<ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يهلم T75 القارورة باستخدام محلول 0.1 ٪ الجيلاتين واحتضان لوحة في 37 درجة مئوية ، و 5 ٪ CO 2 زراعة الأنسجة حاضنة قبل يوم واحد بين عشية وضحاها. </li><li style=";text-align:right;direction:rtl"> أخرج من احتضان خلايا MES ، نضح المتوسط ​​، شطف خلية ثقافة وفاق مع برنامج تلفزيوني ، إضافة 0.05 ٪ إلى حل التربسين معطف أسفل الطبق (2 صفيحة ml/100mm). </li><li style=";text-align:right;direction:rtl"> احتضان عند 37 درجة مئوية لمدة ما يقرب من 1 دقيقة ، حتى الخلايا هي النزع قبالة لوحة. </li><li style=";text-align:right;direction:rtl"> متمزج بلطف (الماصة صعودا وهبوطا) الخلايا trypsinized أربع إلى ست مرات لتفريق الخلايا وفاق مع ماصة باستير توصيله. نقل الخلايا ES فرقت في أنبوب 15 مل تحتوي على أجهزة الطرد المركزي المخروطية prewarmed (37 درجة مئوية) MES المتوسطة. </li><li style=";text-align:right;direction:rtl"> جمع الخلايا بواسطة الطرد المركزي. نضح لطاف ، أضف 10 مل من وزارة التربية والعلم المتوسطة ماصة صعودا وهبوطا لتشكيل تعليق خلية واحدة. </li><li style=";text-align:right;direction:rtl"> نقل تعليق خلية في قارورة T75 قبل المغلفة مع 0.1 ٪ في الجيلاتين واحتضان 37 درجة مئوية مع نسبة 5 ٪ CO 2 لمدة ساعة واحدة * بعد ساعة واحدة ، قد أولت الليفية إلى لوحة الخلايا الجذعية ولكن تبقى في المتوسط. ماصة يصل المتوسط ​​لجمع الخلايا الجذعية. </li><li style=";text-align:right;direction:rtl"> تدور الخلايا في 1000 دورة في الدقيقة لمدة 5 دقائق ، ونضح قبالة المتوسطة زارة التربية والعلم. ثم يضاف 10 مل أخرى من المتوسطة وزارة التربية والعلم تمايز الخلايا عن طريق resuspend pipetting المتكررة حتى يبدو أن هناك تعليق غرامة من الخلايا. </li><li style=";text-align:right;direction:rtl"> عدد الخلايا التي تحتوي على التفريق المتوسطة عدادة الكريات تستخدم لتخفيف تعليق الخلايا الجذعية إلى التركيز بين 400 إلى 500 خلية لكل 20ul (20ul/drop) في حوض العقيمة. </li><li style=";text-align:right;direction:rtl"> رفع الغطاء ، عكس بعناية ووضعه على قمة من صحن يحتوي على 10 مل من برنامج تلفزيوني. باستخدام ماصة الأقنية ، وجعل الصفوف من قطرات 20ul على السطح حتى تحول الداخلي للغطاء من النسيج صحن الثقافة. </li><li style=";text-align:right;direction:rtl"> المكان بعناية الطبق في الحاضنة لمدة 2 ايام. بعد يومين ، بدوره بعناية على مدى غطاء لوحة ، ونضح 180 UL متوسطة التمايز الطازجة ووضع عدة قطرات في البئر لوحة ultralow مرفق 96 - جيدا. ثم تراجع لاقط مع قطرات ماصة ونقلها ، واحدة تلو الأخرى ، لوحة 96 - جيدا. وضع لوحات في الحاضنة لمدة 3 أيام دون عائق. </li><li style=";text-align:right;direction:rtl"> معطف كل بئر لزراعة الأنسجة 48 لوحة جيدا مع 300ul من 0.1 ٪ الجيلاتين. بعد إضافة الجيلاتين ، واحتضان لوحة ليلا 37 درجة مئوية قبل يوم واحد قبل نقل EBS. </li><li style=";text-align:right;direction:rtl"> في اليوم التالي ، نضح في الجيلاتين من 48 لوحة جيدا. ثم يضاف 300 UL متوسطة التمايز إلى كل بئر. نقل الصفائح الحديدية من 96 إلى جانب لوحات 48 الجيلاتين المغلفة جيدا واحدة تلو الأخرى. تغيير المتوسطة في اليوم التالي ثم تغيير المتوسط ​​كل يوم للحفاظ على الخلايا. </li><li style=";text-align:right;direction:rtl"> وينبغي أن تقلصات cardiaomyocyte عفوية يكون واضحا في غضون 7 أيام. </li></ol>

