<meta charset="utf8"></head><body>用于药物筛选和疾病建模的 hiPSC 来源的血管类器官

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<table><tbody><tr><td>2-Mercaptoethanol (1000x)</td><td>Gibco</td><td>21985023</td><td></td></tr><tr><td>Accutase</td><td>Sigma-Aldrich</td><td>A6964</td><td></td></tr><tr><td>Alexa Fluor 488 AffiniPure F(ab&#039;)&lt;sub&gt;2&lt;/sub&gt; Fragment Donkey Anti-Rabbit IgG (H+L)</td><td>Jackson Immuno Research Labs</td><td>711-546-152</td><td>reconstitute with 0.25 mL 50% glycerol,&amp;nbsp; store at -20 &amp;deg;C for up to 1 year, dilution ratio for use: 1:1000</td></tr><tr><td>Alexa Fluor 647 AffiniPure F(ab&#039;)&lt;sub&gt;2&lt;/sub&gt; Fragment Donkey Anti-Goat IgG (H+L)&amp;nbsp;</td><td>Jackson Immuno Research Labs</td><td>705-606-147</td><td>reconstitute with 0.25 mL 50% glycerol,&amp;nbsp; store at -20&amp;deg;C for up to 1 year, dilution ratio for use: 1:1000</td></tr><tr><td>B27 supplement minus vitamin A (50x)</td><td>Gibco</td><td>12587010</td><td>thaw at 4 &amp;deg;C, aliquot and store at -20 &amp;deg;C, avoid freeze-thaw cycle</td></tr><tr><td>BMP4</td><td>ProSpec</td><td>CYT-081</td><td>reconstitute with sterile ddH&lt;sub&gt;2&lt;/sub&gt;O at a concentration of 100 ng/&amp;micro;L, aliquot and store at -20 &amp;deg;C</td></tr><tr><td>CD31 antibody&amp;nbsp;</td><td>abcam</td><td>ab28364</td><td>dilution ratio: 1:400</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>022626001</td><td></td></tr><tr><td>CHIR99021</td><td>Tocris Bioscience</td><td>4423/10</td><td>make the stock solution at 12 mM with DMSO, and store at -20 &amp;deg;C for up to 1 year.</td></tr><tr><td>Chroman 1</td><td>Tocris</td><td>7163/10</td><td>make the stock solution at 100 &amp;micro;M with DMSO, and store at -20 &amp;deg;C for up to 1 year.</td></tr><tr><td>Collagen I&amp;nbsp;</td><td>Corning</td><td>40236</td><td></td></tr><tr><td>Confocal Microscope</td><td>Nikon</td><td>AXRMP/Ti2</td><td></td></tr><tr><td>Corning Elplasia Plates</td><td>Corning</td><td>4441</td><td></td></tr><tr><td>Countess II FL automated cell counter</td><td>Thermo Fisher</td><td>AMQAF1000</td><td></td></tr><tr><td>DMEM/F-12, GlutaMAX supplement</td><td>Gibco</td><td>10565018</td><td></td></tr><tr><td>DMEM/F-12, powder</td><td>Gibco</td><td>12500062</td><td>make 10x stock solution with biograde ddH&lt;sub&gt;2&lt;/sub&gt;O and sterilize it with a 0.2 &amp;micro;m filter. Store at 4 &amp;deg;C.</td></tr><tr><td>Edi042A</td><td>Cedars Sinai</td><td></td><td></td></tr><tr><td>Emricasan</td><td>Medchemexpress LLC</td><td>HY1039610MG</td><td>make the stock solution at 1 mM with DMSO, and store at -20 &amp;deg;C for up to 1 year.</td></tr><tr><td>ERG antibody</td><td>Cell Singali Technology</td><td>97249</td><td>dilution ratio: 1:400</td></tr><tr><td>Fetal Bovine Serum - Premium</td><td>Atlanta Biologicals</td><td>S11150</td><td>thaw at 4 &amp;deg;C&amp;nbsp; and aliquot, store at&amp;nbsp; -20 &amp;deg;C for up to five years, or store at 4 &amp;deg;C for up to a month</td></tr><tr><td>FGF2</td><td>ProSpec</td><td>CYT557</td><td>reconstitute with sterile ddH2O at a concentration of 100 ng/&amp;micro;L, aliquot and store at -20 &amp;deg;C</td></tr><tr><td>Fluorodish Cell Culture Dish</td><td>World Precision Instruments</td><td>FD35-100</td><td></td></tr><tr><td>Forskolin</td><td>Tocris