Additional file 1 of Selective ablation of cochlear hair cells promotes engraftment of human embryonic stem cell-derived progenitors in the mouse organ of Corti
Creators: name: Takeda, Hiroki
name: Dondzillo, Anna
name: Randall, Jessica A.
name: Gubbels, Samuel P.
Publisher: name: figshare
Year: 2021
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Figure S1. Results of semi-qPCR for LT2e cell line. The intensity of each band was quantified. The expression pattern of each gene is similar to that of the WA09 cell line. n= 3. Figure S2. Results of cell counts for immunocytochemistry. The number of each marker positive cells was quantified. The expression pattern of each protein is similar to that of mRNA. n= 1. Figure S3. ABR results at day 28 for WT and Pou4f3DTR/+ mice with or without DT. A hearing threshold of Pou4f3DTR/+ mice that received DT is increased over 90dB while hearing of WT with or without DT and Pou4f3DTR/+ without DT mice are preserved. n=4 for WT without DT, n= 6 for WT with DT, n= 4 for Pou4f3DTR/+ mice without DT and n=5 for Pou4f3DTR/+ with DT. WT; wild type, DTR; diphtheria toxin receptor, DT; diphtheria toxin. Figure S4. Results of cell viability tests. (A) The cell survival rate was assessed after ejecting from the micro-glass pipettes. Cells were completely clogged in the pipette when 12.5 μm sized tip was chosen, while 40-50% of cells were alive when 18.75 μm used. The best survival rate (90-100%) was recorded by 25 μm of tip. n=1 (B)(C) Cell viability test was performed with two different media, DMEM and SF media (B) or different temperature (C) using trypan blue solution in vitro (Thermo Fisher). SF media shows better survivability at 60- 120 mins after cell dissociation than DMEM. There is no difference between each temperature condition. Each n=1. DMEM; Dulbecco's Modified Eagle Medium. Figure S5. The graph showing the percentage of engrafted cells expressed supporting cell marker (Sox2) and hair cell marker (Myoa7a) in the OC at day 28 in a Pou4f3DTR/+ mouse after DT and cell transplantation. 50% of the engrafted cells were positive for Sox2 and 30% of the engrafted cells were Sox2 and Myo7a double-positive, while 20% of the engrafted cells did not express either Sox2 or Myo7a. OC; organ of Corti, DTR; diphtheria toxin receptor, DT; diphtheria toxin. Figure S6. ABR results at day 26-28 for WT and Pou4f3DTR/+ mice after cell transplantation. Hearing thresholds of Pou4f3DTR/+ mice with DT was not improved by cell transplantation. WT mice received both DT injection and cell transplantation shows slight damage to hearing. WT; wild type, DTR; diphtheria toxin receptor, DT; diphtheria toxin. Figure S7. Images of the organ of Corti at day 26-28 after cell transplantation in transgenic mice. A; non-transplanted side of the transgenic mouse cochlea, B; transplanted side of the transgenic mouse cochlea. A’ and B’ shows large images of square part in each image. Green indicates autofluorescence (thin arrow) and engrafted human cells (thick arrow). Red indicates phalloining staining. Bars represent 50 μm. Figure S8. Images of the spiral ganglion at day 14 and day 28 after cell transplantation in transgenic mice. Green indicates engrafted human cells in the mouse spiral ganglion. On day 14, some engrafted cells make cluster and those are Sox2 positive (a, b). At day 28, a very few cells are still engrafted in the spiral ganglion (c) and modiolus (d). Green; naïve GFP signal, white; Sox2, red; MAP2 or Tuj1, yellow; NF-200 and Blue; DAPI staining. Bars represent 50 μm. Table S1. Details on antibodies. Table S2. Primer sequences utilized.