نود أن نشكر أعضاء المختبر لي لإجراء مناقشات قيمة على المخطوطة. وأيد العمل في المختبر لي من المنح المقدمة من روبرتسون جائزة الباحث نيويورك مؤسسة الخلايا الجذعية من ولاية ماريلاند وصندوق أبحاث الخلايا (TEDCO) الجذعية.

1. إعداد اللوحة 96 جيد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مزيج 1 ميكرولتر من خلية واحدة DNase1 إلى 9 ميكرولتر وحيد الخلية تحلل الحل. </li><li style=";text-align:right;direction:rtl"> وضع 10 ميكرولتر حل مختلطة في كل بئر من 96 لوحة جيدا PCR. </li></ol> 2. فصل hESCs لنظام مراقبة الأصول الميدانية تنقية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> فصل OCT4 :: خط الخلية EGFP ES من 60 ملم صحن مع 1 مل Accutase لمدة 20 دقيقة، عند 37 درجة مئوية، والتي كانت متعادلة مع وسائل الاعلام الإنسان ES. </li><li style=";text-align:right;direction:rtl"> إعداد السكان خلية في 1 مل FACS العازلة وضبط خلية إلى 1 × 10 6 خلية / مل. </li><li style=";text-align:right;direction:rtl"> تمرير عينة الخلية من خلال مصفاة الخلية 35 ميكرومتر غطاء الأنبوب. </li><li style=";text-align:right;direction:rtl"> تخزين أنبوب في الجليد قبل الفرز الخلية. </li></ol> 3. تحلل من نظام مراقبة الأصول الميدانية تنقية خلية واحدة في كل بئر من لوحة 96 جيدا <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> فرز عينة للخلايا إيجابية EGFP على فارز الخلية مع المشغل المدربين. وضع خلية واحدة في خلية واحدة Lysis/DNase1 حل في 96 لوحة جيدا PCR. إذا لزم الأمر، و9لوحة 6 جيدا مع عينة يمكن تخزينها في -80 درجة مئوية التجميد العميق أقل من شهر واحد. </li><li style=";text-align:right;direction:rtl"> احتضان العينات 5 دقائق على RT لتحلل الخلية. </li><li style=";text-align:right;direction:rtl"> إضافة 1 ميكرولتر من وقف الحل لوقف رد فعل تحلل. </li><li style=";text-align:right;direction:rtl"> احتضان 2 دقيقة في RT. </li></ol> 4. عكس النسخ <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة إلى كل قسامة 0.5 ميكرولتر من 20 ميكرومتر SMA-T15، SMA-A. </li><li style=";text-align:right;direction:rtl"> إضافة 4 ميكرولتر 5X العازلة، 2 ميكرولتر DTT، 1 ميكرولتر الناسخ العكسي، و 1 ميكرولتر dNTP إلى كل بئر. </li><li style=";text-align:right;direction:rtl"> أداء النسخ العكسي في cycler الحرارية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ضبط البرنامج الحراري في 42 ° C × 90 دقيقة وتعطيل الناسخ العكسي في 85 ° C × 5 دقائق. </li></ol></li></ol> 5. التضخيم <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 4 ميكرولتر من كاشف ExoSAP-IT لكل عكس العينة كتب. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> احتضان العينات عند 37 درجة مئوية لمدة 15 دقيقة و 80 درجة مئوية لمدة 15 دقيقة إلى تعطيل كاشف ExoSAP-IT. </li></ol></li><li style=";text-align:right;direction:rtl"> إعداد PCR مزيج التفاعل ثإيث SMA-P2 (2 نانومتر) </li><li style=";text-align:right;direction:rtl"> إضافة 10 ميكرولتر من مزيج PCR رد فعل على عكس كل عينة كتب. </li><li style=";text-align:right;direction:rtl"> أداء التضخيم، ويتألف من 20 دورات تمسخ (94 درجة مئوية لمدة 30 ثانية)، الصلب (57 درجة مئوية لمدة 30 ثانية)، والإرشاد (68 درجة مئوية لمدة 10 دقيقة). </li></ol> 6. الأداء QRT-PCR <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إضافة 10 ميكرولتر من 2X SYBR الأخضر ميكس ماجستير PCR، 1 ميكرولتر تضخيم [كدنا]، 2 الاشعال نانومتر، و 7 ميكرولتر المياه إلى كل بئر. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ضبط البرنامج تليها 95 درجة مئوية لمدة 3 ثانية، 60 درجة مئوية لمدة 30 ثانية × 40 دورات. </li></ol></li><li style=";text-align:right;direction:rtl"> أداء في مكررة لأخطاء فنية. </li></ol>

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والكتاب ليس لديهم ما يكشف.

