STEMdiff™ 脑类器官试剂盒

用于人脑类器官建立与成熟的培养基试剂盒

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产品号 #08570_C

用于人脑类器官建立与成熟的培养基试剂盒

产品组分包括

STEMdiff™ 脑类器官基础培养基 1,100 mL
STEMdiff™ 脑类器官基础培养基 2，250 mL
STEMdiff™ 脑类器官补充剂 A，10 mL
STEMdiff™ 脑类器官补充剂 B，0.5 mL
STEMdiff™ 脑类器官补充剂 C，0.25 mL
STEMdiff™ 脑类器官补充剂 D，0.5 mL
STEMdiff™ 脑类器官补充剂 E，4.5 mL

立即询价

总览

生成自组织的、由多能干细胞 (PSC) 衍生的神经类器官，其细胞组成和结构组织与发育中的人脑相似。

这些成分明确的无血清细胞培养基和简单的四阶段实验方案基于 Lancaster 等人发表的配方（Lancaster MA 等人，Nature，2013 和 Lancaster MA 等人，Science，2014），旨在更可靠地生成脑类器官。使用 STEMdiff™ 脑类器官试剂盒生成的类器官从胚状体 (EB) 形成步骤开始，随后神经上皮细胞扩增，具有皮质样区域，包括脑室区 (PAX6+/SOX2+/Ki-67+)、外脑室下区 (Ki-67+/p-Vimentin+)、中间区 (TBR2+) 和皮质板 (CTIP2+/MAP2+/TBR1+)，其分层方向与体内观察到的相似。

对于长期培养（> 40 天），成熟所需的成分可作为STEMdiff™ 脑类器官成熟试剂盒购买。

实验数据

Cerebral Organoids Contain Multiple Layered Regions That Recapitulate the Cortical Lamination Process Observed During In Vivo Human Brain Development

Figure 1. Cerebral Organoids Contain Multiple Layered Regions That Recapitulate the Cortical Lamination Process Observed During In Vivo Human Brain Development

(A) A representative phase-contrast image of a whole cerebral organoid at Day 40 generated using the STEMdiff™ Cerebral Organoid Kit. Cerebral organoids at this stage are made up of phase-dark structures that may be surrounded by regions of thinner, more translucent structures that display layering (arrowheads). (B) Immunohistological analysis on cryosections of cerebral organoids reveals cortical regions within the organoid labeled by the apical progenitor marker PAX6 (red) and neuronal marker β-tubulin III+ (TUJ-1) (green). (C-F) Inset of boxed region from (B). (C) PAX6+ apical progenitors (red, enclosed by dotted line) are localized to a ventricular zone-like region. β-tubulin III+ neurons (green) are adjacent to the ventricular zone. (D) CTIP2, a marker of the developing cortical plate, co-localizes with β-tubulin III+ neurons in a cortical plate-like region. Organization of the layers recapitulates early corticogenesis observed during human brain development. (E) Proliferating progenitor cells labeled by Ki-67 (green) localize along the ventricle, nuclei are counterstained with DAPI (blue). (F) An additional population of Ki-67+ cells is found in an outer subventricular zone-like region (arrowheads). Scale bar = (A) 1 mm, (B) 1 mm and (C-F) 200 µm.

Immunocytochemistry image of a cerebral organoid cultured in mTeSR™ Plus and directed to cerebral organoids using the STEMdiff™ Cerebral Organoid Kit.

Figure 2. Cerebral Organoids Can Be Generated from hPSCs Maintained in mTeSR™ Plus

Human ES (H9) cells were cultured with mTeSR™ Plus and directed to cerebral organoids using the STEMdiff™ Cerebral Organoid Kit. Image shows apical progenitor marker SOX2 (magenta) and neuronal marker TBR1 (green).

Figure 3. Cryosectioned Cerebral Organoids Show Stratification of Cortical Plate Neurons and Progenitor Zones

Cerebral organoids were generated using the STEMdiff™ Cerebral Organoid Kit. A 16-μm-thick section of a Day 40 cerebral organoid was stained for CTIP2 (green), PAX6 (magenta), βIII-tubulin/TUJ1 (blue), and DAPI (gray). Cortical regions are defined by progenitor cells (PAX6+) that are radially organized around a pseudo-ventricle (dashed line). These progenitors give rise to cortical plate neurons indicated by CTIP2 and TUJ1 expression. For a detailed cryogenic tissue processing and immunofluorescence protocol, please see the Methods Library.

