20% calf serum, gentamicin (50&#181;g/ml) and fungizone (2.5&#181;g/ml)</td>
		</tr>
		<tr>
			<td>Calf Serum, iron supplemented, from formula-fed calves</td>
			<td>Sigma</td>
			<td>C8056-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>1/2 In plastic bushing</td>
			<td>Iberville</td>
			<td>2704-CP</td>
			<td>SKU:1000120918 (Home Depot)</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>Linde</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin soluton, neutral buffered, 10%</td>
			<td>SIGMA</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60cc syringes</td>
			<td>BD Syringe</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic tubing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Krebs-Ringer-Henseleit stock buffer (KRH)</td>
			<td></td>
			<td></td>
			<td>Prepare stock buffer as following: 25mM HEPES pH7.6, 125mM NaCl, 3.73mM KCl, 5mM CaCl2.2H2O, 2.5mM MgCl2.6H2O, 1mM K2HPO4. Adjust pH to 7.4.</td>
		</tr>
		<tr>
			<td>Krebs-Ringer-Henseleit-Working Buffer (KRH-WB)</td>
			<td></td>
			<td></td>
			<td>Add the following components freshly to KRH buffer: 4% bovine serum albumin, 5mM glucose, 0.1&#181;M adenosine, 560 &#181;M ascorbic acid</td>
		</tr>
		<tr>
			<td>KRH-WB supplemented with Type I collagenase</td>
			<td></td>
			<td></td>
			<td>Add 350U/ml of Type I collagenase</td>
		</tr>
		<tr>
			<td>T25 unvented cap tissue culture flask</td>
			<td>Sarsted or other brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>BD Falcon or other brand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass 22x22</td>
			<td>Fisherbrand</td>
			<td>12-542-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile beakers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of human adipose stem cells through dedifferentiation of mature adipocytes in ceiling cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

שינויים מורפולוגיים גדולים נוצרים להתבגר adipocytes במהלך dedifferentiation (איור 1). כפי שניתן לראות באיור 2, תאים עוברים dedifferentiation הוכתמו נוגדן אנטי FABP4 לניתוח הקרינה. תאים עם מורפולוגיה עגולה הביעו חלבון FABP4 בעוד רוב התאים כמו פיברובלסטים-לא. לאחר dedifferentiation, יכו...

Watch this Scientific Journal Video about Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures at JoVE.com

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

דור של תאי גזע שומן האדם דרך Dedifferentiation של מבוגרים adipocytes בתרבויות תקרה

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

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Research

JoVE Journal

Developmental Biology

11.6K Views.  IUCPQ Research Center. Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

Video: Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

בידוד וההעשרה של אדם שומן המופק מתאי סטרומה לOsteogenesis משופר

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

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 ...

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

דור של גזע תאים המושרה pluripotent מתאי T ההיקפיים אנושיים באמצעות וירוס סנדאי בתנאים ללא מזין

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

דור של תרבויות עור Organotypic מהונדסות גנטי שימוש חסר חיוניות עור אדם

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

בידוד ובידול של תאי גזע נגזר שומן מרקמות שומן תת עורי בשר חזירים

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

דור של תאי גזע מושרים ממלנומה האדם שחדר-גידול לימפוציטים

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

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 ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

מוגדרי דור Scalable של תאי Hepatocyte דמוי מתאי גזע פלוריפוטנטיים האנוש

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing &#946;-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100&#946;/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.

Marrow stromal cells (MSCs) with neural potential exist within the bone marrow. Our protocol enriches this population of cells via hypoxic preconditioning and thereafter directs them to become mature Schwann cells.

Hypoxic Preonditioning של תאים יליד מארו נגזר כמקור לדור של תאים שוואן בוגרת

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

ניתוח חישובית של Germline Caenorhabditis elegans ללמוד הפצה של גרעינים, חלבונים ו את שלד התא

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...