تشريح والعزلة في وقت مبكر من الخلايا الجذعية المكونة للدم من الكيس المحي الفئران والشريان الأورطي ، الغدد التناسلية ، mesonephros المنطقة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قبل أن تبدأ ، فمن المهم أن يكون كل الكواشف والأدوات اللازمة لإجراء العملية : <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مقص </li><li style=";text-align:right;direction:rtl"> ملقط ، حجم 4 ، والجراحية الصف عازمة ذات الرؤوس ملقط ، حجم 5 / 45 (اختياري) </li><li style=";text-align:right;direction:rtl"> إبر عيار 30 مع حقنة </li><li style=";text-align:right;direction:rtl"> أطباق بتري 35mm و 100mm </li><li style=";text-align:right;direction:rtl"> برنامج تلفزيوني مع 10 ٪ و 20 ٪ مصل بقري جنيني </li><li style=";text-align:right;direction:rtl"> وأخيرا ، كولاجيناز </li></ul></li><li style=";text-align:right;direction:rtl"> للبدء في التشريح ، ونحن بحاجة لحصاد القرون الرحم من النساء الحوامل بنسبة 10.5 مفهوم آخر أيام. </li><li style=";text-align:right;direction:rtl"> يصرح باستخدامها في الحيوانات وفقا للاجراءات المعتمدة. نستخدم ثاني أكسيد الكربون الاختناق ، تليها خلع عنق الرحم. </li><li style=";text-align:right;direction:rtl"> بطن الأنثى هو الرطب مع الإيثانول وفتح الجلد مع المقص. </li><li style=";text-align:right;direction:rtl"> القابضة للجلد مع ملقط ، يتم إجراء تخفيضات إضافية لفتح البريتوني. </li><li style=";text-align:right;direction:rtl"> يتم فتح البطن والرحم القرون يمكن تصور. باستخدام الملقط لعقد قرون الرحم ، وقطع كل نهاية القاصي ومركز لاستئصال كل من قرنيه. </li><li style=";text-align:right;direction:rtl"> ثم توضع في الرحم قرون طبق بيتري الباردة التي تحتوي على برنامج تلفزيوني مع FBS 10 ٪ لإزالة أنسجة الرحم. </li><li style=";text-align:right;direction:rtl"> باستخدام حجم 4 ملقط والتي تعمل تحت المجهر ، وإزالة أنسجة بطانة الرحم بلطف حول كل من الحمل المستكن الجنين ، والحرص على عدم ثقب المشيمة. </li><li style=";text-align:right;direction:rtl"> إزالة مرة واحدة ، وتوضع الأجنة في طبق برنامج تلفزيوني جديد مع البرد وFBS 10 ٪. </li><li style=";text-align:right;direction:rtl"> إزالة أنسجة المشيمة بعناية للكشف عن الجنين المغطى في كيس المح باستخدام ملقط الجنين لخفض المغطى الكيس المحي بعيدا عن المشيمة في المرحلة الخاصة بهم. الكيس المحي الشرايين تحتوي على صفار ، وجزء من الحبل السري ، يمكن مثار بلطف بعيدا من الجنين ويترك جانبا لتفارق على الجليد. </li><li style=";text-align:right;direction:rtl"> يتم نقل الجنين الى طبق بيتري صغيرة مع مستويات أدنى من برنامج تلفزيوني مع FBS 10 ٪ -- ما يكفي لالرطب الجنين ولكن ليس كثيرا. المستويات المثلى حفاظ على الجنين ثابتة بينما يتم تحريكها ببطء لوحة ، والحفاظ على الجنين في مكان آمن أثناء تشريح. </li><li style=";text-align:right;direction:rtl"> التبديل إلى إبر عيار 30 ، والتي تستخدم لإزالة الأجزاء العلوية والسفلية من الجنين عن طريق خفض الأمامية فوق وتحت hindlimbs. قد أصبح معتادا على استخدام الإبر وأدوات القطع يستغرق بعض الممارسة. وهناك طريقة استخدام قطع صغيرة متكررة من عبور الإبر تشريح الأنسجة ، مع الإبرة التي عقدت في اليد المهيمنة ليكون بمثابة دليل لقطع الإبرة التي عقدت في يد غير المهيمنة. </li><li style=";text-align:right;direction:rtl"> إزالة الجزء الظهري للجنين. هذه هي الأنسجة التي تحتوي على somites. خفض بلطف بعيدا النسيج الظهري بينما معظم الباقين على مسافة كافية من الشريان الأبهر (خط أحمر) لمنع أي نيكس. وقطع الشريان الأورطي يؤدي إلى فقدان الدم وعدم القدرة على تصور بسهولة موقعه. مرة أخرى ، مما يجعل الجروح الصغيرة مع الإبر أمر حيوي للحفاظ على موقع قطع دقيقة. </li><li style=";text-align:right;direction:rtl"> دفع