نشكر اندريا Parmeggiani (L2C وDIMNP، مونبلييه) وبينوا CHARLOT (IES، مونبلييه) وكذلك أعضاء المختبر لإجراء مناقشات مفيدة، كريستوف Duperray للمساعدة في تحليل نظام مراقبة الأصول الميدانية، ومونبلييه منشأة RIO التصوير (MRI) لتوفير بيئة ملائمة لنظام مراقبة الأصول الميدانية والفحص المجهري متحد البؤر. وأيد البنك المركزي الماليزي من خلال زمالة الدراسات العليا من LabEx Numev (اتفاقية وكالة الاستخبارات الوطنية-10-LABX 20). وأيد AB من قبل الزمالة الجامعية من جامعة وارسو والاتحاد الأوروبي (رقم POKL.04.01.02-00-221 / 12). MLV هو عالم موظفين من المركز الوطني للبحث العلمي (CNRS).

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INSERM (المعهد الوطني للسانتيه آخرون للبحوث ميديكال)، والتي CJ وML.V. تنتسب، وقدمت طلب براءة اختراع تقنية MitoCeption (EP14306154.7).

تظهر عددا متزايدا من الدراسات أن الخلايا يمكن تبادل الميتوكوندريا وأن هذه الميتوكوندريا يكون لها آثار عميقة على استقلاب الخلية المستهدفة وظائف. لذلك، لا بد من السيطرة على أدوات لنقل الكمية الميتوكوندريا من الخلايا المانحة لهذه الخلايا المستهدفة لتمكين دراسة دقيقة...

