The authors wish to thank Carl Hobbs and Lindsey Majoram for their technical assistance, and Professor Pat Doherty for use of microscopy facilities. Dr. Agley was supported by a studentship from King&#8217;s College London. Funding from the Spurrell Trust is also acknowledged.

<ol>
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The authors declare that they have no competing financial interests.

שתארנו הליך מיון immunomagentic להעשרה סלקטיבית של מבשרים המופק משריר אנושיים מדגימות קטנות של חומר ביופסיה של שריר. טכניקה זו כבר לא יסולא בפז במעבדה שלנו להתגברות על האובדן של תרבויות המופק משריר האנושיים לfibroblasts, אלא גם להבנת ההתנהגות הייחודית של אוכלוסיות שונות של אבות...

התיקון וההתחדשות של שריר שלד דורש הפעולה של תאי לווין 1, תאי גזע myogenic 2,3. בvivo תאים אלה קיימים במדינה הפיכה שקטה הממוקמת בין sarcolemma וlamina בסיס של כל myofibre, אלא הופכים להיות מופעל כדי להתרבות, פתיל ולהבדיל כרקמת שריר פגום, תיקון ומחדש 3. יכולים להיות מבודדים תאי לווין מדגימות צעירות וקשישים שריר אנושי ביופסיה באמצעות עיכול אנזימטי 4 ומאפייני myogenic לאחר מכן ניתן יהיה למד בתרבות העיקרית 5. היעילות של תהליך בידוד זה בהקשר לשתי התשואה והטוהר של אוכלוסיית תאים תלויה בשיטות ויכולה להשתנות ממדגם לדגום. שני סוגי התאים חסיד העיקריים המתקבלים מעיכול אנזימטי הם תאי הלווין (תאי myogenic עכשיו כינו או תאי מבשר שריר), שזוהו בתחילה כתאי CD56 + / desmin, וmufibroblasts הנגזר scle, זוהה כCD56 - וTE7 + תאי 5. לפיברובלסטים שיעור שגשוג מהיר ולא יעברו מעצר בלתי הפיך צמיחה ובידול מסוף במגע תאי תאים כמו תאי myogenic; כך באוכלוסיות מעורבות, fibroblasts עשוי מוצף תאי myogenic להשתלט על התרבות. לעתים קרובות נצפה fibroblasts כגירוי לביולוגים שריר, לעומת זאת, יש כיום עניין גובר בfibroblasts כתאים ראויים למחקר בפני עצמם, במיוחד כשהם הוכחו יש להם תפקיד של שיתוף פעולה עם תאי myogenic במהלך תיקון שריר 6 . הבידוד וטיהור סוגי תאים שונים משריר האנושי שלו ובכך שיקול מתודולוגית חשוב כאשר מנסים לחקור את ההתנהגות המולדת של שני סוגי התאים בתרבית. מיון תא הקרינה המופעל (FACS) הוא שיטה שבאמצעותה ניתן למיין תאים למחקר נוסף ו / או וספרניתח. FACS הוכח להעשיר באופן מהימן תאי myogenic אנושיים, אבל התשואה של תאים לתרבות שלאחר מכן עד כה לא הייתה גבוהה 7. לאור פוטנציאל השכפול המוגבל של תאים סומטיים כגון תאים נגזרים תאי לווין myogenic ועני מאוד התפשטות ובידול הקשורים להזדקנות 4, גישות עדינות יותר נדרשות. תרבויות סיב שריר בודדות מציעות אחר, פחות אגרסיווי, אמצעי להשגת תאי לווין עכבריים עדיין תושב בנישה sublaminal ולאחר ההפעלה שלהם בתרבות 8,9. עם זאת, זה בדרך כלל לא אפשרי מחומר ביופסיה של שריר אדם (כי רק לעתים נדירות ניתן להשיג מגיד לגיד סיבים) כלומר טכניקה זו עשויה שלא להיות נגישה למעבדות מחקר רבות מעוניינים ללמוד תאים שמקורם בשרירים אנושיים. יתר על כן, טכניקת הסיב בודדת מספקת רק מספרים סלולריים מוגבלים מאוד. כאן אנו מתארים מערכת של מיון המבוסס על genעיכול אנזימטי tle של תאים באמצעות collagenase וdispase אחרי שני סיבובים רצופים של תא מופעל מגנטי מיון (MACS) אשר נותן שני טוהר גבוה (&gt; תאי myogenic 95%) ותשואה (~ 2.8 x 10 6 ± 8.87 x 10 5 תאים / רקמת ז) לניסויים בתרבות. CD56 נחשב סמן משטח תקן זהב לזיהוי של תאי לווין אנושיים באתר 10 ובמבחנה 11 ומספק מועמד סמן משטח האידיאלי עבור קובץ מצורף חרוז. בגישה זו נוגדני CD56 מוצמדים לתחמוצת ברזל וחרוזים פאראמגנטי המכיל פוליסכריד חייב תאים ועבר דרך עמודת הפרדת תא מגנטית גבוהה שיפוע מונחת בשדה מגנטי חזק 12,13. עמודי ההפרדה מלאים במטריצה ​​של תחומים צמר פלדה או ברזל פרומגנטיים המשמשים להתמקד קווי שדה מגנטיים לכיוון פני השטח שלהם יצירת שיפועים חזקים שדה מגנטי (~ 4tesla) 14. בטורים אלה אפילו תאים מעט מגנטיים נמשכים ושנספחו לפני השטח שלהם 14. Unbound (CD56 -) תאים עוברים דרך הטור ואילו + תאי CD56 שכותרתו עם microbeads המגנטי נשמרים עד לסילוקו מהשדה המגנטי 12,15. השעיות תא מכל שלב של תהליך המיון יכולות להיות מצופים בצפיפות הרצויה לניסויים נוספים. בעקבות התערבות נתון ניתן לזהות את המרכיבים התאיים באמצעות immunocytochemistry, צילם באמצעות שדה רחב או מיקרוסקופ פלואורסצנטי confocal ונותח כמותית באמצעות גישת ניתוח תמונה המאפשרת מדידה אובייקטיבית מהירה של כל התאים שכותרתו בכל תמונת נתונה. במעבדה שלנו יש לנו השתמשתי בגישת מיון immunomagnetic זה כפול ואחרי ניתוח תמונה 16 להפגין CD56 ש-- fibroblasts אדם transdifferentiate בקלות לתוך תאי שומן, תוך תא myogenicים ממוצא הלווין עמיד מאוד בפני המרת adipogenic זה 5.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:169px;">Collagenase&#160; D</td>
			<td style="width:111px;">Roche&#160;</td>
			<td style="width:89px;">11088866001</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:169px;">Dispase II</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:89px;">D4693-1G</td>