الأنسجة خارج بعيدا عن الأنظار وتحويل الجنين في جميع أنحاء لقطع الأنسجة بطني ، وقطع مرة أخرى أقرب إلى الشريان الأورطي ممكن من دون knicking عليه. جعل التخفيضات بعيدا جدا عن مكان أفضل من شأنه أن يؤدي في أنسجة البطن يجري اليوم ، والتي يمكن إزالتها بمجرد أن يتم عزل الجمعية العمومية. </li><li style=";text-align:right;direction:rtl"> موقف الجنين مع الجانب الظهري أسفل ظهرها أو على طبق من ذهب. وسع بلطف الجنين فتح طريق دفع الجانبين إلى أسفل وتصور مكان التلال الغدد التناسلية. وتقع هذه قليلا خارج المركز كما أنبوب يشبه الهياكل على جانبي الشريان الأورطي. </li><li style=";text-align:right;direction:rtl"> تشريح كاملة عن طريق إزالة الأنسجة الزائدة الجانب باستخدام تقنيات خفض نفسه كما كان من قبل. بمجرد إزالة الأنسجة الجانب من الجنين ، ثم تحول الجنين رأسا على عقب لإزالة الأنسجة الجانب المتبقية. </li><li style=";text-align:right;direction:rtl"> عند هذه النقطة ، ويتم تفتيش الجمعية العمومية للأي نوع من الأنسجة الزائدة التي يمكن إزالتها بسهولة مع الإبر دون وقوع إصابات في الشريان الأورطي أو الغدد التناسلية. إزالة الزائدة كما قدر النتائج الممكنة في أفضل عزل الخلايا الجذعية السكان نظرا لأننا نعتمد على مجرد علامات سطح الخلية قليلة للتنقية. </li><li style=";text-align:right;direction:rtl"> باستخدام الملقط عازمة ذات الرؤوس ، مغرفة برفق ومكان كل اجتماع الجمعية العامة العادية في برنامج تلفزيوني الباردة مع FBS 10 ٪ حتى جاهزة للتفكك. </li></ol> تفارق وتحليل FACS <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> أن يفرق بين الأنسجة ، وصفار المقام الأول وبيانات توزيع الأكياس في أنابيب منفصلة. </li><li style=";text-align:right;direction:rtl"> تدور لفترة وجيزة إلى الأنسجة وتجاهل بيليه في برنامج تلفزيوني مع FBS 10 ٪. </li><li style=";text-align:right;direction:rtl"> Resuspend الأنسجة في كولاجيناز 0.25 ٪ في برنامج تلفزيوني مع FBS 20 ٪ ، حيث وصل حجم ما يكفي لتغطية كافية الأنسجة. </li><li style=";text-align:right;direction:rtl"> احتضان الأنسجة عند 37 درجة. 25 - 30mins لبيانات توزيع 40mins و 35 مقابل كيس المح. </li><li style=";text-align:right;direction:rtl"> في نهاية الحضانة ، ماصة الأنسجة بلطف صعودا ونزولا إلى التفكك في تعليق خلية واحدة. </li><li style=";text-align:right;direction:rtl"> غسل المناديل مع واي PBS الزائدةال 10 ٪ FBS ، بيليه الخلايا وresuspend في برنامج تلفزيوني العقيمة جديدة مع FBS 10 ٪. </li><li style=";text-align:right;direction:rtl"> تحليل لنظام مراقبة الأصول الميدانية ، هي الخلايا الملون وفقا لتوصيات الشركة الصانعة. نستخدم FACSAria من العلوم البيولوجية دينار بحريني لأداء التدفق الخلوي. نظرا لحجم والهشاشة من الخلايا الجنينية ، يتم ضبط فتحة الفرز لأكبر قطر للخلايا المكونة للدم من الكبار ، ونحن أيضا دراسة في المختبر. وبالمثل ، يتم ضبط الضغط إلى أن تكون أقل من الشروط المطلوبة من قبل الخلايا المكونة للدم البالغين. على سبيل المثال ، والفرز على FACSAria ، فإن حجم فوهة تكون 100 ميكرون والضغط سيكون فرز 30 رطل. </li><li style=";text-align:right;direction:rtl"> يتم عزل الخلايا الجذعية لتحاليل ، يتم عزل الخلايا إيجابية الثلاثي معربا عن cKit علامات ، CD34 ، CD41 ووفرزها من الكيس المحي وايجابيات مزدوجة (cKit ، CD34) من الجمعية العمومية عن طريق نظام مراقبة الأصول الميدانية. ومن شأن المؤامرة نموذجي تبدو هذه : ويتم تحليل الخلايا إيجابية لcKit CD34 CD41 وبشكل متزامن أو بشكل فردي. وعادة ما تعرض الخلايا لتدرج من الإيجابية لكل علامة مع الخلية الجذعية السكان كما وجدت مجموعة فرعية من السكان إيجابية. هذه المؤامرات تبين لنا أننا قد حققت غلة جيدة مع 100-300 خلية خلية لكل كيس المح ومساعد المدير العام الواحد. </li></ol>

