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.

وصفناها إجراء فرز immunomagentic لتخصيب انتقائية السلائف مأخوذة من العضلات البشرية من عينات صغيرة من المواد خزعة العضلات. وكانت هذه التقنية لا تقدر بثمن في مختبرنا للتغلب على فقدان الثقافات مأخوذة من العضلات الإنسان إلى الخلايا الليفية، ولكن أيضا لفهم سلوك فريد من السكان ...

إصلاح وتجديد العضلات والهيكل العظمي يتطلب العمل من الخلايا الأقمار الصناعية 1، والخلايا الجذعية المنشأ العضلي 2،3. في الجسم الحي وتوجد هذه الخلايا في حالة هادئة عكسية تقع بين غمد الليف العضلي والصفيحة القاعدية من كل myofibre، ولكن تصبح تنشيط لتتكاثر، تلف الصمامات وتفرق كما الأنسجة العضلية، وإصلاح وإعادة إنشاء 3. الخلايا الأقمار الصناعية يمكن أن تكون معزولة من الشباب والمسنين عضلات الإنسان عينات الخزعة باستخدام الهضم الأنزيمي 4 وممتلكاتهم المنشأ العضلي يمكن بعد ذلك أن تدرس في الثقافة الابتدائية 5. كفاءة هذه العملية بمعزل فيما يتعلق بكل من المحصول ونقاء السكان الخلية تعتمد على الأساليب المستخدمة، ويمكن أن تختلف من عينة لعينة. أنواع الخلايا اثنين الرئيسية ملتصقة تم الحصول عليها من عملية الهضم الأنزيمي هي الخلايا الأقمار الصناعية (خلايا المنشأ العضلي يطلق عليه الآن أو خلايا السلائف العضلات)، التي تم تحديدها في البداية باعتبارها خلايا CD56 + / desmin، وموالليفية المستمدة scle ويدعى CD56 - وTE7 + 5 خلايا. الليفية لديها معدل التكاثري السريع ولا تخضع اعتقال النمو لا رجعة فيه والتمايز النهائي على اتصال خلية خلية مثل خلايا المنشأ العضلي. وبالتالي في المختلطة السكان، قد تجاوز الخلايا الليفية خلايا المنشأ العضلي للهيمنة على الثقافة. وغالبا ما كان ينظر الليفية باعتبارها تهيج لعلماء الأحياء العضلات، ومع ذلك، هناك الآن اهتماما متزايدا في الخلايا الليفية إلى خلايا تستحق الدراسة في حد ذاتها، لا سيما وقد ثبت أنها ليكون لها دور تعاوني مع خلايا المنشأ العضلي خلال إصلاح العضلات 6 . عزل وتنقية من أنواع مختلفة من الخلايا من العضلات الإنسان تشكل بالتالي اعتبار المنهجي المهم عند محاولة للتحقيق في السلوك الفطري لكل من أنواع الخلايا في الثقافة. مضان تنشيط الخلايا الفرز (FACS) هو الطريقة التي يمكن فرز الخلايا لمزيد من الدراسة و / أو عدها وتحليل. وقد تبين FACS لإثراء موثوق خلايا المنشأ العضلي الإنسان، ولكن لم يكن العائد من الخلايا للثقافة اللاحقة حتى الآن 7 عالية. نظرا لإمكانية تكرار محدودة من الخلايا الجسدية مثل الخلايا المشتقة خلايا المنشأ العضلي الأقمار الصناعية وانتشار سيئة للغاية والتمايز المرتبطة الشيخوخة 4، ثمة حاجة إلى نهج أكثر لطيف. ثقافات الألياف العضلية واحدة تقدم آخر، أقل عدوانية، يعني الحصول على الخلايا الأقمار الصناعية الفئران لا يزال مقيما في مكانها المناسب sublaminal وبعد تفعيلها في الثقافة 8،9. ومع ذلك، وهذا هو في كثير من الأحيان غير ممكن من الإنسان المواد خزعة العضلات (لأن الألياف نادرا ما يمكن الحصول عليه من وتر إلى وتر) وهذا يعني أن هذه التقنية قد لا تكون في متناول العديد من مختبرات الأبحاث المهتمة في دراسة الخلايا المشتقة من عضلات الإنسان. وعلاوة على ذلك، فإن تقنية الألياف واحدة لا يوفر سوى أعداد الخلايا محدودة للغاية. نحن هنا وصف نظام الفرز على أساس جنرالالهضم الأنزيمي TLE من الخلايا باستخدام كولاجيناز وdispase تليها اثنين جولات متتالية من خلية تنشيط المغناطيسية الفرز (MACS) الذي يعطي كل من نقاء عالية (&gt; 95٪ خلايا المنشأ العضلي) والعائد (~ 2.8 × 10 6 ± 8.87 × 10 5 خلايا / ز الأنسجة) للتجارب في الثقافة. ويعتبر CD56 الذهب علامة سطح القياسية لتحديد الخلايا الأقمار الصناعية الإنسان في الموقع 10 و 11 في المختبر، ويوفر المثالي سطح مرشح علامة لمرفق حبة. في هذا النهج الأجسام المضادة CD56 مترافق إلى أكسيد الحديد وملزمة الخرز مغنطيسية مسايرة فائقة superparamagnetic التي تحتوي على السكاريد إلى الخلايا ومرت عالية التدرج عمود فصل الخلية المغناطيسي وضعها في مجال مغناطيسي قوي 12،13. تمتلئ الأعمدة الفصل مع مصفوفة من المغناطيسية الصوف الصلب أو الحديد المجالات التي تعمل على تركيز خطوط المجال المغناطيسي نحو سطحها توليد التدرجات حقل مغناطيسي قوي (~ 4tesla) 14. في هذه الأعمدة تنجذب حتى الخلايا المغناطيسية قليلا، وكثف إلى سطحها 14. غير منضم (CD56 -) خلايا تمر عبر عمود في حين يتم الاحتفاظ CD56 + الخلايا المسمى مع ميكروبيدات المغناطيسية حتى إزالة من الحقل المغناطيسي 12،15. تعليق خلية من أي مرحلة من مراحل عملية الفرز يمكن مطلي في كثافة المطلوب لمزيد من التجارب. وبعد تدخل معين يمكن التعرف على المكونات الخلوية باستخدام مناعية، تصوير باستخدام واسعة المجال أو متحد البؤر المجهري مضان وتحليلها كميا باستخدام نهج تحليل الصور التي تسمح القياس الموضوعي السريع لجميع الخلايا المسمى في أي صورة معينة. في مختبرنا وقد استخدمنا هذا النهج الفرز immunomagnetic مزدوج تليها تحليل الصور 16 إلى إثبات أن CD56 - الخلايا الليفية الإنسان تحورها بسهولة في الخلايا الشحمية، في حين خلية المنشأ العضليالصورة الأصلية الأقمار الصناعية هي شديدة المقاومة لهذا التحويل مكون الشحم 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

