المؤلفين أشكر شيوى بينغ وانغ للمساعدة أولية مع الثقافات DESC. تمول من الكتاب في جزء من المنح الدراسية والمنح المقدمة من المعاهد الوطنية للصحة (K99-DE022059 لAHJ، K12-GM081266 إلى MGC، K08-DE022377-02 لOH، وR01-DE021420 إلى ODK).

1. تشريح السفلى هيمي-الفك السفلي من الكبار ماوس <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قبل تنفيذ هذا البروتوكول، والحصول على موافقة المؤسسية اللازمة والتأكد من الامتثال لجميع المبادئ التوجيهية رعاية الحيوان. الموت ببطء الحيوان باستخدام معيار وافق IACUC الإجراءات. لهذا العمل كان يستخدم CO 2 الخنق تليها خلع عنق الرحم. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> اختياري: رئيس منفصلة عن بقية الجسم باستخدام شفرة حلاقة. </li></ol></li><li style=";text-align:right;direction:rtl"> إزالة الجلد من الشفة السفلى لمستوى خط الرقبة. </li><li style=";text-align:right;direction:rtl"> باستخدام مشرط، وجعل شق بين اثنين من القواطع السفلية، والتي ترتبط بشكل فضفاض. عند الضغط سيتم تقسيم الفك السفلي إلى نصفين. </li><li style=";text-align:right;direction:rtl"> إسفين مشرط بين اللقمة من الفك ومفصل الفك الصدغي وقطع جنبا إلى جنب مع العضلات الفك لفصل الفك. </li><li style=";text-align:right;direction:rtl"> إزالة جميع العضلات والأوتار، والأربطة حتى يبقى سوى العظام. </li></ol> 2. عزل القاطعة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> باستخدام مشرط رقم 15، يحلق بعناية قبالة العظام في زاوية 45 درجة من النهاية البعيدة للنهاية القريبة من المنطقة الغشائية. استخدام غيض من مشرط لاختيار بعيدا أي العظام المتبقية بما في ذلك شظايا العظام عند الحافة. تبدأ ببطء لالتقاط بعيدا في لوحة القشرية اللغات للفك السفلي، بدأت للتو تحت الرحى الثالثة 3. </li><li style=";text-align:right;direction:rtl"> تستمر حتى القاطعة نظيفة، والعمل بعناية تجاه المنطقة التي تحتوي على CL. </li><li style=";text-align:right;direction:rtl"> إزالة حزمة الأعصاب السنخية السفلية. </li><li style=";text-align:right;direction:rtl"> اجراء خفض نظيفة لإزالة الأولى العظام والأنسجة الأقرب إلى CL. </li><li style=";text-align:right;direction:rtl"> اجراء خفض الثانية بشكل أقصى مع مشرط، تقريبا تحت الرحى الثانية 2. ثم يتم تشريح المنطقة القريبة القاطعة للخروج عن طريق إدراج مشرط بين العظام والسطح الإنسي من القاطعة الداني. هذا يجب أن يتم بعناية، حتى لا تمزق الأنسجة. الحركة شق الداني القاصي-. الخطوة البديلة: إزالة أكبر قدر ممكن العظام على طول المنطقة فوق طncisor. مرة واحدة يتم مسح المنطقة، وإزالة بعناية القاطعة بأكمله باستخدام حجم 5 ملقط للتعامل مع جزء المينا الأقرب إلى المنطقة CL. </li><li style=";text-align:right;direction:rtl"> وضع الأنسجة تشريح (CL إما معزولة كما في الشكل 3E، أو القاطعة بأكملها كما هو الحال في البديل الخطوة 2.5) في 2٪ كولاجيناز في برنامج تلفزيوني 1X لمدة 3-4 ساعة في 4 درجات مئوية في لوحة التقيد منخفضة. ل1-10 القواطع، استخدم 100 ميكرولتر لكل بئر في لوحة 12 جيدا. لأكبر من 10 القواطع، استخدم 500 ميكرولتر لكل بئر في أدنى مستوى 6 جيدا لوحة الالتزام. </li></ol> 3. Microdissect في عنق الرحم حلقة شفوي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إزالة المنطقة CL من كولاجيناز ومكان في وسائل الاعلام DMEM/F12 الباردة. </li><li style=";text-align:right;direction:rtl"> برفق في الطرف الأدنى من CL باستخدام ملقط حجم 5 أو حقنة الأنسولين (1 سم