isolation of early hematopoietic stem cells from murine yolk sac and agm

في جنين الفأر ، في وقت مبكر تكون الدم يحدث في وقت واحد في الهيئات المتعددة ، التي تشمل كيس المح والمنطقة التناسلية ، الشريان الأورطي ، mesonephros. هذه المناطق تشكل عاملا حاسما في إنشاء نظام الدم في الأجنة ويؤدي في نهاية المطاف لحركة الخلايا الجذعية الجنينية في الكبد ومن ثم تطوير خلايا جذعية للبالغين في bonemarrow. يمكن عزل الخلايا الجذعية المكونة للدم في وقت مبكر من هذه الأجهزة من خلال microdissection الجنين تليها فرز cytometric تدفق السكان من الحصول على أكثر نقاء. يبقى من غير الواضح كيف يمكن لهذه الخلايا الجذعية تسهم السكان إلى تجمع الخلايا الجذعية الجنينية والكبار. أيضا ، تحقق في مختبرنا كيف تختلف الخلايا الجذعية في وقت مبكر وظيفيا من الخلايا الجنينية والبالغين الجذعية المكونة للدم. علاوة على ذلك ، يفرز مختبرنا مجموعات مختلفة من الخلايا الجذعية المكونة للدم واختبار دورها الوظيفي في سياق مجموعة متنوعة من النماذج الوراثية. في هذا الفيديو ، ونحن لشرح تشريح الداخلي الدقيقة التي نستخدمها عادة ، وتظهر أيضا نتائج plotfter FACS نموذجية عزل هؤلاء السكان نادرة ، فمن الممكن لتنفيذ مجموعة متنوعة من المقايسات الفنية بما في ذلك : المقايسات مستعمرة وزرع نخاع العظام.

Watch this Scientific Journal Video about Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM at JoVE.com

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

هذا الفيديو يوضح كيفية الجزئي تشريح كيس المح والشريان الأورطي ، الغدد التناسلية ، mesonephros المنطقة من الأجنة واستخدام التدفق الخلوي لفرز الخلايا الجذعية المكونة للدم.

عزل الخلايا الجذعية المكونة للدم في وقت مبكر من الكيس المحي الفئران وAGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

isolation-early-hematopoietic-stem-cells-from-murine-yolk-sac

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Biology

14.6K Views.  Brigham and Women's Hospital and Harvard Medical School. هذا الفيديو يوضح كيفية الجزئي تشريح كيس المح والشريان الأورطي ، الغدد التناسلية ، mesonephros المنطقة من الأجنة واستخدام التدفق الخلوي لفرز الخلايا الجذعية المكونة للدم.

Video: Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

العزلة وزرع الخلايا الجذعية المكونة للدم (HSCs)

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

اشتقاق خلايا الجذعية المكونة للدم من الفئران خلايا الجذعية الجنينية

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

العزلة وتحليل الخلايا الجذعية المكونة للدم من المشيمة

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

عزل الخلايا الجذعية من الإنسان Xenografts سرطان البنكرياس

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

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 ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

المظهري التحليل وعزل الخلايا الجذعية المكونة للدم الفئران والأسلاف النسب، الملتزم

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

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 ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

عزل الخلايا الجذعية من دليل الدهنية المشتقة من Lipoaspirates الإنسان

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

عدوى فيروسات الفئران الجنينية الجذعية المستمدة خلية خلايا الجسم مضغي لتحليل التمايز لدم

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

عزل الخلايا العضلية الفطرية من الغدد الفكه البالغة الدمعية

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...