Robustly generate three-dimensional, patterned spinal cord organoid cultures from human pluripotent stem cells without matrix embedding by using STEMdiff™ Spinal Cord Organoid Differentiation Kit. When paired with AggreWell™800, this serum-free cell culture media can prevent organoid fusion and support the generation of up to 300 organoids per kit for higher-powered statistical replicates and more detailed longitudinal study.
Adapted from protocols by Dr. Sergiu Paşca (Andersen J et al. Cell, 2020; Yoon SJ et al. Nature Methods, 2018), the spinal cord organoids generated with this kit are 3D in vitro models with a cellular composition and structural organization representative of the developing human cervical spinal cord. They are consistently sized with reproducible morphology between various cell lines and provide a reliable model for the study of motor neurons within a functional 3D system.
Organoids generated with this kit can also be co-cultured as AssemBloids™ to study brain region interactions (Andersen J et al. Cell, 2020). For extended periods of organoid culture (> 50 days), organoids can be maintained with STEMdiff™ Neural Organoid Maintenance Kit to support long-term culture survival for predictive assays, high-throughput phenotypic screening, and neurotoxicity assays.
Subtype Specialized Media
Cell Type Neural Cells, PSC-Derived, Neural Stem and Progenitor Cells, Pluripotent Stem Cells
Area of Interest Disease Modeling, Drug Discovery and Toxicity Testing, Neuroscience, Organoids
Formulation Category Serum-Free
Data Figures
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 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 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
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
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
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