			<td style="width:140px;">Must be filter sterilized before use</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:169px;">Trypsin/EDTA</td>
			<td style="width:111px;">(Gibco) Invitrogen</td>
			<td style="width:89px;">15400-054</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:169px;">100 micron cell strainer</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:89px;">352360</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:169px;">Collagen solution ( 3 mg/ml in 0.1% acetic acid)</td>
			<td style="width:111px;">Sigma, Dorset, UK</td>
			<td style="width:89px;">C8919&#160;</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:169px;">Minisart SRP15 syringe filter (0.2 micron)</td>
			<td style="width:111px;">Sartorius</td>
			<td style="width:89px;">17573ACK</td>
			<td style="width:140px;">Polytetrafluorethylene (PTFE) membrane</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:169px;">CD56 human Microbeads</td>
			<td style="width:111px;">Miltenyi Biotech</td>
			<td style="width:89px;">130-050-401</td>
			<td style="width:140px;">Be aware of the limited shelf life of microbeads</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:169px;">Anti- fibroblast Microbeads, human</td>
			<td style="width:111px;">Miltenyi Biotech</td>
			<td style="width:89px;">130-050-601</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:169px;">40 &#956;m Pre-separation filters</td>
			<td style="width:111px;">Miltenyi Biotech</td>
			<td style="width:89px;">130-041-407</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="129">
			<td height="129" style="height:129px;width:169px;">Large Cell Collumns</td>
			<td style="width:111px;">Miltenyi Biotech</td>
			<td style="width:89px;">130-042-202</td>
			<td style="width:140px;">These columns come with a flow resistor. Use of the flow resistor is not necessary to obtain the high myogenic purities described here.</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:169px;">LS columns&#160;</td>
			<td style="width:111px;">Miltenyi Biotech</td>
			<td style="width:89px;">130-042-401</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:169px;">MiniMacs Seperator</td>
			<td style="width:111px;">Miltenyi Biotech</td>
			<td style="width:89px;">130-042-102</td>
			<td style="width:140px;">This separator fits the large cell column but not the LS column.&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:169px;">MidiMACS</td>
			<td style="width:111px;">Miltenyi Biotech&#160;</td>
			<td style="width:89px;">130-042-302</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:169px;">MACS multistand</td>
			<td style="width:111px;">Miltenyi Biotech</td>
			<td style="width:89px;">130-042-303</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:169px;">BSA</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:89px;"></td>
			<td style="width:140px;">Must be filter sterilized before use</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:169px;">Oil Red O</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:89px;">O0625</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:169px;">Triethyl phosphate</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:89px;">538728</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="166">
			<td height="166" style="height:166px;width:169px;">Whatman Paper</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:89px;">Z241121-1PAK</td>
			<td style="width:140px;">No. 42, Ashless. To prepare the filter fold the circular filter paper to make a semi circle, then fold the semi-circle in half again to form a cone shape. Fit the cone into a funnel for filtering.&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:169px;"></td>
			<td style="width:111px;"></td>
			<td style="width:89px;"></td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="74">
			<td height="74" style="height:74px;width:169px;">ProLong Gold Antifade Reagent</td>
			<td style="width:111px;">Molecular Probes, Invitrogen</td>
			<td style="width:89px;">P36930</td>
			<td style="width:140px;">This can be purchased with or without DAPI and does not quench initial fluorescence.</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:169px;">AxioVision</td>
			<td style="width:111px;">Carl Zeiss</td>
			<td style="width:89px;">Contact Zeiss</td>
			<td style="width:140px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:169px;">Adobe Photoshop CS5 Extended</td>
			<td style="width:111px;">Adobe (purchased from Pugh Computers)</td>
			<td>ADPH16982*&#160;</td>
			<td style="width:140px;"></td>
		</tr>
	</tbody>
</table>

