نشكر أعضاء المختبر للحصول على مساعدة مارياني تحرير البروتوكول. وقد تم تمويل هذا العمل من قبل على منحة ما بعد الدكتوراه التدريب CIRM (JLF)، والتدريب الجسور CIRM (TZTT)، وروبرت E. ومايو R. رايت في مؤسسة (FVM)، وجامعة جنوب كاليفورنيا (FVM).

<ol>
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الإعلان عن أي تضارب في المصالح.

بصمات هامة من موت الخلايا المبرمج وتشمل الكشف عن الحمض النووي من التلف مع مقايسة TUNEL، جنبا إلى جنب مع الكشف عن النشاط كاسباس (الكشف عن MABS أو مع جزيء ملزمة مثل ZVAD-ماركا)، ومراقبة التغيرات الخلوية، وطريقة عرض فسفاتيديل (PS ) على سطح الغشاء (الكشف عنها بواسطة Annexin V ملزمة). هن...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td></td><td> كتالوج رقم </td></tr><tr><td> LysoTracker الأحمر DND-99 </td><td> إينفيتروجن </td><td> # L-7528 </td><td></td></tr><tr><td> هانكس BSS </td><td> إينفيتروجن </td><td> 14025-076 </td><td></td></tr><tr><td> بارافورمالدهيد </td><td> EMD </td><td> EM-PX0055-3 </td><td></td></tr><tr><td> Vectashield </td><td> VECTOR </td><td> H-1200 </td><td></td></tr><tr><td> DMEM </td><td> Cellgro </td><td> 10-013-CV </td><td></td></tr><tr><td> غير الأحماض الأمينية الأساسية </td><td> Cellgro </td><td> 25-025-CI </td><td></td></tr><tr><td> البيروفات الصوديوم </td><td> Cellgro </td><td> 25-000-CI </td><td></td></tr><tr><td> FBS </td><td> Hyclone </td><td> SH30071.02 </td><td></td></tr><tr><td> القلم بكتيريا </td><td> إينفيتروجن </td><td> 15140-122 </td><td></td></tr><tr><td> B-المركابتويثانول، 50 مم </td><td> إينفيتروجن </td><td> 21985-023 </td><td></td></tr><tr><td> LabTek-II الشرائح غرفة (8-أيضا) </td><td> Nalge نونك الدولية </td><td> 154534 </td><td></td></tr><tr><td> 0.1٪ الجيلاتين </td><td> ميليبور </td><td> ES-006-B </td><td></td></tr><tr><td> Dulbecco في PBS (D-PBS) </td><td> Cellgro </td><td> 21-031-CV </td><td></td></tr><tr><td></td><td></td><td></td><td> حل وصفات 4٪ لامتصاص العرق ل100 مل: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> خلط 4 غرام بارافورمالدهيد، 90 مل H 2 O، وهيدروكسيد الصوديوم (هيدروكسيد الصوديوم بنسبة انخفاض قدرها 2N). سوف لن أخوض في بارافورمالدهيد حل حتى تقوم بإضافة بعض هيدروكسيد الصوديوم لزيادة درجة الحموضة. </li><li style=";text-align:right;direction:rtl"> اثارة والحرارة عند 60 درجة مئوية حتى كل المسحوق هو في الحل (~ 10-20 دقيقة). لا افراط. </li><li style=";text-align:right;direction:rtl"> إضافة 10 مل PBS ~ 10X لتحقيق الحجم النهائي من 100 مل. </li><li style=";text-align:right;direction:rtl"> المحل في -20 درجة مئوية في مريحة (ML 10 ~ ~ 40 مل أو) مأخوذة. </li></ol> تحذير: لامتصاص العرق في شكل &quot;هدب&quot; (مضغوط الكريات الصغيرة) أقل مساحيق وبالتالي يمكن قياس خارج غطاء. ومع ذلك لا يزال يجب عليك ارتداء قناع الغبار واقية (N95على الأقل) أثناء التداول. EB الثقافة وسائل الإعلام وسائل الإعلام لمدة 500 مل EB: <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> DMEM: </td><td> 404،5 مل </td></tr><tr><td> FBS: </td><td> 75،0 مل </td></tr><tr><td> الجلوتامين L-: </td><td> 5،0 مل </td></tr><tr><td> البنسلين / الستربتوميسين: </td><td> 5،0 مل </td></tr><tr><td> غير الأحماض الأمينية الأساسية: </td><td> 5،0 مل </td></tr><tr><td> البيروفات الصوديوم: </td><td> 5،0 مل </td></tr><tr><td> β-المركابتويثانول: </td><td> 500ميكرولتر </td></tr></tbody></table></td></tr></tbody></table>

use of lysotracker to detect programmed cell death in embryos and differentiating embryonic stem cells