مكعب، 28 G ½)؛ سوف اللحمة المتوسطة ينهار بينما يبقى ظهارة سليمة. سوف CL وظهارة المجاورة التي تمتد أفقيا كهيكل &quot;الجناحين مثل&quot; تكون مرئية. </li><li style=";text-align:right;direction:rtl"> نزعبرعم قمية من ظهارة المجاورة عن طريق إجراء شق على شكل حرف V على جانبي، ووضع على الفور في DMEM/F12 الباردة على الجليد. </li><li style=";text-align:right;direction:rtl"> أكرر لجميع CLS، وجمع في 1.5 مل microfuge أنبوب. </li><li style=";text-align:right;direction:rtl"> تدور باستمرار microfuge أنبوب مع العينات، وإزالة وسائل الإعلام، مع استبدال 100 ميكرولتر حل مفرزة الخلية. </li><li style=";text-align:right;direction:rtl"> احتضان الأنسجة في حل مفرزة خلية لمدة 30 دقيقة عند 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> فصل الأنسجة لإنتاج تعليق خلية واحدة بواسطة pipetting بلطف صعودا وباستخدام 1،000 ميكرولتر المنخفضة التصاق ماصة أسفل. يمكن أيضا أن توضع الخلايا من خلال مصفاة الخلية العقيمة لتحقيق التفكك. </li><li style=";text-align:right;direction:rtl"> عد الخلايا مع عدادة الكريات، لوحة من البلاستيك على زراعة الأنسجة أو المواد المطلوبة الأخرى. </li></ol> 4. الثقافة الابتدائية من DESCs <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> خلايا لوحة في لوحة 6 جيدا في 1-6 × 10 4 خلية / مل في وسائل الإعلام DESC، والذي يتألف من DMEM/F12 تستكمل مع الماوس EGF المؤتلف البروتين بتركيز 20 نز / مل، جمعية جيل المستقبل المؤتلف البروتين بتركيز 25 نانوغرام / مل، 1X B27 الملحق، و 1٪ حل المضادات الحيوية (البنسلين، و 100 U / مل، الستربتوميسين، 50 ميكروغرام / مل). </li><li style=";text-align:right;direction:rtl"> تنمو الخلايا مطلي على لوحة 6 جيدا عند 37 درجة مئوية في 5٪ CO 2 في 1 مل من DESC سائل الإعلام. لا تخل الثقافات الأولية لمدة 5 أيام بعد الطلاء للسماح التصاق الخلايا وتشكيل مستعمرة. </li><li style=";text-align:right;direction:rtl"> لتغير وسائل الإعلام الأولى، يستعاض عن نصف حجم (500 ميكرولتر) من وسائل الإعلام القديمة مع نصف حجم (500 ميكرولتر) من وسائل الإعلام الجديدة. تحقق المستعمرات تحت المجهر عند تغيير وسائل الإعلام. </li><li style=";text-align:right;direction:rtl"> بعد التغيير الأول وسائل الإعلام، والاستمرار في تغيير وسائل الإعلام (1-2 مل) كل يوم 2. تحقق المستعمرات تحت المجهر عند تغيير وسائل الإعلام. </li></ol> 5. DESCs الممر <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وسائل الاعلام نضح وغسل الخلايا مع برنامج تلفزيوني 3x أخرى قبل تحسنت. </li><li style=";text-align:right;direction:rtl"> إضافة قبل تحسنت 0.25٪ التربسين EDTA-في محلول ملحي واحتضان عند 37 درجة مئوية لمدة 10-15 دقيقة لDESCs إلى فصل. </li><li style=";text-align:right;direction:rtl"> إضافة وسائط DESC لتحييد التربسين، ماصة بلطف على طول لوحة زراعة الأنسجة لجمع الخلايا، ومكان في أنبوب 15 مل المخروطية. </li><li style=";text-align:right;direction:rtl"> تدور باستمرار الخلايا في 800 x ج لمدة 2 دقيقة. </li></ol>

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+FGF+signaling+loop+sustains+the+generation+of+differentiated+progeny+from+stem+cells+in+mouse+incisors.">An FGF signaling loop sustains the generation of differentiated progeny from stem cells in mouse incisors.</a> Development. 135, 377-385 (2008).</li></ol>

والكتاب ليس لديهم تضارب المصالح في الكشف عنها.