isolation and quantitative immunocytochemical characterization of primary myogenic cells and fibroblasts from human skeletal muscle

התיקון וההתחדשות של שריר שלד דורש הפעולה של תאי לווין, שהם תאי גזע שריר התושב. אלה יכולים להיות מבודדים מדגימות שריר אנושי ביופסיה באמצעות עיכול אנזימטי ומאפייני myogenic למדו בתרבות. כמותית, שני סוגי התאים חסיד העיקריים המתקבלים מעיכול אנזימטי הם: (i) תאי הלווין (המכונים תאי myogenic או תאי מבשר שריר), שזוהו בתחילה כCD56 + ובהמשך כCD56 + / desmin ותאים + (ii) שריר fibroblasts נגזר, שזוהה כCD56 - וTE-7 +. Fibroblasts להתרבות ביעילות רבה בתרבות ובאוכלוסיות תאים מעורבות תאים אלה עשויים מוצפים תאי myogenic להשתלט על התרבות. הבידוד וטיהור סוגי תאים שונים משריר האנושי שלו ובכך שיקול מתודולוגית חשוב כאשר מנסים לחקור את ההתנהגות המולדת של כל אחד מסוגי התאים בתרבית. כאן אנו descrIBE מערכת של מיון המבוסס על עיכול אנזימטי העדין של תאים באמצעות collagenase וdispase אחריו תא מופעל מגנטי מיון (MACS) אשר נותן שני טוהר גבוה (&gt; תאי myogenic 95%) ותשואה טובה (~ 2.8 x 10 6 ± 8.87 x 10 5 תאים / g רקמות לאחר 7 ימים במבחנה) לניסויים בתרבות. גישה זו מבוססת על דוגרים אוכלוסיית תאים המעורבת נגזר שריר עם חרוזים microbeads מגנטיים המוצמדים לנוגדנים נגד CD56 ולאחר מכן עוברים תאים למרות ששדה מגנטי. CD56 + תאים חייבים microbeads נשמרים על ידי השדה ואילו CD56 - תאים עוברים ללא הפרעה דרך העמודה. השעיות תא מכל שלב של תהליך המיון יכולות להיות מצופים ותרבותית. בעקבות התערבות נתון, מורפולוגיה של תאים, והביטוי והלוקליזציה של חלבונים כוללים גורמי שעתוק גרעיניים ניתן לכמת באמצעות תיוג immunofluorescent עם נוגדנים ספציפיים וp תמונהrocessing וחבילת ניתוח.

תאי myogenic מטוהרים וfibroblasts יכולים להיות מתורבת במדיום בידול adipogenic במשך שלושה ימים ואחריו בינוני תזונת adipogenic מכל מקום בין 7-30 ימים כדי להעריך את הפוטנציאל שלהם לadipogenesis. שימוש באוכלוסיות התאים המטוהרים, מכתים השמן האדום O בשילוב עם immunostaining עבור סמני שושלת adipogenic וmyogenic הרא?...