إصلاح وتجديد العضلات والهيكل العظمي يتطلب العمل من الخلايا الأقمار الصناعية، والتي هي الخلايا الجذعية العضلية المقيمين. هذه يمكن أن تكون معزولة من عينات خزعة العضلات الإنسان باستخدام الهضم الأنزيمي وممتلكاتهم المنشأ العضلي درس في الثقافة. كميا، وهما أنواع الخلايا الملتصقة الرئيسية التي تم الحصول عليها من عملية الهضم الأنزيمي هي: (أ) الخلايا الأقمار الصناعية (وهو ما يسمى خلايا المنشأ العضلي أو خلايا السلائف العضلات)، التي تم تحديدها في البداية كما CD56 + CD56 وفيما بعد + / + desmin خلايا و (ثانيا) muscle- الليفية المشتقة ويدعى CD56 - وTE-7 +. الليفية تتكاثر بشكل فعال جدا في الثقافة والسكان الخلية المختلطة هذه الخلايا قد تجاوز خلايا المنشأ العضلي للهيمنة على الثقافة. عزل وتنقية من أنواع مختلفة من الخلايا من العضلات الإنسان تشكل بالتالي اعتبار المنهجي المهم عند محاولة للتحقيق في السلوك الفطري إما نوع من الخلايا في الثقافة. نحن هنا أن descrالمكتب الدولي للتربية نظام الفرز على أساس الهضم الأنزيمي لطيف من الخلايا باستخدام كولاجيناز وdispase تليها المغناطيسية خلية تنشيط الفرز (MACS) الذي يعطي كل من نقاء عالية (&gt; خلايا المنشأ العضلي 95٪) والعائد الجيد (~ 2.8 × 10 6 ± 8.87 5 × 10 خلية / ز الأنسجة بعد 7 أيام في المختبر) للتجارب في الثقافة. ويستند هذا النهج على احتضان مختلطة السكان خلية مأخوذة من العضلات مع ميكروبيدات المغناطيسية الخرز مترافق إلى الأجسام المضادة ضد CD56 ومن ثم تمرير الخلايا على الرغم من مجال مغناطيسي. يتم الاحتفاظ CD56 + الخلايا بد أن ميكروبيدات حسب الحقل في حين CD56 - خلايا تمر دون عوائق من خلال العمود. تعليق خلية من أي مرحلة من مراحل عملية الفرز يمكن مطلي ومثقف. وبعد تدخل معين، مورفولوجيا الخلايا، والتعبير وتوطين البروتينات بما في ذلك عوامل النسخ النووية يمكن قياسها كميا باستخدام الوسم مناعي مع الاجسام المضادة المحددة وصورة عو rocessing وحزمة التحليل.