Bioscience</td><td>1099</td><td>reconstitute with DMSO at a concentration of 12 mM, aliquot and store at -20 &amp;deg;C</td></tr><tr><td>Gentamicin (50 mg/mL)</td><td>Gibco</td><td>15710064</td><td></td></tr><tr><td>GlutaMAX supplement (100x)</td><td>Gibco</td><td>35050061</td><td></td></tr><tr><td>Heparin, 100 kU</td><td>MilliporeSigma</td><td>375095100KU</td><td>reconstitute with sterile ddH2O at a concentration of 4 kU/mL</td></tr><tr><td>HEPES (1 M)</td><td>Gibco</td><td>15630080</td><td></td></tr><tr><td>Incubator</td><td>Thermo Scientific</td><td>13998123</td><td></td></tr><tr><td>Knockout Serum Replacement</td><td>Gibco</td><td>10828028</td><td>thaw at 4 &amp;deg;C, aliquot and store at -20&amp;deg;C, avoid freeze-thaw cycle</td></tr><tr><td>Matrigel, GFR Basement Membrane Matrix</td><td>Corning</td><td>356230</td><td>thaw at 4 &amp;deg;C, aliquot and store at -80&amp;deg;C, avoid freeze-thaw cycle</td></tr><tr><td>MEM Non-Essential Amino Acids Solution (NEAA, 100x)</td><td>Gibco</td><td>11140050</td><td></td></tr><tr><td>Molecular Biology Grade Water</td><td>Corning</td><td>46000CV</td><td></td></tr><tr><td>mTeSR plus kit</td><td>STEMCELL Technologies</td><td>1001130</td><td>thaw at 4 &amp;deg;C, aliquot and store at -20&amp;deg;C, avoid freeze-thaw cycle</td></tr><tr><td>N2 supplement (100x)</td><td>Gibco</td><td>17502048</td><td>thaw at 4 &amp;deg;C, aliquot and store at -20&amp;deg;C, avoid freeze-thaw cycle</td></tr><tr><td>NaOH solution (1 M)</td><td>Cytiva&amp;nbsp;HyClone</td><td>SH31088.01</td><td></td></tr><tr><td>NeuroBasal medium</td><td>Gibco</td><td>21103049</td><td></td></tr><tr><td>NIS-Elements, ver 5.42</td><td>Nikon</td><td></td><td></td></tr><tr><td>Normal Donkey Serum</td><td>Jackson Immuno Research Labs</td><td>17000121</td><td>reconstitute with 10 mL sterile ddH2O, store at 4 &amp;deg;C for up to two weeks</td></tr><tr><td>paraformaldehyde, 20%</td><td>Electron Microscopy Sciences</td><td>15713S</td><td>dilute with PBS</td></tr><tr><td>PBST, 20x</td><td>Thermofisher</td><td>28352</td><td></td></tr><tr><td>PDGFR&amp;beta; antibody</td><td>R&amp;amp;D</td><td>AF385</td><td>dilution ratio: 1:400</td></tr><tr><td>polyamine supplement (1000x)</td><td>Sigma-Aldrich</td><td>P8483</td><td></td></tr><tr><td>Rapiclear clearing reagent, 1.49</td><td>SUNJIN LAB</td><td>RC149001</td><td></td></tr><tr><td>Sodium Bicarbonate (7.5%)</td><td>Gibco</td><td>25080094</td><td></td></tr><tr><td>Sodium deoxycholate monohydrate</td><td>Thermofisher</td><td>J6228822</td><td>dissolve with ddH2O for 1% (wt/v) stock solution</td></tr><tr><td>TBST, 20x</td><td>Thermofisher</td><td>28360</td><td></td></tr><tr><td>trans-ISRIB</td><td>Tocris Bioscience</td><td>5284/10</td><td>make the stock solution at 1 mM with DMSO, and store at -20&amp;deg;C for up to 1 year.</td></tr><tr><td>Tritin X-100</td><td>Sigma-Aldrich</td><td>T8787-50ML</td><td></td></tr><tr><td>Trypan Blue Stain (0.4%)</td><td>Thermo Fisher</td><td>15250061</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P7949-100ML</td><td></td></tr><tr><td>VEGF-A</td><td>ProSpec</td><td>CYT-116</td><td>reconstitute with sterile ddH2O at a concentration of 100 ng/&amp;micro;L, aliquot and store at -20&amp;deg;C</td></tr></tbody></table>

