Animal model and deafening procedure
Animal work was approved by the Institutional Animal Care and Use Committee of the University of Colorado Anschutz Medical Campus.
Pou4f3tm1.1(HBEGF)Jsto mice on a C57BL/6J background (from now on, we refer to them as Pou4f3DTR/+ mice) were a kind gift from Dr. Rubel’s lab. Additional Pou4f3tm1.1(HBEGF)Jsto mice were purchased from the Jackson Laboratory (stock no: 028673). The coding region of human diphtheria toxin receptor (HBEGF) was introduced upstream of the translation initiation site of the mouse Pou4f3 (POU domain, class 4, transcription factor 3) gene. The human diphtheria toxin receptor increases the sensitivity of mouse cells to the toxin. Given that the Pou4f3 genes are only expressed in the inner ear hair cells DT injected into a mouse specifically ablates inner ear hair cells and spares all other cells in the organ of Corti and beyond. The mouse colony was maintained by cross-breeding: Pou4f3DTR/+ and Pou4f3DTR/+, or Pou4f3DTR/+ and wild type (WT) mice. Mice at postnatal day (P) 1–2 were genotyped. The following primers were used for genotyping: wild type reverse [ATT GTT CTG GGC GAC ATG A], common [AAG AAG CAG GTG GGG GAG AG] and mutant reverse [CAG AAA GAG CTT CAG CAC CAC]. In order to ablate hair cells, 2–4 ng/g DT was intramuscularly injected into right caudal thigh muscles for both WT and Pou4f3DTR/+ mice at P1-2 after genotyping.
Cell culture of human ES cells
We used two cell lines; WA09 (H9 cell line from WiCell) for in vitro cell differentiation study, and LT2e-H9CAGGFP (GFP-expressing H9 cell line from WiCell)  for mainly in vivo cell transplantation study. In the LT2e-H9CAGGFP cell line, the green fluorescent protein (GFP) gene is driven by a CAG promoter, which results in constitutive and robust GFP expression for a prolonged time period. Both cell lines were maintained on matrigel-coated plates in mTeSR1 medium (STEMCELL, Vancouver, Canada) until the start of the day of differentiation (day 0). The medium was changed daily except for one double feed during a seven day period. Cells were passaged every three to four days.
For differentiation of the stem cells to the pre-placodal ectoderm (PPE) state, we used Leung et al.’s protocol . For 48–72 hours cells were cultured on matrigel covered plates, and maintained in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) supplemented with 20% KnockOut serum replacement, 1 x Non-Essential Amino Acid (NEAA), 1 x L-Glutamine, 100uM Beta-mercaptoethanol, and 8 ng/ml basic Fibroblast Growth Factor (bFGF). Ten µM Rho kinase inhibitor (Y27632, Calbiochem, San Diego, CA) was included for the first 24 hours and then removed. On day 3, the medium was changed to serum-free (SF) media containing 1% N2 supplement, 2% B27 supplement, 1 × NEAA, 1 × L-glutamine, 100 µM β-mercaptoethanol in DMEM/F12 and the culture was maintained in this media until the day of harvest. This medium was changed daily for the first six days and every other day thereafter.
For transplantation, LT2e hESCs were differentiated for 7–8 days prior to transplantation with the same protocol as WA09 hESCs described above. On the day of transplantation, differentiated cells were dissociated into a single-cell suspension by incubating with Accutase (StemCell, Vancouver, Canada) for 6 minutes at room temperature and then triturated (cell suspension was pipetted up and down several times). Cells were centrifuged and adjusted to the concentration of 5.0–10.0 × 106 /ml in SF media.
mRNA was extracted from cells at each day of differentiation using a RNeasy Mini Kit® (QIAGEN, Germantown, MD). mRNA was then quantitated using Qubit and 1ug of mRNA was converted to DNA using the qScript cDNA Synthesis Kit ® (Quanta bio, Beverly, MA). Subsequently, DNA was PCR amplified (ProFlex PCR System; Thermo Fisher, Waltham, MA) with GoTaq® master mix (Promega, Madison, WI). The amplified DNA was then electrophoresed on 2% agarose gels and PCR bands were visualized with an ultraviolet transilluminator. Images were captured and stored on a computer. For quantification of gene expression levels, the detected bands were analyzed with Image J software (NIH, Maryland, MD) and we obtained relative intensity by dividing the intensity of each band by that of a 500 bp ladder band on the same gel in order to reduce bias in combining data from each sample taken with different UV light exposure time.
Technical considerations of the transplant procedure
To assess cell viability for cell transplantation, we examined cell survival rate at the three technical stages: 1) after cell dissociation and collection, 2) in a suspension during surgery time until injection, 3) after ejection from the glass pipettes (using several different tip sizes and the same pressure setting as our cell transplantation experiment). We found that when the pipette tip size was 12.5 µm, cells completely clogged the tip, and with a tip size of 18.75 µm, out of ejected cells about half of the cells died. Almost all cells survived, however, when ejected through the tip of 25 µm diameter (Supplemental Fig. 3A). Thus we used glass pipettes with a 25 µm diameter tip opening for all the following transplantation studies. Furthermore, we found that SF media is comparable or perhaps better as a solution for a short term cell storage during the surgery just before the injection (Supplemental Fig. 3B). Moreover, we found there was no real difference in cell viability when incubating the cells at various temperatures (n = 1) (Supplemental Fig. 3C). Therefore, we kept cells at room temperature from the time of cell dissociation, and during the surgery until the time of transplantation.