Watch this Scientific Journal Video about Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle at JoVE.com

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

בידוד וכמוני Immunocytochemical אפיון של תאים ראשוניים myogenic וFibroblasts משרירי שלד אדם

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

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15.8K Views.  King's College London. The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells. 

Video: Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

באמצעות מבודד המיטוכונדריה מכמויות מינימליות של שרירי שלד עכבר למדידות נשימה microplate תפוקה הגבוה

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

בידוד ואפיון של תאים בודדים מעוברים דג הזברה

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

הקמת תאי tetraploid Proliferative מ Nontransformed אדם פיברובלסטים

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

הכנה והתרבות של תאים מבשר myogenic / ראשי myoblasts מן שרירי השלד של מבוגר ובני אדם בני

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

בידוד ואפיון של תאי סטרומה mesenchymal מן טבורי אדם העובר שליה

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Isolation of Human Myoblasts, Assessment of Myogenic Differentiation, and Store-operated Calcium Entry Measurement

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for muscle regeneration. Upon injury, which occurs frequently in skeletal muscles, SCs are activated. They proliferate as myoblasts and differentiate to repair muscle lesions. Among many events that take place during muscle differentiation, cytosolic Ca2+ signals are of great importance. These Ca2+ signals arise from Ca2+ release from internal Ca2+ stores, as well as from Ca2+ entry from the extracellular space, particularly the store-operated Ca2+ entry (SOCE). This paper describes a methodology used to obtain a pure population of human myoblasts from muscle samples collected after orthopedic surgery. The tissue is mechanically and enzymatically digested, and the cells are amplified and then sorted by flow cytometry according to the presence of specific membrane markers. Once obtained, human myoblasts are expanded and committed to differentiate by removing growth factors from the culture medium. The expression levels of specific transcription factors and in vitro immunofluorescence are used to assess the myogenic differentiation process in control conditions and after silencing proteins involved in Ca2+ signaling. Finally, we detail the use of Fura-2 as a ratiometric Ca2+ probe that provides reliable and reproducible measurements of SOCE.

Here, we describe a methodology to obtain a pure population of human myoblasts from adult muscle tissue. These cells are used to study in vitro skeletal muscle differentiation and, in particular, to study proteins involved in Ca2+ signaling.

בידוד של Myoblasts האדם, הערכה של בידול Myogenic, ו-מופעלות סידן מופעל מדידה

Satellite cells (SC) are muscle stem cells located between the plasma membrane of muscle fibers and the surrounding basal lamina. They are essential for ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

זיהוי של שרירי השלד תאים הלוויין על-ידי Immunofluorescence עם Pax7 ו- Laminin נוגדנים

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

הבידוד והתרבות של תאי Epicardial הראשית נגזר מן הלב האנושי למבוגרים ו עוברית דגימות

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

מבוסס-RNA תכנות מחדש של האנושי Fibroblasts הראשי לתוך תאי גזע Pluripotent המושרה

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally isolated and characterized from bone marrow (BM), then acquired from tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC). MSCs are a heterogeneous cell population with the capacity for (1) adherence to plastic in standard culture conditions, (2) surface marker expression of CD73+/CD90+/CD105+/CD45-/CD34-/CD14-/CD19-/HLA-DR- phenotypes, and (3) trilineage differentiation into adipocytes, osteocytes, and chondrocytes, as currently defined by the International Society for Cellular Therapy (ISCT). Although BM is the most widely used source of MSCs, the invasive nature of BM aspiration ethically limits its accessibility. Proliferation and differentiation capacity of MSCs obtained from BM generally decline with the age of the donor. In contrast, fetal MSCs obtained from UC have advantages such as vigorous proliferation and differentiation capacity. There is no ethical concern for UC sampling, as it is typically regarded as medical waste. Human UC starts to develop with continuing growth of the amniotic cavity at 4-8 weeks of gestation and keeps growing until reaching 50-60 cm in length, and it can be isolated during the whole newborn delivery period. To gain insight into the pathophysiology of intractable diseases, we have used UC-derived MSCs (UC-MSCs) from infants delivered at various gestational ages. In this protocol, we describe the isolation and characterization of UC-MSCs from fetuses/infants at 19-40 weeks of gestation.

Human umbilical cord (UC) can be obtained during the perinatal period as a result of preterm, term, and postterm delivery. In this protocol, we describe the isolation and characterization of UC-derived mesenchymal stem cells (UC-MSCs) from fetuses/infants at 19-40 weeks of gestation.