واحد التنميط الجيني الخلايا يمكن أن تكون أداة رئيسية للتنبؤ وظيفة من خلية واحدة أو شعب بأكمله. بسبب قيود تقنية، وقد تم تقييد التحليل التنميط الجيني بأكمله إلى المتوسطات السكان. وقد اقترحت الاختلافات في أنماط التعبير الجيني ومستويات بين الخلايا الفردية والمجموعات ا?...

السكان حقيقي النواة معظم العالي غير متجانسة وبالتالي مع تحليل من سكان المجمعة، غالبا ما يكون من الصعب تفسير خصائصها الخلوية. الخلايا الفردية ضمن مجموعة من السكان قد تكون مختلفة بمهارة، ويمكن لهذه الاختلافات لها عواقب هامة للخاصية وظيفة من إجمالي عدد السكان 1، 2. خاصة، ومن المعروف عن الخلايا الجذعية الجنينية البشرية (hESCs) أن تكون غير متجانسة، والذي يسبب مستويات مختلفة من تعدد القدرات والإمكانات المتنوعة لمواصفات النسب في طرق مميزة بدقة 3 و 4. على سبيل المثال، تختلف مستضدات سطح الخلية يمكن استخدامها لتصنيف الخلايا الجذعية المحفزة غير متمايزة، (5) ومجموعة أوستن سميث المقترحة مستويات مختلفة من تعدد القدرات في الخلايا الجذعية الجنينية، استنادا على التشكل، الميل التمايز وتبعية يشير مسار 6. وقد افترض هذه الظاهرة في الجنين البشريالخلايا الجذعية شركة الاستثمارات الوطنية 7. في حين أجريت الدراسات الشاملة بين مختلف خطوط الخلايا الجذعية، وليس الخلايا الجذعية فرد واحد، ويمكن أن تكون مثيرة للغاية لتحليل مستويات مختلفة من تعدد القدرات على مستوى خلية واحدة، والتي يحتمل أن يؤثر على قدرات التمايز تجاه كل الأنساب الخلية الجسدية. يمكن أملت عدم التجانس الخلوية والجزيئية من خلال التنميط النسخ، وهو ما يسمى &#39;Transcriptome على خلية واحدة&#39; ويؤكد نهج جديدة لقياس مستويات التعبير الجيني 8-10. لتحليل مستويات التعبير الجيني في الخلايا الفردية، وضعنا بروتوكولا بسيطة، ولكن قوية من خلية واحدة الكمي RT-PCR. أكدنا على فعالية وجدوى من بروتوكول لدينا من خلال مقارنة كل نصف لست] خلية واحدة، وكذلك مجموع الرنا المخفف متسلسل من hESCs، مما أدى إلى الاختلافات التقنية والحد الأدنى من الخلافات. علاوة على ذلك، استخدمنا خط مراسل الوراثية لعزل ع متجانسةopulation من hESCs باستخدام نظام استهداف الجينات. استخدمت ناقلات المانحة لاستهداف OCT4 موضع (OCT4-2A-EGFP-PGK-بورو بناء) وزوج من البلازميدات TALEN 11. أدخلت ناقلات المانحة وزوج من البلازميدات TALEN في hESCs (H9، WA09) باستخدام لدينا nucleofection وبروتوكول اختيار نسيلي وصيانة أجريت hESCs استنادا لدينا بروتوكول روتين 12. أكدنا هذا الخط مراسل الوراثية التعبير عن EGFP لOCT4 التعبير في OCT4 :: EGFP hESCs. لدينا نتيجة يدل على أن hESCs الفردية (تم الفرز حسب OCT4 :: EGFP الخلايا إيجابية بقوة) عقد مستويات عالية من OCT4 التعبير، ولكن مستويات مختلفة من NANOG التعبير. لذلك، يجب أن يكون لدينا واحد الجينات الخلية التعبير فحص مفيدة لدراسة التغاير السكان من الخلايا الجذعية المحفزة.