Figure 4. Zones of Active Proliferation in Cerebral Organoids Are Preserved Following the Protocol for Tissue Processing

Organoid tissue was processed for immunofluorescence and stained for TBR2 (intermediate precursors, green) and phosphorylated vimentin (PVIM, dividing cells, magenta). Cells actively divide at the apical border of cortical regions along the border of the pseudo-ventricle (dashed line). A population of these dividing cells will express TBR2 and then migrate (arrows) from the progenitor zone to form a layer of intermediate progenitors. For a detailed cryogenic tissue processing and immunofluorescence protocol, please see the Methods Library.

Figure 5. Immunofluorescence from Cryosectioned Cerebral Organoids Indicates Preserved Organization of Cortical Neurons

Organoid tissue was processed for immunofluorescence and stained for CTIP2 (green), TBR1 (layer 5/6 cortical neurons, magenta), and DAPI (white). Deep layer neuronal markers CTIP2 and TBR1 are expressed in cells around presumptive progenitor zones (dashed line) toward the outside or apical surface of organoids. For a detailed cryogenic tissue processing and immunofluorescence protocol, please see the Methods Library.

Cryogenic Tissue Processing and Immunofluorescence Captures Arrangement of Neural Progenitors Around Pseudo-Ventricles in Cerebral Organoids

Figure 6. Cryogenic Tissue Processing and Immunofluorescence Captures Arrangement of Neural Progenitors Around Pseudo-Ventricles in Cerebral Organoids

Organoid tissue was processed for immunofluorescence and stained for (A) FOXG1 (forebrain cells, green) or (B) SOX2 (neural progenitors, magenta). Organoids derived from STEMdiff™ Cerebral Organoid Kit generate forebrain-type tissue as indicated by FOXG1 expression. Neural progenitors expressing SOX2 are radially arranged around a pseudo-ventricle area (dashed line). For a detailed cryogenic tissue processing and immunofluorescence protocol, please see the Methods Library.

Cerebral Organoids Generated with the STEMdiff™ Cerebral Organoid Kit Are Transcriptionally Similar to Those from Published Protocols

Figure 7. Cerebral Organoids Generated with the STEMdiff™ Cerebral Organoid Kit Are Transcriptionally Similar to Those from Published Protocols

Principal component analysis of hPSC and cerebral organoid transcriptomes. Cerebral organoids generated using the STEMdiff™ Cerebral Organoid Kit (filled blue circles) cluster together, and cluster with previously published (C Luo et al. Cell Rep, 2016) cerebral organoids (open blue circles). The first principal component accounts for the majority of variance seen (PC1; 80%) and distinguishes the cerebral organoid samples from the hPSCs (green circles). The second principal component accounts for only 9% of the variation, and highlights the modest expression differences between cultured organoids and primary embryonic fetal brain samples (19 post-conceptional weeks, brown circles).

Figure 8. Neural Organoids Generated with STEMdiff™ Cerebral Organoid Kit Express Expected Key Markers

Heatmap of expression levels for genes associated with synaptic transmission function and neurogenesis in Day 40 organoids. These data show that gene expression of cerebral organoids generated from the STEMdiff™ Cerebral Organoid Kit are similar to published results (C Luo et al. Cell Rep, 2016).

scRNA seq data for STEMdiff™ Cerebral Organoid Kit

Figure 9. Neural Organoids Generated with STEMdiff™ Cerebral Organoid Kit Contain Diverse Cell Types

Unguided cerebral organoids were generated from H9 ESCs using STEMdiff™ Cerebral Organoid Kit. scRNA-seq gene expression data captures the cellular diversity of these neural organoids which can be further explored using the Single Cell RNA Sequencing Data Visualization Tool for Neural Organoids . At Day 50, the organoids were dissociated into a single-cell suspension. The library was prepared using Chromium Single Cell 3ʹ v1 protocol with Feature Barcoding technology (10x Genomics) following surface protein staining with TotalSeq™–B (BioLegend). The barcoded processing, gene counting, and aggregation were done using the Cell Ranger software v3.1.0. Further processing and demultiplexing was done with Seurat v4.1.1. The data have been made publicly available on GEO: GSE218457. ESC = embryonic stem cell

Time-lapse images and heatmap showing calcium signals spreading across a Day 91 cerebral organoid, indicating synchronized neuronal activity.

