Journal: Cell Transplantation
Article Title: Neuronal Cell Sheets of Cortical Motor Neuron Phenotype Derived from Human iPSCs
Figure Lengend Snippet: In vitro characterization of neuronal cell sheet derived from human induced pluripotent stem cells (hiPSCs). (A) A schematic representation of neuronal sheet formation from hiPSCs and transplantation. Embryoid bodies were developed from undifferentiated hiPSCs in a floating condition for 4 d. Then the cells were cultured in fibronectin-coated dishes for 4 d during which retinoic acid (RA), noggin (NOG), and cyclopamine (CyP; 3 factors) were introduced twice (on days 5 and 7). In some experiments, sonic hedgehog (SHH) were introduced instead of adding CyP, together with RA and NOG in order to compare the effects of CyP with those of SHH. Thereafter, the cells cultured with RA, NOG, and CyP were harvested on day 8. An aliquot of the cells was cultured in 24-well culture plates for 12 to 16 d and then harvested using trypsin/ethylenediaminetetraacetic acid (EDTA) solution for transplantation. The remaining cells were cultured on temperature-responsive gelation polymer-coated plate (temp. resp. polymer) for 12 to 16 d depending on the neuronal maturation, where they extended axon-like processes, leading to the formation of neuronal cell sheet. The neuronal cell sheet was recovered by lowering the temperature of culture plate below 22°C. Schedule for brain injury, transplantation, and motor function test was reported previously. 10 , 20 , 28 , 29 The cells on day 8 were used as neural stem/progenitor cells for comparison. (B) Stereomicroscopic view of the neuronal cell sheet cultured in a 48-well (diameter 12 mm) culture plate. Right lower part of the sheet detached from the bottom of the plate. (C) Stereomicroscopic view of the sheet in a 10-cm culture dish (diameter 90 mm). (D, E) Hematoxylin and eosin staining of the neuronal cell sheet (D, lower; E, higher magnification). Flow cytometric analysis of the cells in the sheet treated with trypsin/EDTA revealed that more than 75% of the cells expressed human NCAM (data not shown). (F, G) The neuronal cell sheet at day 19 was stained with anti-human nuclei (F) and anti-β-tubulin antibodies (G). (H) Real-time polymerase chain reaction analyses of the Figure 1. (continued) . neuronal cell sheet. For comparison, neural cells on day 8 (neural cells were cultured in the same manner except that SHH was added instead of CyP and were harvested on day 8 before making cell sheet) were included. The cell sheet expressed Foxp2, Fezf2, Igfbp4, CTIP2, and synaptophysin mRNA predominantly. CRIM1 mRNA was expressed throughout the culture period. Thus, the cell sheet expressed mRNAs of cortical motoneuron–associated proteins. A white horizontal bar represents 200 μm in panel D, 100 μm in panels E and F, and 50 μm in panel G. UiPSCs, undifferentiated-induced pluripotent stem cells; CRIM1, cysteine-rich motor neuron 1 protein precursor; MEF, mouse embryonic fibroblasts; floating indicates floating condition; Foxp2, forkhead box p2; Fezf2, forebrain embryonic zinc finger family zinc finger 2; Igfbp4, insulin-like growth factor-binding protein 4; CTIP2, COUP-TF-interacting protein 2; mRNA, messenger RNA.
Article Snippet: An aliquot of the cells was cultured in 24-well culture plates for 12–16 d, which were then harvested using trypsin/ethylenediaminetetraacetic acid (EDTA) (Thermo Fisher Scientific, Inc.) solution for transplantation.
Techniques: In Vitro, Derivative Assay, Transplantation Assay, Cell Culture, Staining, Flow Cytometry, Real-time Polymerase Chain Reaction, Binding Assay