<ol>
	<li>Wobus, A. M., Wallukat, G., Hescheler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+mouse+embryonic+stem+cells+are+able+to+differentiate+into+cardiomyocytes+expressing+chronotropic+responses+to+adrenergic+and+cholinergic+agents+and+Ca2++channel+blockers.">Pluripotent mouse embryonic stem cells are able to differentiate into cardiomyocytes expressing chronotropic responses to adrenergic and cholinergic agents and Ca2+ channel blockers.</a> Differentiation. 48, 173-182 (1991).</li><li>Metzger, J. M., Lin, W. I., Samuelson, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transition+in+cardiac+contractile+sensitivity+to+calcium+during+the+in+vitro+differentiation+of+mouse+embryonic+stem+cells.">Transition in cardiac contractile sensitivity to calcium during the in vitro differentiation of mouse embryonic stem cells.</a> J Cell Biol. 126, (1994).</li></ol>

مساوئ أسلوب قطرة شنقا هي كما يلي : يقتصر حجم السائل لتنخفض إلى أقل من 50ul بسبب الحفاظ على قطرات المعلقة على غطاء من التوتر السطحي ، وانه من المستحيل تغيير وسيلة لتعليق قطرات. الملاحظة تشكيل EBS في قطرات مباشرة مع المجهري هو أيضا من الصعب جدا خلال زراعة. مزيد المزيد ، وطريقة قطرة شنقا...

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in vitro differentiation of mouse embryonic stem (mes) cells using the hanging drop method

الخلايا الجذعية لديها القدرة على تطوير ملحوظ في العديد من أنواع مختلفة من الخلايا. عندما يقسم الخلايا الجذعية ، فإن كل خلية جديدة لديها القدرة على البقاء إما أو تصبح الخلايا الجذعية نوع آخر من الخلايا ذات وظيفة أكثر تخصصا ، وهذا العلم هو واعد كبار العلماء لدراسة إمكانية خلية مقرها العلاجات لعلاج المرض. عندما الثقافة في الوقف بدون عوامل antidifferentiation ، والخلايا الجذعية الجنينية تفرق بشكل عفوي وشكل ثلاثي الأبعاد مجاميع متعددة الخلايا. وتسمى هذه المجاميع الخلية الهيئات مضغي الشكل (EB). اعدام ثقافة الإفلات من تشكيل EB المستخدمة على نطاق واسع طريقة الاستقراء. الجزء السفلي من قطرة شنقا تقريب يسمح تجميع الخلايا ES الخلايا التي يمكن أن توفر بيئة جيدة زارة التربية والعلم لتشكيل EBS. يمكن السيطرة على عدد من الخلايا ES aggregatied في انخفاض شنقا متفاوتة عدد من الخلايا في خلية تعليق أولي على أن تكون معلقة على قطرة من غطاء طبق بيتري. باستخدام هذا الأسلوب يمكن أن نشكل بتكاثر EBS متجانسة من عدد محدد مسبقا من الخلايا ES.

Watch this Scientific Journal Video about In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method at JoVE.com

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

هذا الفيديو يوضح كيفية السلوك في المختبر من تمايز الخلايا الجذعية الجنينية للهيئات مضغي الشكل باستخدام طريقة قطرة شنقا.

التمايز في المختبر من خلايا الجنينية الماوس (MES) الجذعية باستخدام أسلوب إسقاط الشنق

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

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In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

31.3K Views.  Stanford University. هذا الفيديو يوضح كيفية السلوك في المختبر من تمايز الخلايا الجذعية الجنينية للهيئات مضغي الشكل باستخدام طريقة قطرة شنقا.

Video: In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.

We demonstrate a simple method for placing cells at desired locations on a substrate. This method patterns cells by flipping a silicone chip containing microwells filled with cells onto the substrate. This method provides a new way to modulate diffusible and juxtacrine signaling between cells.

الزخرفة من الخلايا الجذعية الجنينية باستخدام رقاقة بيو فليب

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

في المختبر والمجراة في ضوء بارد مراسل التصوير الجيني للخلايا منشأ جنينية بشرية

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

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 ...

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

لإعداد ذبابة الفاكهة الأجنة لايف التصوير باستخدام بروتوكول إسقاط الشنق

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

العزلة واشتقاق خلايا الفأر الجنينية بيرشوت

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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 ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

كفاءة تمايز الخلايا الجذعية الجنينية في الخلايا العصبية الحركية

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

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

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

جيل من الفئران المستمدة من الخلايا الجذعية المحفزة المستحثة

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

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

التفريق بين الأطفال حديثي الولادة ماوس خلايا الجلد الجذعية المشتقة في مثل جرثومة خلايا<em&gt; في المختبر</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

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 ...