vascular organoid generation from human-induced pluripotent stem cells

<meta charset="utf8"></head><body>作者聚焦：使用 ROCK 抑制剂和微孔限制提高血管类器官的可重复性

从人诱导多能干细胞生成血管类器官

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human ...

vascular-organoid-generation-from-human-induced-pluripotent-stem-cells

Research

JoVE Journal

Bioengineering

Vascular Organoid Generation from Human-Induced Pluripotent Stem Cells

1.4K 次观看.  Columbia University. 本研究介绍了一种改进的方法，用于从人 IPSC 中可扩展生产均质血管类器官。通过使用新型 ROCK 抑制剂和微孔限制，该方法提高了可重复性并最大限度地减少了批次间的变化，使其适用于下游形态学分析和疾病建模应用。单细胞测序技术已广泛用于类器官研究，以揭示细胞群内的基因组、表观基因组和转录组异质性和动力学。组织透明化和光片...

视频: 作者聚焦：使用 ROCK 抑制剂和微孔限制提高血管类器官的可重复性

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

人诱导多能干细胞的前脑型脑 Organoids 标准化和可再生性的生成

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

科研

发育生物学

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

一种从人类胚胎干细胞中生成脑器官的静态自导方法

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in vitro. Developing more efficient organ-like platforms that can model complex in vivo phenotypes, such as organoids and organs-on-a-chip, will enhance the ability to study human heart development and disease. This paper describes a protocol to generate highly complex human heart organoids (hHOs) by self-organization using human pluripotent stem cells and stepwise developmental pathway activation using small molecule inhibitors. Embryoid bodies (EBs) are generated in a 96-well plate with round-bottom, ultra-low attachment wells, facilitating suspension culture of individualized constructs.
The EBs undergo differentiation into hHOs by a three-step Wnt signaling modulation strategy, which involves an initial Wnt pathway activation to induce cardiac mesoderm fate, a second step of Wnt inhibition to create definitive cardiac lineages, and a third Wnt activation step to induce proepicardial organ tissues. These steps, carried out in a 96-well format, are highly efficient, reproducible, and produce large amounts of organoids per run. Analysis by immunofluorescence imaging from day 3 to day 11 of differentiation reveals first and second heart field specifications and highly complex tissues inside hHOs at day 15, including myocardial tissue with regions of atrial and ventricular cardiomyocytes, as well as internal chambers lined with endocardial tissue. The organoids also exhibit an intricate vascular network throughout the structure and an external lining of epicardial tissue. From a functional standpoint, hHOs beat robustly and present normal calcium activity as determined by Fluo-4 live imaging. Overall, this protocol constitutes a solid platform for in vitro studies in human organ-like cardiac tissues.

Here, we describe a protocol to create developmentally relevant human heart organoids (hHOs) efficiently using human pluripotent stem cells by self-organization. The protocol relies on the sequential activation of developmental cues and produces highly complex, functionally relevant human heart tissues.

产生来自多能干细胞的自组装人心脏类器官

The ability to study human cardiac development in health and disease is highly limited by the capacity to model the complexity of the human heart in ...