الميتوكوندريا هي عضيات توجد في الخلايا حقيقية النواة حيث أنها تلعب دورا رئيسيا في امتصاص المواد الغذائية وكذلك في إنتاج الطاقة والأيض. هذه العضيات تحتوي على الحمض النووي دائرية (و mtDNA)، 16.6 كيلوبايت طويل، الذي يشفر البروتينات المجمعات سلسلة نقل الإلكترون، tRNAs وrRNAs 1. وظيفة هذه العضيات هو أمر حاسم لارتبطت توازن الخلايا والعديد من الأمراض مع الميتوكوندريا اختلال وظيفي 1، 2، 3. وعلى سبيل المثال تم ربط مركز الميتوكوندريا إلى التهاب والأمراض المعدية والسرطان، في هذه الحالة الأخيرة مع عواقب للورم خبيث ومقاومة للعلاج 4، 5، 6، 7. الميتوكوندريا عرض القدرة اللافتة ل<html> الحصول على نقلها بين &quot;المانحة&quot; وخلايا &quot;الهدف&quot;. وهذا يؤدي إلى تغييرات في عملية الأيض نشطة من الخلايا المستهدفة وكذلك في التعديلات الفنية الأخرى مثل إصلاح الأنسجة ومقاومة للعوامل العلاج الكيميائي، كما هو مبين في الآونة الأخيرة من قبل مختلف المختبرات 8، 9، 10، 11، 12، 13، 14، 15 16. الخلايا الجذعية الوسيطة الإنسان (اللجان الدائمة) تعرض هذه القدرة على نقل الميتوكوندريا إلى مجموعة واسعة من الخلايا المستهدفة، بما في ذلك العضلية، والخلايا البطانية، الرئوية الخلايا الظهارية السنخية، والخلايا أنبوبي كلوي والخلايا السرطانية، مما أدى إلى إدخال تعديلات على الخصائص الوظيفية لهذه الخلايا 8،&gt; 9، 10، 12، 17، 18. يبدو الصرف الميتوكوندريا الآن كآلية المستخدمة على نطاق واسع أن تسمح لعدد من أنواع مختلفة من الخلايا على التواصل مع بعضها البعض، وتعديل خصائصها البيولوجية. يمكن أن يحدث هذا التبادل الميتوكوندريا من خلال نفق الأنابيب النانوية (تي ان تي) تشكيل، التي تنطوي على connexin 43 التي تحتوي على تقاطعات الفجوة 8 أو M-ثانية / TNFaip2 وexocyst المعقدة 19. بدلا من ذلك، تم عرض نقل الميتوكوندريا أيضا أن بوساطة arrestin microvesicles التي تحتوي على نطاق البروتين 1 بوساطة (ARMMs) 20. ومن المثير للاهتمام، وارتبط فعالية نقل الميتوكوندريا إلى معدل تعبير عن رو GTPase 1 MIRO1 21، عاملا رئيسيا لتفسير الاختلافات في كفاءات نقل الميتوكوندريا بين iPSC-اللجان الدائمة والكبار BM-اللجان الدائمة 22. وعلى الرغم من هذه الثروة من البيانات المتعلقة الصرف الميتوكوندريا خلية إلى خلية، لا يعرف إلا القليل نسبيا عن التمثيل الغذائي ونتائج البيولوجية لهذا النقل الميتوكوندريا. وبالتالي، فإنه يستحق تماما وضع الأدوات المناسبة لإجراء تقييم كامل للآثار البيولوجية من هذا النقل. على مر السنين، العديد من الطرق التقنية لنقل الميتوكوندريا من الجهات المانحة للخلايا متقبل وقد اقترحت. وهذا يشمل الحقن المباشر للالميتوكوندريا في البويضات 23، 24، 25، انصهار الخلية لتوليد cybrids transmitochondrial 26، 27 ومؤخرا، نقل الميتوكوندريا المعزولة باستخدام ضوئي؛ ضوحراري nanoblades 28. نحن وآخرون أظهرت سابقا قدرة mitochond معزولةريا على أن يكون داخليا من قبل الخلايا الحية، كما لوحظ على حد سواء في التجارب المختبرية والحية 29، 30، 31، من خلال الآليات المقترحة لإشراك macropinocytosis 32. نحن تطويرها وسيلة، ودعا MitoCeption، لنقل الكمية الميتوكوندريا المعزولة (من اللجان الدائمة) لاستهداف الخلايا، كما يتضح مع (ملتصقة) MDA-MB-231 سرطان الثدي خط الخلية 31. وقد تم تكييف هذا البروتوكول هنا لنقل معزولة الميتوكوندريا MSC الإنسان إلى خلايا ورم أرومي الجذعية (GSCs). ورم أرومي والأورام الخبيثة العدوانية من الدماغ الذي أصبح سريعا مقاومة للعلاج، ويرجع ذلك أساسا إلى خلايا ورم أرومي الجذعية (GSC) الموجودة داخل الورم 33. تنمو هذه GSCs كما neurospheres في المختبر، وتوليد أورام في نماذج طعم أجنبي. الخلايا السرطانية داخل ورم أرومي لديهاالقدرة على إجراء اتصالات خلية إلى خلية، كما هو مبين في الآونة الأخيرة لخلايا نجمي ورم في المخ المتصلة بعضها ببعض عبر microtubes الموسعة، التي من خلالها الميتوكوندريا (وكذلك الكالسيوم وخلية نوى) يمكن أن تهاجر، مما أدى إلى شبكات نجمي مقاومة للعلاج بالأشعة 34. ورم أرومي يمكن تجنيد العديد من الخلايا المختلفة داخل المكروية ورم، بما في ذلك اللجان الدائمة 35 و 36. أظهرنا أن اللجان الدائمة يمكن إجراء اتصالات خلية خلية مع GSCs في coculture ونقل الميتوكوندريا الخاصة بهم (لا تظهر البيانات)، التي من المتوقع أن تعديل الخصائص الفنية GSC. يصف هذا البروتوكول كيف تقنية MitoCeption يمكن استخدامها لنقل الميتوكوندريا، قبل معزول من اللجان الدائمة الإنسان، إلى GSCs البشرية بهدف تحديد نتائج البيولوجية الوظيفية. تم استخدام متعدد القدرات ومكون للأورام للغاية خط Gb4 GSC 37 في هذه الدراسة.

