STEMdiff™ 腹侧前脑类器官分化试剂盒

用于从人多能干细胞高效生成腹侧前脑神经类器官的细胞培养基试剂盒

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产品号 #08630_C

用于从人多能干细胞高效生成腹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，减少操作频次与培养基浪费
在不同细胞系及单个类器官之间保持形态一致性，提高疾病表型的分析灵敏度
使用 AggreWell™800 每套可生成 500 个以上类器官，支持更有力的统计重复与详细的纵向研究
无需基质嵌入步骤，采用无基质配方与简化流程
易于处理与切片，获得大小一致的类器官
支持长期培养，降低 caspase-3 表达，适用于神经毒性与神经退行性疾病模型
可将不同区域类器官模块化组合为先进的 AssemBloids™，用于疾病建模与再生医学应用

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
STEMdiff™ 神经类器官补充剂 D，0.5 mL

立即询价

总览

使用STEMdiff™腹侧前脑类器官分化试剂盒，从人多能干细胞中高效稳定地生成定向神经类器官。与AggreWell™ 800配合使用时，这种无基质、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抑制性中间神经元提供了可靠模型。

STEMdiff™腹侧前脑类器官分化试剂盒可生成早期发育的腹侧大脑皮层下组织，而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。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）。对于需要长期培养（>50天）的类器官，其维持培养所需的成分可通过STEMdiff™神经类器官维持培养试剂盒获得。

实验数据

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

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

hPSC-derived ventral 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 ventral forebrain. For the long-term maintenance and further maturation of ventral forebrain organoids, see the PIS. hPSC = human pluripotent stem cell; EBs = embryoid bodies.

Schematic protocol diagram to generate patterned ventral forebrain organoids from iPSCs

Figure 2. STEMdiff™ Ventral Forebrain Organoid Differentiation Kit Supports the Generation of Homogeneous Organoids

(A) Neural aggregates formed in AggreWell™800 microwell plates exhibit uniform size and shape at day 6. H9-derived ventral 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) Ventral 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).

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. Ventral Forebrain Organoids Exhibit Brain-Region-Specific Marker Expression as They Mature

(A) Ventral forebrain organoids at day 25 exhibit a high level of expression of NKX2.1 (magenta; DAPI, gray). (B) NKX2.1-positive neurons (magenta; DAPI, cyan) are plentiful by Day 50 (DAPI, cyan), but Somatostatin-positive GABAergic interneurons (SST, green) are rare. (C) By day 75, SST-positive interneurons (green; NKX2.1, magenta) are observable.

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.

scRNA seq data for STEMdiff™ Ventral Forebrain Organoid Differentiation Kit

Figure 8. Neural Organoids Generated with STEMdiff™ Ventral Forebrain Organoid Differentiation Kit Contain Ventral Cell Populations

Guided ventral forebrain organoids were generated from H9 ESCs using STEMdiff™ Ventral 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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