موت الخلية المبرمج (PCD) ويحدث في الكبار للحفاظ على التوازن الطبيعي الأنسجة الجنينية والتنمية خلال لتشكيل الأنسجة والأعضاء 1،2،6،7. خلال التنمية، يمكن أن المواد الكيميائية السامة أو تغيرات جينية تسبب زيادة في PCD PCD أو تغيير الأنماط مما أدى إلى تشوهات النمو والعيوب الخلقية 3-5. لفهم مسببات هذه العيوب، يمكن أن تستكمل دراسة الأجنة في المقايسات مع المختبر التي تستخدم التفريق الجذعية الجنينية (ES) الخلايا. موت الخلايا المبرمج هو شكل مدروسة من PCD التي تنطوي على حد سواء الذاتية وخارجي مما يشير إلى تفعيل تتالي كاسباس الانزيم. التغييرات الخلية مميزة تشمل blebbing الغشاء، وتقلص النووية، وتجزئة الحمض النووي. أشكال أخرى من PCD لا تنطوي على تفعيل كاسباس وربما تكون النتيجة النهائية من الالتهام الذاتي لفترات طويلة. بغض النظر عن المسار PCD، الخلايا الميتة تحتاج إلى إزالة. في البالغين، والخلايا المناعية PERFORM هذه الوظيفة، بينما في الأجنة، حيث لم الجهاز المناعي تطور بعد، وإزالة يحدث من قبل آلية بديلة. هذه الآلية تنطوي على الخلايا المجاورة (وتسمى &quot;غير المهنية البالعات&quot;) أخذ على أكلة الدور يعترفون &#39;أكل لي&#39; إشارة على سطح الخلية الموت وتبتلع من 8-10. بعد الإحاطة، يتم جلب الحطام إلى يحلول للتدهور. بغض النظر بالتالي آلية PCD، يمكن يرتبط زيادة في النشاط الليزوزومية مع زيادة موت الخلايا. يمكن لدراسة PCD، وهو فحص بسيط لتصور الجسيمات الحالة في الأنسجة سميكة ومتعدد الطبقات الثقافات يكون من المفيد التمييز. صبغ LysoTracker هو جزيء صغير جدا للذوبان التي يتم الاحتفاظ بها في حجرات التحت خلوية الحمضية مثل يحلول 11-13. يتم أخذ صبغة بنسبة نشرها من خلال والدورة الدموية. منذ اختراق ليست عائقا، والتصور من PCD في الأنسجة سميكة والثقافات متعددة الطبقات من الممكن 12،13 </سوب&gt;. في المقابل، ويقتصر TUNEL (الطرفية deoxynucleotidyl ترانسفيراز نهاية نيك dUTP التوسيم) تحليل 14، إلى عينات صغيرة، المقاطع النسيجية، والثقافات أحادي الطبقة لأن الإجراء يتطلب إدخال / نفاذية محطة ترانسفيراز. وعلى النقيض من الزرقاء الأنيلين، الذي ينشر ويذوب من المذيبات، LysoTracker الأحمر DND-99 هو قابل للتثبيت، ومشرق، ومستقرة. يمكن تلطيخ يمكن تصور المجهر الفلورسنت القياسية مع مبائر أو في جبل لكليا أو القسم أو باستخدام المذيبات المائية القائمة على وسائل الإعلام تصاعد 12،13. نحن هنا وصف البروتوكولات باستخدام هذه الصبغة للنظر في PCD في الحالات العادية والماوس سونيك القنفذ الأجنة فارغة. وبالإضافة إلى ذلك، علينا أن نبرهن تحليل PCD في التفريق مزارع الخلايا ES وتقديم طريقة بسيطة الكمي. وباختصار، يمكن تلطيخ LysoTracker يكون تكملة كبيرة إلى أساليب أخرى للكشف عن PCD.

Watch this Scientific Journal Video about Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells at JoVE.com

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

نقدم بروتوكول بسيط لتصور مناطق موت الخلايا المبرمج (PCD) في أجنة الفئران الجنينية والتمييز الثقافات الخلايا الجذعية (ES) باستخدام صبغة قابلة للذوبان للغاية ودعا LysoTracker.

استخدام LysoTracker لكشف موت الخلية المبرمج في الأجنة والخلايا الجذعية الجنينية التفريق

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

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Unit 1

Cell Death

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23.0K Views.  University of Southern California. نقدم بروتوكول بسيط لتصور مناطق موت الخلايا المبرمج (PCD) في أجنة الفئران الجنينية والتمييز الثقافات الخلايا الجذعية (ES) باستخدام صبغة قابلة للذوبان للغاية ودعا LysoTracker.

Video: Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

تشريح 6.5 أجنة الفئران DPC

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

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 ...

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

الخلايا الجذعية الجنينية المستمدة من الخلايا البطانية لعلاج نقص التروية Hindlimb

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

إعداد الأجنة المجهري للإلكترون من ذبابة الفاكهة أنبوب القلب الجنينية

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

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 ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

الفيديو المعلوماتية الحيوية تحليل النمو البشري الخلية الجذعية الجنينية كولوني

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

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 ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

رسم مصير الخلايا الجذعية الجنينية البشرية من خلال تكوين ورم مسخي

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.