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			<td>Mitochondria Isolation Kit for Tissue&#160;</td>
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			<td>Peprotech&#160;</td>
			<td>AF-100-15&#160;</td>
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			<td>H3149&#160;</td>
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			<td>MERCK</td>
			<td>2382</td>
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			<td>Trypsine Inhibitor&#160;</td>
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			<td>T9003</td>
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			<td>Trypsin</td>
			<td>Gibco&#160;</td>
			<td>15090-046</td>
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			<td>Protease inhibitors EDTA free</td>
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			<td>4693159001</td>
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			<td>Ciprofloxacine&#160;</td>
			<td>Sigma</td>
			<td>17850-5G-F</td>
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			<td>Fungine&#160;</td>
			<td>Invivogen</td>
			<td>ant-fn-1</td>
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			<td>&#160;M7512</td>
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			<td>&#160;M22426&#160;</td>
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			<td>C12881</td>
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			<td>Santa Cruz</td>
			<td>sc-24948</td>
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			<td>World Precision Instruments</td>
			<td>FD35-100</td>
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			<td>Fisher Scientific</td>
			<td>267110</td>
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			<td>Beckman Coulter</td>
			<td>2,523,749</td>
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			<td>Gallios&#160;</td>
			<td>&#160;3L 10C</td>
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			<td>LC FAST START DNA MASTER PLUS&#160;</td>
			<td>Roche</td>
			<td>3515885001</td>
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mitoception: transferring isolated human msc mitochondria to glioblastoma stem cells

الميتوكوندريا تلعب دورا محوريا لاستقلاب الخلية، وإنتاج الطاقة والسيطرة على موت الخلايا المبرمج. وقد تم العثور على وظيفة الميتوكوندريا عدم كفاية مسؤولة عن أمراض متنوعة جدا، بدءا من الأمراض العصبية والسرطان. ومن المثير للاهتمام، وقد ثبت الميتوكوندريا مؤخرا لعرض القدرات على أن يتم تحويلها بين أنواع الخلايا، وخاصة من الخلايا الجذعية الوسيطة الإنسان (MSC) إلى الخلايا السرطانية في ظروف coculture، مع عواقب والتمثيل الغذائي والوظيفية للخلايا الميتوكوندريا المتلقي، ومواصلة تعزيز الاهتمام الحالي لالخصائص البيولوجية لهذه العضيات. تقييم الآثار المترتبة على الميتوكوندريا MSC المنقولة في الخلايا المستهدفة هي من الأهمية بمكان لفهم نتائج البيولوجية لهذه التفاعلات خلية خلية. بروتوكول MitoCeption الموصوفة هنا يسمح بنقل الميتوكوندريا المعزولة مسبقا من الخلايا المانحة إلى الخلايا المستهدفة، وذلك باستخدام MSC الميتوكوندرياوالخلايا الجذعية ورم أرومي (GSC) كنظام نموذج. وقد سبق استخدام هذا البروتوكول لنقل الميتوكوندريا، معزولة عن اللجان الدائمة، لمتمسكا الخلايا السرطانية MDA-MB-231. ويتم تكييف هذا البروتوكول نقل الميتوكوندريا هنا لGSCs التي تقدم خصوصية محددة من تنامي كما neurospheres في المختبر. نقل الميتوكوندريا المعزولة يمكن اتباعها من قبل مضان تنشيط الخلايا الفرز (FACS) والتصوير متحد البؤر باستخدام الميتوكوندريا الأصباغ الحيوية. استخدام الميتوكوندريا المانحة والهدف الخلايا مع النسخ المتنوعة مميزة (النيوكلوتايد) كما يتيح الكشف عن الميتوكوندريا نقل على أساس تركيز الحمض النووي التعميم الخاصة بهم (و mtDNA) في الخلايا المستهدفة. مرة واحدة وقد تم التحقق من صحة بروتوكول مع هذه المعايير، فإن خلايا إيواء الميتوكوندريا نقل يمكن تحليلها لتحديد الآثار المترتبة على الميتوكوندريا الخارجية على الخصائص البيولوجية مثل استقلاب الخلية، والمطاوعة، وانتشار واستجابة للعلاج.