كانت الخلايا الظهارية الأولى بنجاح مثقف 40 عاما مضت أكثر من 21-24، وفي الآونة الأخيرة، والعزلة الناجحة من الخلايا الجذعية الظهارية 25-27 وقد تقدمت معرفتنا البيولوجيا الظهارية. نفيدكم بروتوكول للعزل DESCs من القاطعة الماوس الكبار، يبلغ عدد سكانها سلوكه نسبيا من ...

واحدة من الخصائص الفريدة من الفقاريات هو تطور الأسنان. أصبح الأسنان نظام نموذجا هاما للمناطق عديدة من البحث، حيث تم التحقيق في المسارات الجزيئية والمورفولوجية التخصصات ذات الصلة لهذا الجهاز من عدة زوايا، بما في ذلك علماء البيولوجيا التطورية والتطورية 5. وفي الآونة الأخيرة، بدأ مجال الطب التجديدي للحصول على معلومات قيمة باستخدام الأسنان كنموذج. على وجه الخصوص، وقد تم اكتشاف الخلايا الجذعية الظهارية الأسنان تقدما هاما 6-13. جميع القوارض تمتلك القواطع تزايد مستمر الذي يغذيه نمو الخلايا الجذعية، مما يجعل هذه الأسنان نظام نموذج الوصول إليها لدراسة تنظيم الخلايا الجذعية للبالغين. ، وقد أثبتت التجارب وضع العلامات في 1970s 10،11، تليها النسب الوراثية التجارب التي تتبع 8،9،12،14 DESCs يقيمون في المنطقة القريبة من القاطعة. جذعذرية خلية في مقصورة الظهارية على هذه الخطوة الجانب شفوي من مكانة المفترضة، والمعروفة باسم حلقة عنق الرحم شفوي (CL)، والمساهمة في السكان من الخلايا تسمى (TA) العبور تضخيم الخلايا (الشكل 1). على وجه التحديد، DESCs يقيمون في ظهارة الخارجي المينا (OEE) وشبكية النجمية (SR) 8،9،14، والمينا ظهارة الداخلية (IEE) يثير الخلايا TA التي تقدم من خلال عدد محدود من دورات الخلية ثم الخطوة بشكل أقصى طول طول القاطعة (الشكل 1). تواصل خلايا تكوين المينا التفريق في القاطعة الماوس للتحرك بشكل أقصى طول القاطعة بمعدل ملحوظ من حوالي 350 ميكرومتر في يوم واحد 15،16. لأنها تتحرك، فإن الخلايا تتمايز إلى خلايا تكوين المينا ناضجة والطبقة intermedium (SI) الخلايا. بعد إيداع كامل سماكة المينا المصفوفة، فإن العديد من خلايا تكوين المينا الخضوع لموت الخلايا المبرمج، والخلايا المتبقية في تقليص حجم وتنظيم نضوج المينا &lt;سوب&gt; 17. سلالة من أنواع الخلايا الأخرى في CL شفوي، مثل SR، هو أقل وضوحا، والبيانات المتعلقة الخلايا الجذعية في اللحمة المتوسطة 18 و CL في اللغات ليست سوى بداية في الظهور. باستخدام نموذج القاطعة الماوس، عملت عدد من المجموعات لتوضيح مسارات الجينية والعمليات البيولوجية الخلايا الجذعية تشارك في تجديد الجهاز القائم على الخلايا الطبيعية. ومع ذلك، فإن CL شفوي يحتوي على عدد قليل نسبيا من الخلايا، تشير التقديرات إلى أن حوالي 5،000 في القاطعة الماوس، مما يجعل العمل مع الخلايا الأولية التحدي. لذلك، بذلت جهود للثقافة وتوسيع هذه الخلايا في المختبر 6،16،19،20 من أجل فتح أبواب جديدة للمناهج التجريبية التي ليست قابلة للتحقيق في الجسم الحي. وقد أظهرت الدراسة الأخيرة أن هذه الخلايا يمكن أن كلا تجديد الذات والتمايز إلى أميلوجينين معربا عن الخلايا عند مثقف 13. هنا، نحن تصف ويبرهن على وجود طريقة لالالبريد ثقافة موثوقة ومتسقة من الخلايا من شفوي الماوس CL.

