STEMdiff™ Spinal Cord Organoid Differentiation Kit

总览

使用 STEMdiff™ 脊髓类器官分化试剂盒，可无需基质嵌入，稳健地从人多能干细胞生成三维、模式化的脊髓类器官培养物。配合AggreWell™800使用时，该无血清细胞培养体系可防止类器官融合，每套试剂盒最多可生成 300 个类器官，从而实现更高精度的统计重复和更详细的纵向研究。

该试剂盒基于 Dr. Sergiu Paşca的实验方法开发（Andersen J 等，Cell，2020；Yoon SJ 等，Nature Methods，2018），所生成的脊髓类器官是三维体外模型，具有人类颈部脊髓发育阶段所代表的细胞组成和结构特征。它们在不同细胞系之间具有一致的大小和可复制的形态，为在功能性三维系统中研究运动神经元提供了可靠模型。

使用该试剂盒生成的类器官也可以作为AssemBloids™共培养，以研究脑区相互作用（(Andersen J et al. Cell, 2020)。对于较长时间（> 50 天）的类器官培养，可以使用STEMdiff™神经类器官维持试剂盒进行维持，以支持类器官的长期培养存活，从而进行预测性分析、高通量表型筛选和神经毒性分析。

亚型
专用培养基

细胞类型
神经细胞，PSC衍生，神经干/祖细胞，多能干细胞

应用
细胞培养，鉴定，分化，功能学筛选，免疫荧光，类器官培养，表型鉴定，球状体培养

品牌
STEMdiff

研究领域
疾病建模，药物发现和毒理检测，神经科学，类器官

制剂类别
无血清

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实验数据

Schematic protocol diagram to generate patterned spinal cord organoids from hPSCs

Figure 1. Schematic for the STEMdiff™ Spinal Cord Organoid Differentiation Kit

hPSC-derived spinal cord organoids can be generated in 43 days with the STEMdiff™ Spinal Cord Organoid Differentiation Kit. Organoids are initially formed in AggreWell™800 plates. After 6 days, the organoids are transferred and cultured in suspension, allowing growth and subsequent patterning to the spinal cord. Long-term maintenance and further maturation of spinal cord organoids can be achieved using STEMdiff™ Neural Organoid Maintenance Kit, see the Product Information Sheet (PIS) for details. hPSC = human pluripotent stem cell

Spinal cord organoids generated with the STEMdiff Spinal Cord Organoid Kit expressing expected PAX6 and OLIG2 progenitor markers at Day 19

Figure 2. STEMdiff™ Spinal Cord Organoids Express Expected Progenitor Markers

Spinal cord organoids were generated from ES cell line H1 with STEMdiff™ Spinal Cord Organoid Differentiation Kit and express expected progenitor markers via immunostaining. (A) At Day 19, the organoids express elevated levels of the cortical progenitor marker PAX6 in the interior and exterior regions of the organoid as well as (B) motor neuron progenitor marker OLIG2 in the exterior region of the organoid. ES = embryonic stem

Spinal cord organoids generated with the STEMdiff Spinal Cord Organoid Kit expressing expected motor neuron and glutamatergic interneuron markers at Day 30 as well as astrocyte and oligodendrocyte markers at Day 75.

Figure 3. STEMdiff™ Spinal Cord Organoids Express Expected Markers

Spinal cord organoids were generated from hPSCs with STEMdiff™ Spinal Cord Organoid Differentiation Kit and express expected markers via immunostaining. (A) At Day 30, motor neuron markers ISL1 (magenta), HB9 (green), and CHAT (cyan) are expressed, as seen in the upper panel (see insets), as well as motor neuron marker FOXP1 (cyan) and V2a glutamatergic interneuron marker CHX10 (red), as seen in the lower panel (see insets). (B) From Day 75 onwards, the STEMdiff™ spinal cord organoids display astrocyte markers GFAP (magenta) and S100β (green), as well as oligodendrocyte marker MBP (cyan) (see insets). hPSCs = human pluripotent stem cells

Spinal cord organoids generated with STEMdiff Spinal Cord Organoid Differentiation Kit show functional activity on an MEA system

Figure 4. STEMdiff™ Spinal Cord Organoids Are Functionally Active

Spinal cord organoids were generated from iPSC line STiPS-M001 and ES cell line H9 using the STEMdiff™ Spinal Cord Organoid Differentiation Kit. At Day 30, the organoids were plated on an MEA (CytoView MEA 48, Axion Biosystems) and activity from 16 electrodes per well were recorded once per week for 5 minutes using a Maestro MEA system (Axion Biosystems). (A) Representative brightfield images of STEMdiff™ spinal cord organoids on the MEA and representative spike heat maps for the corresponding wells (white = 81 and 74 spikes/sec) for STiPS-M001 and H9 are shown respectively. (B) Raster plots of spike activity show the Day 70 STEMdiff™ spinal cord organoids increase in network bursting (pink lines) after 5 weeks of culture on the MEA. (C) Graphs comparing the number of spikes, mean firing rate, synchrony index, and number of active electrodes from Week 1 to 5. The data was normally distributed by D’Agostino & Pearson test and statistics was calculated with paired t-test. ** p ≤ 0.01. iPSC = induced pluripotent stem cell; ES = embryonic stem; MEA = multielectrode array; MFR = mean firing rate

Spinal cord organoids generated with STEMdiff Spinal Cord Organoid Differentiation Kit express high levels of cervical motor neuron markers compared to controls as shown by qPCR analysis

Figure 5. STEMdiff™ Spinal Cord Organoids Express High Levels of Cervical Motor Neuron Markers by qPCR

Spinal cord organoids were generated from hPSCs with the STEMdiff™ Spinal Cord Organoid Differentiation Kit and compared to organoids generated with the STEMdiff™ Dorsal Forebrain Organoid Differentiation Kit. RNA from duplicate organoids was harvested for qPCR analysis. High levels of cervical motor neuron markers are expressed in STEMdiff™ spinal cord organoids (A) at Day 19 and (B) at Day 30, as compared to STEMdiff™ dorsal forebrain organoids. Fold change is reported relative to their parent hPSC and the TBP housekeeping gene control. HOXA4 and HOXA5 graphs use a log10 scale on the y-axis, all other y-axes use a split linear scale. The data is not normally distributed by D’Agostino & Pearson test and statistics were calculated with Mann-Whitney test. **** p ≤ 0.0001. hPSCs = human pluripotent stem cells; qPCR = quantitative polymerase chain reaction