وتظهر الخطوات الإجرائية تحدد عزل الميتوكوندريا من الخلايا الجذعية الوسيطة (MSC) ونقلها إلى الخلايا الجذعية ورم أرومي المستهدفة (GSC) من خلال MitoCeption في الشكل 1. GSCs هي الخلايا الجذعية السرطانية كما نمت neurospheres للحفاظ على خصائص الخلايا الجذعية الخ...

Watch this Scientific Journal Video about MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells at JoVE.com

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

هنا، يقدم بروتوكول (MitoCeption) لنقل الميتوكوندريا، معزولة عن الخلايا الجذعية الوسيطة الإنسان (MSC)، إلى خلايا ورم أرومي الجذعية (GSC)، بهدف دراسة التأثيرات البيولوجية على GSC الأيض ووظائف. ويمكن تكييف بروتوكول مماثل لنقل الميتوكوندريا بين أنواع الخلايا الأخرى.

MitoCeption: نقل معزولة الإنسان MSC الميتوكوندريا إلى خلايا الجذعية ورم أرومي دبقي

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found ...

mitoception-transferring-isolated-human-msc-mitochondria-to

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12.7K Views.  Montpellier University.  هنا، يقدم بروتوكول (MitoCeption) لنقل الميتوكوندريا، معزولة عن الخلايا الجذعية الوسيطة الإنسان (MSC)، إلى خلايا ورم أرومي الجذعية (GSC)، بهدف دراسة التأثيرات البيولوجية على GSC الأيض ووظائف. ويمكن تكييف بروتوكول مماثل لنقل الميتوكوندريا بين أنواع ا?...

Video: MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

انتشار الخلايا الجنينية البشرية (ES) الجذعية

Counting Human Neural Stem Cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

عد الخلايا الجذعية العصبية البشرية

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

الركض الخلايا الجذعية العصبية البشرية

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Immunocytochemistry: Human Neural Stem Cells

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of an antigen of interest, most commonly a protein, in cultured cells. This protocol presents an easy-to-follow series of steps that will enable researchers to conserve primary and secondary antibodies while getting high quality, reproducible qualitative and quantitative data out of their staining. There are two aspects of this protocol that help to conserve the volume of antibody necessary for staining. For one, the cells are grown on small, circular coverslips that are placed in wells of a tissue culture plate. After fixation, the cells on coverslips can be removed from the wells of the plate. For antibody staining, the coverslip with cells is inverted onto a small drop of antibody solution on parafilm and is covered with a second piece of parafilm to prevent drying. Using this method, only ~25 &mu;l of antibody solution is needed for each coverslip (or sample) to be stained. This protocol describes immunostaining of human neural stem/precursor cells (hNSPCs), but can be used for many other cell types.

Immunocytochemistry is a powerful method to determine the presence, subcellular localization, and relative abundance of an antigen of interest in cultured cells. This protocol presents an easy-to-follow series of steps that will enable one to conserve antibodies and get the most out of one's staining.

كيمياء سيتولوجية مناعية : الخلايا الجذعية العصبية البشرية

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

مناعي لونين من الخلايا الجذعية الجنينية البشرية

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

تنقية الميتوكوندريا من خلايا الخميرة

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

تجميد والذوبان الخلايا الجذعية الجنينية البشرية

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

تمايز الخلايا الجذعية الجنينية في السلائف Oligodendrocyte

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

وبنقل العدوى إلى الإنسان Nucleofecting الجذعية المحفزة المستحثة خلايا

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

متعدد المعلمة قياس نفاذية المسام افتتاح المرحلة الانتقالية في القلب ماوس متفرقة الميتوكوندريا

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...