Figure 10. STEMdiff™ Cerebral Organoids Exhibit Synchronous Calcium Activity

Cerebral organoids were generated using the STEMdiff™ Cerebral Organoid Kit and matured with the STEMdiff™ Cerebral Organoid Maturation Kit. On Day 91, organoids were imaged using the ImageXpress® Confocal HT.ai High-Content Imaging System (Molecular Devices) with MetaXpress® High-Content Image Acquisition and Analysis Software. (A) Time-lapse images of a representative region within a cerebral organoid. Arrows indicate areas of elevated calcium signal intensity. (B) Corresponding heatmap representation of calcium intensity from the regions shown in (A) over time. Signal propagation from the initial peak to surrounding areas suggests the presence of a functional neuronal network.

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Product Information Sheet

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STEMdiff™ Cerebral Organoid Kit

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08570

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English

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Safety Data Sheet 1

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STEMdiff™ Cerebral Organoid Kit

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08570

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English

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STEMdiff™ Cerebral Organoid Kit

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08570

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STEMdiff™ Cerebral Organoid Kit

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08570

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English

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STEMdiff™ Cerebral Organoid Kit

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08570

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STEMdiff™ Cerebral Organoid Kit

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08570

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English

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STEMdiff™ Cerebral Organoid Kit

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08570

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STEMdiff™ Cerebral Organoid Kit

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文献 (32)

Generation of human vascularized brain organoids M. T. Pham et al. NeuroReport 2018

Abstract

The aim of this study was to vascularize brain organoids with a patient's own endothelial cells (ECs). Induced pluripotent stem cells (iPSCs) of one UC Davis patient were grown into whole-brain organoids. Simultaneously,iPSCs from the same patient were differentiated into ECs. On day 34,the organoid was re-embedded in Matrigel with 250 000 ECs. Vascularized organoids were grown in vitro for 3-5 weeks or transplanted into immunodeficient mice on day 54,and animals were perfused on day 68. Coating of brain organoids on day 34 with ECs led to robust vascularization of the organoid after 3-5 weeks in vitro and 2 weeks in vivo. Human CD31-positive blood vessels were found inside and in-between rosettes within the center of the organoid after transplantation. Vascularization of brain organoids with a patient's own iPSC-derived ECs is technically feasible.

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Cerebral organoids at the air–liquid interface generate diverse nerve tracts with functional output S. L. Giandomenico et al. Nature Neuroscience 2019 apr

Abstract

Neural organoids have the potential to improve our understanding of human brain development and neurological disorders. However,it remains to be seen whether these tissues can model circuit formation with functional neuronal output. Here we have adapted air–liquid interface culture to cerebral organoids,leading to improved neuronal survival and axon outgrowth. The resulting thick axon tracts display various morphologies,including long-range projection within and away from the organoid,growth-cone turning,and decussation. Single-cell RNA sequencing reveals various cortical neuronal identities,and retrograde tracing demonstrates tract morphologies that match proper molecular identities. These cultures exhibit active neuronal networks,and subcortical projecting tracts can innervate mouse spinal cord explants and evoke contractions of adjacent muscle in a manner dependent on intact organoid-derived innervating tracts. Overall,these results reveal a remarkable self-organization of corticofugal and callosal tracts with a functional output,providing new opportunities to examine relevant aspects of human CNS development and disease.

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Mechanisms of hyperexcitability in Alzheimer's disease hiPSC-derived neurons and cerebral organoids vs isogenic controls. S. Ghatak et al. eLife 2019

Abstract

Human Alzheimer's disease (AD) brains and transgenic AD mouse models manifest hyperexcitability. This aberrant electrical activity is caused by synaptic dysfunction that represents the major pathophysiological correlate of cognitive decline. However,the underlying mechanism for this excessive excitability remains incompletely understood. To investigate the basis for the hyperactivity,we performed electrophysiological and immunofluorescence studies on hiPSC-derived cerebrocortical neuronal cultures and cerebral organoids bearing AD-related mutations in presenilin-1 or amyloid precursor protein vs. isogenic gene corrected controls. In the AD hiPSC-derived neurons/organoids,we found increased excitatory bursting activity,which could be explained in part by a decrease in neurite length. AD hiPSC-derived neurons also displayed increased sodium current density and increased excitatory and decreased inhibitory synaptic activity. Our findings establish hiPSC-derived AD neuronal cultures and organoids as a relevant model of early AD pathophysiology and provide mechanistic insight into the observed hyperexcitability.

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STEMdiff™ 脑类器官试剂盒

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