STEMdiff™ 背侧前脑类器官分化试剂盒

用于从人多能干细胞高效生成背侧前脑神经类器官的细胞培养基试剂盒

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产品号 #08620_C

用于从人多能干细胞高效生成背侧前脑神经类器官的细胞培养基试剂盒

产品优势

使用无融合生长培养基减少操作和培养基浪费
在不同细胞系和单个类器官之间具有可重复的形态，从而获得更高的疾病表型分析灵敏度
使用AggreWell™800，每个试剂盒可生成200个或更多大小一致的类器官，用于更强大的纵向研究
采用无基质配方和方案，无需基质包埋步骤
可与不同区域分化的模块化类器官结合，生成先进的 AssemBloids™，用于疾病建模和再生应用

产品组分包括

STEMdiff™ 神经类器官基础培养基 1，20 mL
STEMdiff™ 神经类器官基础培养基 2，500 mL
STEMdiff™ 神经类器官补充剂 A，10 mL
STEMdiff™ 神经类器官补充剂 B，0.25 mL
STEMdiff™ 神经类器官补充剂 C，0.25 mL

立即询价

总览

使用STEMdiff™背侧前脑类器官分化试剂盒，从人多能干细胞中稳定地生成定向神经类器官。与AggreWell™ 800配合使用时，该款无基质、无血清的细胞培养基试剂盒可支持每个试剂盒生成200个或更多类器官，从而用于更高效的纵向研究。

本试剂盒通过基于Sergiu Paşca（Birey et al., 2017）的实验方案生成脑区特异性类器官，是具有人类前脑发育特征的三维体外模型，具有代表性的细胞组成和结构组织。这些类器官在不同细胞系之间具有一致的尺寸和可重复的形态，为在功能性三维系统中研究皮层神经元提供了可靠模型。

STEMdiff™背侧前脑类器官分化试剂盒生成早期发育的背侧大脑皮层组织，而STEMdiff™腹侧前脑类器官分化试剂盒生成早期发育的腹侧大脑皮层下组织。使用这些试剂盒生成的类器官也可以作为组装体共培养，以研究脑区间的相互作用（Birey et al., 2017）。若需进行超过 50 天的长期类器官培养，推荐使用STEMdiff™神经类器官维持培养试剂盒中的相关培养成分。

实验数据

Schematic protocol diagram to generate guided dorsal forebrain organoids from iPSCs.

Figure 1. Schematic for the STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit

hPSC-derived dorsal forebrain organoids can be generated in 43 days. Embryoid bodies can be created in 6 days with AggreWell™800 plates. The EBs are then cultured in suspension, allowing growth and subsequent patterning to the dorsal forebrain. For the long-term maintenance and further maturation of dorsal forebrain organoids, see the PIS. hPSC = human pluripotent stem cell; EBs = embryoid bodies.

Figure 2. STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit Supports the Generation of Homogeneous, Reproducible Neural Organoids

(A) Neural aggregates formed in AggreWell™800 microwell plates exhibit uniform size and shape at day 6. H9-derived dorsal forebrain organoids from a single batch have spherical morphology at days (B) 15 (C) 25 (D) 50 (E) 75 and (F) 100. Scale bar = 1 mm. (G) Dorsal forebrain organoids exhibit homogeneous size over multiple cell lines (average ± SD, using 11 cell lines with 3 - 5 organoids counted per cell line and time point). (H-N) Day 25 organoids generated by a novice user with STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit show reproducibility across different cell lines.

Figure 3. STEMdiff™ Dorsal and Ventral Forebrain Organoid Differentiation Kits Reduce Unwanted Organoid Fusion

Whole-well images of dorsal forebrain organoid cultures derived from the H9 cell line in STEMdiff™ Forebrain Organoid Expansion Medium (bottom row) vs. the control published formulation (top row) show the extent of fusion typical in control media without orbital shaking. At Day 10 post-seeding in the control formulation, 12/12 organoids that were seeded are developing, but by Day 20 a single, larger organoid can be seen in the well. In STEMdiff™ Forebrain Organoid Expansion Medium, 20/20 seeded organoids are developing and 20/20 are still developing at Day 20 without shaking. While organoids in both culture conditions grow larger between 10 and 20 days, the reduction in organoids and enlarged size in the control formulation suggest a reduction in organoid yield due to organoid fusion.

