The progressive alignment of same-donor hiPSC-derived SCPs and hiPSC-derived peripheral neurons. A) Phase images show alignment morphology over two weeks. White arrows indicate single SCPs, black arrows indicate single neurons, and red arrows indicate alignment of the two cell types. Scale bar = 100 um. B) Immunocytochemical staining showing the co-cultures of hiPSC-SCPs with hiPSC-motor neurons at 14 DIV. S100b+ and SOX10+ SCPs align with TUJ1+ axons of hiPSC-motor neurons derived from the same doner. Scale bar = 100 um.
Screening to Accelerate Maturation of Schwann Cell Precursors
Screening for compounds that accelerate maturation of SCPs. Cryopreserved SCPs were plated out at a density of 5,000 cells/cm2 and various compounds were applied to SCPs for one week. Both immunocytochemical staining and phase-contrast images showcase differences in cellular morphology and amount of proliferation between different treatments. This highlights the ability of hiPSC derived SCPs to be used as a tool to identify different compounds that drive schwann cells maturation. Scale bars = 200 uM.
Maturation of Schwann Cell Precursors
Maturation of SCPs over two weeks. A) Time course qPCR was performed to characterize the hiPSC-derived SCPs with seven different key genes related to development (SOX10, S100b, Oct6) and maturation (P0, MBP, PMP22, Krox20) of Schwann cells. B) Phase images showing SCP maturation over 2 weeks post differentiation. Cryopreserved SCPs were plated out at 5,000 cells/cm2 and show proliferation along with morphological changes during maturation in vitro. Over time, the SCPs elongate into a bipolar morphology characteristic of mature Schwann cells. Scale bar = 20 um
hiPSC-SCPs show myelination capabilities in 5 weeks
SCP and sensory neuron co-cultures show myelination phenotype in vitro. A) TEM images showcasing wrapping morphology of sensory neurons-SCP co-cultures in vitro. B) Immunocytochemical staining of week 5 co-cultures of hiPSC-derived SCPs and hiPSC-derived sensory neurons displaying expression of TUJ1 (beta-3 tubulin) and P0 (myelin protein zero). Co-expression of P0 and TUJ1 occurs within the culture, indicating successful maturation of SCPs into myelinating Schwann cells.
MEA analysis of hiPSC-SCP and hiPSC-Motor Neuron co-cultures
Microelectrode array analysis of Schwann cell precursor and motor neuron co-cultures. Motor neurons and SCPs were plated at an optimized ratio and subsequently analyzed for differences in spontaneous baseline electrical activity. A) Raster plots at W2 timepoint showcasing increased baseline activity within co-culture conditions as compared to motor neurons alone. B-D) Quantification of percent active electrodes, MFR, and wMFR. In general, co-culture conditions increase activity shown by the number of active electrodes and firing rate. D) Cultures switched into optimized MEA media 24 hours prior to recording show an increase in burst spiking activity, as shown by interspike interval (ISI).
SCP cell aligment with sensory and motor neurons in vitro
The progressive alignment of same-donor hiPSC-derived SCPs and hiPSC-derived peripheral neurons. A) Phase images show alignment morphology over two weeks. White arrows indicate single SCPs, black arrows indicate single neurons, and red arrows indicate alignment of the two cell types. Scale bar = 100 um. B) Immunocytochemical staining showing the co-cultures of hiPSC-SCPs with hiPSC-motor neurons at 14 DIV. S100b+ and SOX10+ SCPs align with TUJ1+ axons of hiPSC-motor neurons derived from the same doner. Scale bar = 100 um.