生物化学

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney disease and injury, developing cell-based therapies, and testing new therapeutics. For such applications, large numbers of uniform organoids and highly reproducible assays are needed. We have built upon our previously published kidney organoid protocol to improve the overall health of the organoids. This simple, robust 3D protocol involves the formation of uniform embryoid bodies in minimum component medium containing lipids, insulin-transferrin-selenium-ethanolamine supplement and polyvinyl alcohol with GSK3 inhibitor (CHIR99021) for 3 days, followed by culture in knock-out serum replacement (KOSR)-containing medium. In addition, agitating assays allows for reduction in clumping of the embryoid bodies and maintaining a uniform size, which is important for reducing variability between organoids. Overall, the protocol provides a fast, efficient, and cost-effective method for generating large quantities of kidney organoids.

Here we describe a protocol to generate kidney organoids from human pluripotent stem cells (hPSCs). This protocol generates kidney organoids within two weeks. The resulting kidney organoids can be cultured in large-scale spinner flasks or multi-well magnetic stir plates for parallel drug-testing approaches.

一种从人类多能干细胞中生成肾类器官的简化方法

Kidney organoids generated from hPSCs have provided an unlimited source of renal tissue. Human kidney organoids are an invaluable tool for studying kidney ...

Generation, Maintenance, and Characterization of Human Pluripotent Stem Cell-derived Intestinal and Colonic Organoids

Intestinal regional specification describes a process through which unique morphology and function are imparted to defined areas of the developing gastrointestinal (GI) tract. Regional specification in the intestine is driven by multiple developmental pathways, including the bone morphogenetic protein (BMP) pathway. Based on normal regional specification, a method to generate human colonic organoids (HCOs) from human pluripotent stem cells (hPSCs), which include human embryonic stem cells (hES) and induced pluripotent stem cells (iPSCs), was developed. A three-day induction of BMP signaling sufficiently patterns mid/hindgut tube cultures into special AT-rich sequence-binding protein 2 (SATB2)-expressing HCOs containing all of the main epithelial cell types present in human colon as well as co-developing mesenchymal cells. Omission of BMP (or addition of the BMP inhibitor NOGGIN) during this critical patterning period resulted in the formation of human intestinal organoids (HIOs). HIOs and HCOs morphologically and molecularly resemble human developing small intestine and colon, respectively. Despite the utility of HIOs and HCOs for studying human intestinal development, the generation of HIOs and HCOs is challenging. This paper presents methods for generating, maintaining, and characterizing HIOs and HCOs. In addition, the critical steps in the protocol and troubleshooting recommendations are provided.

Here, detailed methods for generating, maintaining, and characterizing human pluripotent stem cell-derived small intestinal and colonic organoids are described. These methods are designed to improve reproducibility, expand scalability, and decrease the working time required for plating and passaging of organoids.

Intestinal regional specification describes a process through which unique morphology and function are imparted to defined areas of the developing ...

Brain Organoid Generation from Induced Pluripotent Stem Cells in Home-Made Mini Bioreactors

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology has emerged recently. It is still in its infancy and has some limitations unsolved yet. The current protocols do not allow obtaining organoids to be consistent enough for drug discovery and preclinical studies. The maturation of organoids can take up to a year, pushing the researchers to launch multiple differentiation processes simultaneously. It imposes additional costs for the laboratory in terms of space and equipment. In addition, brain organoids often have a necrotic zone in the center, which suffers from nutrient and oxygen deficiency. Hence, most current protocols use a circulating system for culture medium to improve nutrition.
Meanwhile, there are no inexpensive dynamic systems or bioreactors for organoid cultivation. This paper describes a protocol for producing brain organoids in compact and inexpensive home-made mini bioreactors. This protocol allows obtaining high quality organoids in large quantities.

Here we describe a protocol for generating brain organoids from human induced pluripotent stem cells (iPSCs). To obtain brain organoids in large quantities and of high quality, we use home-made mini bioreactors.

在自制迷你生物反应器中从诱导多能干细胞中生成脑类器官

The iPSC-derived brain organoid is a promising technology for in vitro modeling the pathologies of the nervous system and drug screening. This technology ...