Figure 4. STEMdiff™ Dorsal Forebrain Organoids Exhibit Cortical Layering and Brain-Region-Specific Marker Expression as They Mature

(A) Day 25 dorsal forebrain organoids display multiple cortical-like regions marked by radialized PAX6+ cells surrounded by MAP2 neurons. (B) Day 50 dorsal forebrain organoids continue to display multiple cortical-like regions marked by PAX6 (green) and MAP2 (magenta). (C) Dorsal forebrain organoids cultured for 100 - 200 days show increasing separation of deep-layer neurons (CTIP2, green; TBR1, cyan) from upper-layer neurons (SATB2, magenta).

Figure 5. Neural Organoids Generated with STEMdiff™ Dorsal and Ventral Forebrain Organoid Kits Express Key Markers of Brain-Region-Specific Patterning

RNA from single organoids was harvested at a series of time points and subsequently assayed using bulk RNA-seq (1 data column = 1 organoid). (A) Heat map of select genes shows that both dorsal forebrain organoids (DFO) and ventral forebrain organoids (VFO) express the forebrain-specific marker FOXG1 while showing a shift from neural progenitor fates to neuronal cell types. (B) DFO express increasing levels of cortex- and glutaminergic neuron-specific genes from day 25 to 75. (C) Day 25 VFO exhibit high expression of markers of the medial ganglionic eminence and of GABAergic neurons. Heat map scale quantifies gene expression across each row with a normalized z-score for each gene.

Figure 6. Dorsal Forebrain Organoids, Not Ventral Forebrain Organoids, Display Early Network Bursting Activity

Day 50 dorsal and ventral forebrain organoids were plated on a microelectrode array (MEA; CytoView MEA 96, Axion Biosystems) coated with 0.1% polyethyleneimine in borate buffer and 20 µg/mL CellAdhere™ Laminin-521. Activity from 8 electrodes per well was recorded once per week for 4 weeks using a Maestro MEA system (Axion Biosystems). (A) Representative bright field image of dorsal and ventral forebrain organoids on the MEA. Insets show representative spike rate heat maps for the corresponding well (red = 12 spikes/sec). Scale bar = 1 mm. (B) Raster plots of spike activity show increasing network bursting (pink lines) for dorsal forebrain organoids between week 1 and week 4, whereas no network bursting is observed in the ventral forebrain organoid (lower panels) over the same time period. (C) Mean firing rate (average ± SEM; 3 - 6 organoids per time point) increases for dorsal forebrain organoids but not ventral forebrain organoids over 4 weeks of measurements.

Figure 7. Fluorescent Imaging in BrainPhys™ Imaging Optimized Medium Improves Signal-to-Background Ratios of 3D Neural Cultures

GFP-labeled ventral forebrain organoids were co-cultured and merged with unlabeled dorsal forebrain organoids for one week prior to live imaging in Forebrain Organoid Differentiation Medium from STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit (right) or BrainPhys™ Imaging Optimized Medium (BPI, left). Interneuron migration can be visualized more clearly in BPI. Scale bar top panels = 300μm; scale bar bottom = 100μm. Adapted from Zabolocki et al. 2020, Nature Communications, available under a Creative Commons 4.0 License. For experimental details on generating AssemBloids™ from dorsal and ventral forebrain organoid co-cultures, see the protocol in our Methods Library.

Figure 8. hPSC-Derived Microglia Incorporate into Brain Organoids After 10 Days and Display an Activated Morphology upon Injury

(A, C) Representative microglia and brain organoid co-cultures after 10 days, sectioned and stained with IBA1 for microglia (green) and MAP2 for neurons (magenta). The microglia integrate among the neurons and display an unactivated morphology with extended processes. (B, D) The microglia display an activated amoeboid morphology upon injury as shown by IBA1 staining.

scRNA seq data for STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit

Figure 9. Neural Organoids Generated with STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit Contain Dorsal Cell Populations

Guided dorsal forebrain organoids were generated from H9 ESCs using STEMdiff™ Dorsal Forebrain Organoid Differentiation 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

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文献 (3)

Examining the NEUROG2 lineage and associated gene expression in human cortical organoids Development (Cambridge,England) 2025 Jan