الخلايا والخلايا الليفية المنشأ العضلي المنقى يمكن تربيتها في مكون الشحم متوسطة التمايز لمدة ثلاثة أيام تليها مكون الشحم المتوسطة التغذية من أي مكان ما بين 7-30 أيام لتقييم قدرتها على تكون الشحم. باستخدام السكان الخلية المنقى، وأظهر النفط الأحمر O تلطيخ في تركيبة مع ا...

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.

العزلة والكمية Immunocytochemical توصيف الخلايا عضلي الابتدائية والخلايا الليفية من العضلات الهيكلية البشرية

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.

عن طريق عزل الميتوكوندريا من كميات الحد الأدنى من الفأر العضلات الهيكلية لارتفاع صفيحة ميكروسكوبية الإنتاجية قياسات الجهاز التنفسي

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.

عزل وتوصيف الخلايا واحدة من اسماك الزرد الأجنة

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.

إنشاء خلايا التكاثري رباعي الصيغة الصبغية من Nontransformed الخلايا الليفية الإنسان

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.

إعداد والثقافة من الخلايا عضلي السلائف / Myoblasts الابتدائية من العضلات الهيكلية من الكبار والمسنين البشر

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.

عزل وتوصيف الخلايا الجذعية الوسيطة اللحمية من الحبل السري البشري والجنين المشيمة

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.

عزل ميوبلاستس الإنسان، تقييم التمايز العضلي، وقياس دخول الكالسيوم التي تديرها المخازن

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.

تحديد خلايا الهيكل العظمى والعضلات الأقمار الصناعية قبل الفلورة مع Pax7 والأجسام المضادة لامينين

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.

بالعزلة وثقافة الخلايا Epicardial الأولية المستمدة من عينات من قلب الكبار والأجنة البشرية

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.

الجيش الملكي النيبالي على إعادة برمجة الليفية الأولية البشرية داخل الخلايا الجذعية المستحثة Pluripotent

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.