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isolation and culture of dental epithelial stem cells from the adult mouse incisor

فهم الآليات الخلوية والجزيئية التي تكمن وراء تجديد الأسنان والتجديد أصبح موضوع اهتمام كبير 1-4، ويوفر الماوس القاطعة نموذجا لهذه العمليات. هذا الجهاز الرائع تنمو باستمرار طوال حياة الحيوان ويولد كل أنواع الخلايا اللازمة من أحواض نشطة من الخلايا الجذعية البالغة يضم في الشفة (الشفة نحو) واللغات (نحو اللسان) حلقة عنق الرحم (CL) المناطق. فقط الخلايا الجذعية الأسنان من CL شفوي تؤدي إلى خلايا تكوين المينا التي تولد المينا، والغطاء الخارجي للأسنان، على سطح شفوي. هذا التشكيل المينا غير المتماثلة يسمح التآكل في الطرف القاطعة، والأسلاف والخلايا الجذعية في القاطعة الداني ضمان أن أنسجة الأسنان تتجدد باستمرار. القدرة على عزل وزراعة هذه الخلايا الاصلية أو الجذعية في المختبر يسمح توسعها وتفتح الأبواب أمام العديد من التجارب لا يمكن تحقيقه في الجسم الحي، مثل حIGH اختبار الإنتاجية من العوامل التنظيمية الخلايا الجذعية المحتملة. هنا، نحن تصف ويبرهن على وجود طريقة موثوقة ومتسقة لخلايا الثقافة من CL شفوي من القاطعة الماوس.

وتضم الماوس نصفي الفك السفلي واحد تزايد مستمر القاطعة وثلاثة الأضراس الجذور (الشكل 1A). تتكون جميع الأسنان من عاج والمينا، واثنين من المكونات المعدنية للتاج الأسنان (أرقام 1A 1A و&#39;). المنازل القاطعة اثنين من محاريب الخلايا الجذعية تسمى CL شفوي واللغات...

Watch this Scientific Journal Video about Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor at JoVE.com

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

يوفر القاطعة الماوس تزايد مستمر نموذجا لدراسة تجديد أنسجة الأسنان من الخلايا الجذعية الظهارية الأسنان (DESCs). وتفيد التقارير ان نظام قوي للباستمرار وبشكل موثوق الحصول على هذه الخلايا من القاطعة وتوسيعها في المختبر هنا.

العزلة وثقافة الأسنان طلائي الخلايا الجذعية من الكبار ماوس القاطعة

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

isolation-culture-dental-epithelial-stem-cells-from-adult-mouse

Research

JoVE Journal

Biology

15.0K Views.  University of California, San Francisco.  يوفر القاطعة الماوس تزايد مستمر نموذجا لدراسة تجديد أنسجة الأسنان من الخلايا الجذعية الظهارية الأسنان (DESCs). وتفيد التقارير ان نظام قوي للباستمرار وبشكل موثوق الحصول على هذه الخلايا من القاطعة وتوسيعها في المختبر هنا. 

Video: Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

عزل الخلايا الجذعية من شبكية العين بالعين ماوس

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

العزلة وثقافة الكبار الخلايا الجذعية الظهارية من الجلد البشري

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

جيل من الفئران المستمدة من الخلايا الجذعية المحفزة المستحثة

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

العزلة وثقافة فردية وMyofibers زنزاناتهم القمر الصناعي من العضلات الهيكلية الكبار

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

السذاجة الكبار الخلايا الجذعية عزل من المرحلة الابتدائية الثقافات الخلايا الليفية الإنسان

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

الاستقراء وتحليل الظهارية الوسيطة الانتقالية ل

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

عزل وثقافة الكرياتينية الماوس الرئيسية من حديثي الولادة والفأر الكبار الجلد

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

عزل وتوصيف والتفريق في القلب من الخلايا الجذعية من قلب الماوس الكبار

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

العزلة المتزامنة وثقافة Myocytes الأذينية، myocytes البطينية، وغير Myocytes من قلب فأرة البالغين

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

عزل وثقافة الكريات الكيراتينية الفموية الأولية من الحنك الفأري البالغ

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...