Abstract

ABSTRACTProneural genes are conserved drivers of neurogenesis across the animal kingdom. How their functions have adapted to guide human-specific neurodevelopmental features is poorly understood. Here,we mined transcriptomic data from human fetal cortices and generated from human embryonic stem cell-derived cortical organoids (COs) to show that NEUROG1 and NEUROG2 are most highly expressed in basal neural progenitor cells,with pseudotime trajectory analyses indicating that NEUROG1-derived lineages predominate early and NEUROG2 lineages later. Using ChIP-qPCR,gene silencing and overexpression studies in COs,we show that NEUROG2 is necessary and sufficient to directly transactivate known target genes (NEUROD1,EOMES,RND2). To identify new targets,we engineered NEUROG2-mCherry knock-in human embryonic stem cells for CO generation. The mCherry-high CO cell transcriptome is enriched in extracellular matrix-associated genes,and two genes associated with human-accelerated regions: PPP1R17 and FZD8. We show that NEUROG2 binds COL1A1,COL3A1 and PPP1R17 regulatory elements,and induces their ectopic expression in COs,although NEUROG2 is not required for this expression. Neurog2 similarly induces Col3a1 and Ppp1r17 in murine P19 cells. These data are consistent with a conservation of NEUROG2 function across mammalian species. Summary: Analysis of human cortical organoids reveals that NEUROG1 lineages prevail early and NEUROG2 lineages later,and that NEUROG2 targets include COL genes and PPP1R17,a human-accelerated region-associated gene.

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The ADCY1-mediated cAMP signaling pathway mediates functional effects of montelukast treatment in brain organoids Cellular and Molecular Life Sciences: CMLS 2025 Jun

Abstract

Montelukast (MTK) is a drug widely used for treating allergic rhinitis and asthma. However,severe neuropsychiatric adverse events related to MTK have been reported,with limited understanding of the underlying mechanisms. Here we leveraged human forebrain organoids (hFOs) and showed that MTK exposure in hFOs downregulated the expression of genes associated with multiple neuronal functions and neuropsychiatric disorders. The following integrative analysis highlighted adenylate cyclase 1 (ADCY1),a main regulator of the cAMP signaling pathway,as a hub gene mediating the functional effects of MTK exposure. We also showed that MTK exposure resulted in a reduction of cAMP and neuroactivities,and caused neural maturation defects. These cellular phenotypes could be recapitulated by treating hFOs with ST034307,a selective ADCY1 inhibitor,or partially rescued by ADCY1 overexpression in hFOs. Together,this study underscored that MTK exposure caused neuropsychiatric effects through inhibiting the ADCY1-mediated cAMP signaling pathway.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-025-05764-z.

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Modeling Synaptic Maturation From Growth Cone to Synapse in Human Organoids M. S. Ã˜hlenschlÃ¦ger et al. Journal of Neurochemistry 2026 May

Abstract

Human neural organoids (NOs) provide a powerful platform for investigating synaptic development and dysfunction during early neurodevelopment. However,methodologies for isolating functional synaptic structures from these models remain limited. Here,we present a differential centrifugation protocol enabling the enrichment of growth cone particles (GCPs) and immature synaptosomes from airâ€liquid interface cerebral organoids (ALIâ€COs) at distinct developmental stages (Day 90 and 150). Notably,the method avoids density gradients,requires minimal starting material while maintaining reproducibility across human and murine tissues. Quantitative proteomic profiling revealed significant enrichment of growth cone markers (e.g.,GAP43) and classical synaptosomal proteins (e.g.,PCLO,BSN,SYN1). Transmission electron microscopy (TEM) confirmed the presence of membraneâ€enclosed GCPs with fibrous content and mitochondria in Day 90 isolates,and immature synaptosomes containing synaptic vesicles on day 150. Functional viability of both types of synaptic structures was demonstrated through KClâ€induced depolarization,which triggered phosphorylation changes in growth cone proteins (GAP43,MARCKS,MARCKSL1),cytoskeletal regulators (DCLK1,SHTN1,MARK4,MAP1B) and protein kinases (CAMK2G,PRKCE) in Day 90 GCPs,as well as classical synaptic vesicle cycle proteins (SYN1,DNM1,RPH3A) at Day 150. Overall,this study establishes a centrifugationâ€based protocol for isolating growth cones and immature synapses from human organoids,capturing key stages of synaptic development and enabling scalable,patientâ€compatible models to study synaptic function and dysfunction in neurodevelopmental and neurodegenerative disorders. Synapses are implicated in several neurological disorders and psychiatric diseases. The emergence and wide use of neural organoids provide a new opportunity to study human synapses in healthy and disease settings. Therefore,we developed a simple method for the enrichment of synaptosomes and growth cone particles from forebrain organoids. The method is based on differential centrifugation,works with small tissue amounts,and is highly reproducible. We validated the functionality of the isolated structures using KCl stimulation and phosphoproteomics. The method enables detailed mapping of protein composition and function during growth cone pathfinding,synaptogenesis,and establishment of neural circuits in organoids.

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产品号 #08620_C

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STEMdiff™ 背侧前脑类器官分化试剂盒

产品号 #08620

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产品号 #08620_C

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