특징
- Pan-neuroanl marker, Sensory marker를 발현합니다.
- 주요 nociceptor marker를 발현합니다.
- 기능성 nociceptor phenotype을 보입니다.
- 7일 내에 neuronal network를 형성합니다.
- >99% population의 sensory neuron을 나타낼 정도로 순도가 높습니다.
사양
- Starting material : Human iPSC line
- Karyotype : Normal (46, XY)
- Seeding compatibility : 6, 12, 24, 96 and 384 well plates
- Shipping info : Dry ice
- Donor : Caucasian adult male (skin fibroblast)
- Vial size :
- Small: >2 x 10⁶ viable cells
- Quality control : Sterility, protein expression (ICC) and gene expression (RT-qPCR)
- Differentiation method : opti-ox cellular reprogramming
- Recommended seeding density : 60,000 cells/cm²
- User storage : LN2 or -150°C
- Format : Cryopreserved cells
- Product use : ioCells are for research use only
- Applications :
- Chronic pain research & drug development
- MEA analysis
- Calcium imaging
- Transcriptome analysis
- Neurotoxicology
실험 결과
그림 1. Immunofluorescent staining of ioSensory Neurons at day 14 post-revival. The upper panel shows that ioSensory Neurons are positive for BRN3A (red), the pan-neuronal marker MAP2 (green), and the DAPI counterstain (blue), and demonstrates that all MAP2 positive neurons have a sensory neuronal identity. The lower panel shows that ioSensory Neurons are positive for ISL1 (magenta), PRPH (red), the pan-neuronal marker TUBB3 (green), and the DAPI counterstain (blue).
그림 2. Upon reprogramming, rapid morphological changes are observed in the cells, with neurons identified by day 4 post-revival. Visible neuronal networks are observed by day 7 post-thaw. Images show day 1 to 14 post-thawing; 10X magnification; scale bar: 200 μm.
그림 3. Single cell RNA-sequencing analysis was performed with ioSensory Neurons at four specific timepoints (days 7, 10, 14 and 17). By day 7, the population has a distinct expression profile indicating a pure population (>99%) of post-mitotic sensory neurons. Gene expression was assessed by 10x Genomics scRNA-sequencing. Note, this data is from cells in continuous culture and not cryopreserved cells.
그림 4. Single cell RNA-sequencing analysis was performed with ioSensory Neurons at four specific timepoints (days 7, 10, 14 and 17). By day 7, the expression of pan-sensory neuron marker genes, ISL1, ISL2, PRPH, BRN3A, together with the pan-neuronal markers TUBB3 and MAP2, could be detected in post-mitotic sensory neurons. Gene expression was assessed by 10x Genomics scRNA-sequencing. Note, this data is from cells in continuous culture and not cryopreserved cells.
그림 5. Single cell RNA-sequencing analysis was performed with ioSensory Neurons at four specific timepoints (days 7, 10, 14 and 17). By day 7, the expression of key nociceptor marker genes (NTRK1 and TRPV1) could be detected in post-mitotic sensory neurons. Gene expression was assessed by 10x Genomics scRNA-sequencing. Note, this data is from cells in continuous culture and not cryopreserved cells.
그림 6. Multi electrode array (MEA) recordings of ioSensory Neurons at days 6 and 17. The activity maps show firing rate (A), spike amplitude (B) and % of active electrodes (C). Results demonstrate a time-dependent increase of spontaneous activity during neuronal maturation from day 6 to day 17. Analysis was performed on a Maxwell Biosystem's MaxTwo multi-well system. Note, this data is from cells in continuous culture and not cryopreserved cells.
그림 7. Calcium mobilisation imaging upon stimulation of ioSensory Neurons with pharmacological agonists targeting thermosensitive TRP channels such as TRPV1 (capsaicin), TRPM3 (CIM0216) and TRPM8 (WS-12). Active traces represent the increase in intracellular calcium mobilisation of individual cells upon exposure to noxious stimuli but not to vehicle at day 17. This indicates that cells display a functional nociceptor phenotype within 17 days. Note, this data is from cells in continuous culture and not cryopreserved cells.