<table border="0" cellpadding="0" cellspacing="0" width="807">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">96 wll PCR Plate</td>
			<td style="width:123px;">USA scientific</td>
			<td style="width:119px;">1402-8900</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Ambion Cell Lysis Kit</td>
			<td style="width:123px;">Life Technologies</td>
			<td align="right" style="width:119px;">4458235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">SMARTScribe Reverse Transcriptase</td>
			<td style="width:123px;">Clonetech</td>
			<td align="right" style="width:119px;">639536</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">ExoSAP-IT</td>
			<td style="width:123px;">USB</td>
			<td align="right" style="width:119px;">78200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Platinum Taq DNA polymerase High Fidelity</td>
			<td style="width:123px;">Invitrogen</td>
			<td align="right" style="width:119px;">11304</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">10mM dNTP Mix, PCR Grade</td>
			<td style="width:123px;">Invitrogen</td>
			<td align="right" style="width:119px;">18427</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">SYBR Universal 2X Master Mix</td>
			<td style="width:123px;">Kapa biosystem</td>
			<td style="width:119px;">KR0389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Accutase</td>
			<td>Innovative Cell Tech</td>
			<td>S-1100-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">FACS buffer</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td>45 ml PBS, 5 ml a-MEM, 100 ul DNase, filter sterilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">35 &#956;m cell strainer cap tubes</td>
			<td style="width:123px;">BD Biosciences</td>
			<td align="right" style="width:119px;">352235</td>
			<td></td>
		</tr>
	</tbody>
</table>

profiling individual human embryonic stem cells by quantitative rt-pcr

عدم تجانس السكان الخلايا الجذعية يعوق فهم مفصل لبيولوجيا الخلايا الجذعية، مثل التمايز ميلها نحو الأنساب مختلفة. وحيد الخلية Transcriptome على الفحص يمكن أن يكون نهجا جديدا لتشريح الاختلاف الفردية. قمنا بتطوير خلية واحدة طريقة QRT-PCR، وأكد أن هذا الأسلوب يعمل بشكل جيد في العديد من ملامح التعبير الجيني. في مستوى خلية واحدة، كل خلية الجذعية الجنينية البشرية، تم الفرز حسب OCT4 :: الخلايا إيجابية EGFP، لديه عالية في التعبير OCT4، ولكن على مستوى مختلف من NANOG التعبير. وينبغي أن يكون لدينا واحد الجينات الخلية التعبير فحص مفيدة لاستجواب التغاير السكان.

كفاءة وقوية واحدة تضخيم الحمض النووي الريبي الخلية لتقليل التباين بين النسخي hESCs، استخدمنا OCT4 :: EGFP HESC استنساخ لتنقية نظام مراقبة الأصول الميدانية. بعد فرز OCT4 :: الخلايا إيجابية EGFP في لوحة 96 جيدا، وهي lysed كل خلية ...

Watch this Scientific Journal Video about Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR at JoVE.com

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

وهناك حاجة واحدة الجين الخلية التعبير مقايسة لفهم التغاير الخلايا الجذعية.

التنميط الفردية الخلايا الجذعية الجنينية البشرية من خلال الكمي RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

profiling-individual-human-embryonic-stem-cells-by-quantitative-rt-pcr

Research

JoVE Journal

Biology

11.4K Views.  Johns Hopkins University School of Medicine.  وهناك حاجة واحدة الجين الخلية التعبير مقايسة لفهم التغاير الخلايا الجذعية. 

Video: Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

انتشار الخلايا الجنينية البشرية (ES) الجذعية

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 ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

اشتقاق الخلايا الجذعية الجنينية البشرية التي Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

سبر للنشاط مجمع الميتوكوندريا في خلايا المنشأ الجنينية البشرية

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

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 ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

وصفها في المختبر من خلايا المنشأ الجنينية البشرية لتصوير بالرنين المغناطيسي

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

الثقافة والمحافظة على الخلايا الجذعية الجنينية البشرية

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

الكروموسومات التحضير انتشار الخلايا الجذعية الجنينية البشرية لKaryotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

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 ...