使用诱导多能干细胞悬液生产肾脏类器官

Generating Kidney Organoids in Suspension from Induced Pluripotent Stem Cells

Kidney organoids can be generated from induced pluripotent stem cells (iPSCs) through various approaches. These organoids hold great promise for disease modeling, drug screening, and potential therapeutic applications. This article presents a step-by-step procedure to create kidney organoids from iPSCs, starting from the posterior primitive streak (PS) to the intermediate mesoderm (IM). The approach relies on the APEL 2 medium, which is a defined, animal component-free medium. It is supplemented with a high concentration of WNT agonist (CHIR99021) for a duration of 4 days, followed by fibroblast growth factor 9 (FGF9)/heparin and a low concentration of CHIR99021 for an additional 3 days. During this process, emphasis is given to selecting the optimal cell density and CHIR99021 concentration at the start of iPSCs, as these factors are critical for successful kidney organoid generation. An important aspect of this protocol is the suspension culture in a low adherent plate, allowing the IM to gradually develop into nephron structures, encompassing glomerular, proximal tubular, and distal tubular structures, all presented in a visually comprehensible format. Overall, this detailed protocol offers an efficient and specific technique to produce kidney organoids from diverse iPSCs, ensuring successful and consistent results.

作者聚焦：优化 iPSC 分化以实现高效生产以生成肾脏类器官 

This protocol presents a comprehensive and efficient method for producing kidney organoids from induced pluripotent stem cells (iPSCs) using suspension culture conditions. The primary emphasis of this study lies in the determination of the initial cell density and the WNT agonist concentration, thereby benefiting investigators interested in kidney organoid research.

从诱导多能干细胞中产生悬浮液中的肾脏类器官

Kidney organoids can be generated from induced pluripotent stem cells (iPSCs) through various approaches. These organoids hold great promise for disease ...

Integrative Protocol for Generation of iPSC-Derived 3D Human Hepatic Organoids

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing human induced pluripotent stem cells (hiPSCs) to generate 3D cultures of human hepatic organoids (HHOs). The utilization of HHOs offers a valuable approach to understanding liver development, unraveling liver diseases, conducting high-throughput studies for drug development, and exploring the potential for liver transplantation. In the former investigation, through immunofluorescence and quantitative RT-PCR techniques, the progression was monitored, identifying the presence of various cell populations, such as hepatoblasts and the two types of hepatoblast-derived cells: cholangiocytes or hepatocyte-like cells, across different developmental stages. This report presents a straightforward 3D protocol starting from hiPSC to acquire HHOs that mirror the stages of human embryo development. The protocol, spanning 46-50 days, encompasses several steps: (i) meticulous management of hiPSC culture to generate HHOs, (ii) initiation of cell differentiation in 2D and the subsequent transition to 3D, and (iii) an optimized dissociation strategy to break down HHOs into single cells for single-cell RNA sequencing. As an illustration of the broad applications of this approach, the present protocol was previously applied to unravel the role of thyroid hormone signaling in developing liver cells.

Human iPSC-derived 3D hepatic organoids constitute a potential tool for understanding the thyroid hormone's action on liver development.

用于生成 iPSC 衍生的 3D 人肝类器官的综合方案

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing ...

从人诱导多能干细胞生成血管类器官

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人诱导多能干细胞的前脑型脑 Organoids 标准化和可再生性的生成

一种从人类胚胎干细胞中生成脑器官的静态自导方法

产生来自多能干细胞的自组装人心脏类器官

一种从人类多能干细胞中生成肾类器官的简化方法

Generation, Maintenance, and Characterization of Human Pluripotent Stem Cell-derived Intestinal and Colonic Organoids

在自制迷你生物反应器中从诱导多能干细胞中生成脑类器官

从诱导多能干细胞中产生悬浮液中的肾脏类器官

从诱导多能干细胞中产生悬浮液中的肾脏类器官

从诱导多能干细胞中产生悬浮液中的肾脏类器官

用于生成 iPSC 衍生的 3D 人肝类器官的综合方案