Differentiation of LT2e hESCs started 7–8 days prior to the day of transplantation to match the timing of cell differentiation and transplantation. Glass pipettes (Harvard Apparatus, holloston, MA; 30–00570) were pulled with micropipette pullers P-1000 (Sutter Instrument, Novato, CA), then the tips of the glass pipettes were beveled with a Microelectrode Beveler BV-10 (Sutter Instrument, Novato, CA) and adjusted to 25 um tip opening before the day of transplantation. On the day of transplantation, neonatal mice at 3 days after DT injection were anesthetized on ice (hypothermia). After the induction of anesthesia and once the anesthetized had reached an adequate anesthetic depth, the skin on the left side of the post-auricular region was sterilized with Betadine 1% iodine solution. The right ear served as an untreated control. Under a stereomicroscope (Leica, Allendale, NJ), a 0.5 cm post-auricular incision was made behind the left ear, then subcutaneous muscles were incised and separated to expose the temporal bone. The left otic bulla was opened using forceps to expose the round window niche, then the hole was widened sufficiently to visualize the round window membrane (RWM). The cell suspension was injected from a glass pipette into the scala tympani through the round window membrane by a Picoliter microinjector PLI-100A (Warner Instrument, Hamden, CT) for one to two minutes. A total of approximately 0.5 ul (5.0–10.0 × 106 cells/ml, 2.5-5.0 × 103 cells/injection) was delivered to the cochlea. The punctured RWM was sealed quickly after pulling out the pipette with a small piece of muscle or adipose tissue harvested from the cervical area during the approach to prevent leakage from the round window at the puncture site. The hole in the auditory bulla was also covered with adipose tissue and the wound was sutured in layers with a 6 − 0 absorbable chromic suture. The animals were kept on a heated pad until they recovered and put back in the cage with their mothers.
At each day of harvest, the treated animals were sacrificed with an overdose of the intraperitoneal administration of xylazine and ketamine-HCl mix followed by decapitation. Cochleae were extracted and fixed with 4% paraformaldehyde overnight at 4 °C. EDTA solution (0.12M) was used for decalcification of the bone structure (P8-9: for 2–4 hrs, P15 to P16: overnight, P27 to P30: for 2–3 days). For frozen serial sections, the cochleae were mounted in optimal cutting temperature compound (OCT) and frozen at -80 °C freezer then stored at -20 °C. The cochleae were cut on a cryostat (LEICA CM1850, Allendale, NJ) into 8–10 µm slices that were collected onto the glass slides. The entire cochlea was sliced and sections collected on the Superfrost Excell microscope slides (Fisherbrand, Cat # 22-034-985).. Mounted slides were washed with PBS, and to increase tissue adherence to the glass, a 4% paraformaldehyde was applied on each slide for 10 minutes and washed three times. After blocking (0.3% TritonX, 1% bovine serum albumin and 5% normal donkey or goat serum) for 20 minutes, the slides were incubated with primary antibodies (Supplemental Table 1) overnight at 4 °C. The following day, slides were washed 3X with PBS and then incubated with secondary anti-rabbit/mouse/goat Alexa 488/546/647 (Invitrogen, Carlsbad, CA) antibodies for 2 hours at room temperature in the dark. For Phalloidin or neurofilament (NF) staining, the secondary antibody was changed to anti-Phalloidin conjugated with AF 547 or 647 (1:200 in PBS), or anti-NF antibody conjugated with AF 555 (1:500 in PBS) for 30 minutes at room temperature. The slides were then washed with PBS and coverslipped with DAPI (Southern Biotech, Birmingham, AL or Vector, Burlingame, CA).
For whole-mount preparations, cochleae were dissected into the whole turns of organ of Corti under the microscope and stored at 4 °C in PBS until further processing. After three PBS washes, blocking solution was added (1% TritonX, 1% bovine serum albumin and 10% normal donkey or goat serum) and tissue was incubated at room temperature (RT) for 1 hour on a rotator. Next blocking solution was exchanged with primary antibody solution (Supplemental Table 1) (0.3% TritonX, 1% bovine serum albumin and 5% normal donkey or goat serum) overnight at 4 °C on the rotator. The next day, tissue was washed 3X with PBS and then incubated with secondary anti-rb/ms/gt conjugated with Alexa Fluor 488/546/647 (Invitrogen, Carlsbad, CA) antibodies for 2 hours at RT on the rotator protected from light. For Phalloidin or neurofilament (NF) staining another 30 minutes RT incubation with anti-Phalloidin conjugated with AF 547 or 647 (1:200 in PBS), or anti-NF antibody conjugated with AF 555 (1:500 in PBS) was added after the secondary antibody incubation. The tissues were then washed with PBS and mounted on slides with DAPI (Southern Biotech, Birmingham, AL or Vector, Burlingame, CA). For capturing images, we used light fluorescent filter microscope (Zeiss, Qberkochen, Germany) or confocal microscope (Olympus, Center Valley, PA FV 1000, oil obj x63, NA 1.4).
Cell count and Statistical analyses
We defined transplanted human cells as those cells that express GFP (because LT2e hESCs line is expressing GFP constitutively) and are also positive for DAPI nuclear stain. We sampled the entire cochlea and analyzed all the sections by manually counting the number of those double-positive transplanted cells for both cross-sections and whole-mount tissue in each area. For the statistical analysis we included only those mice in which we found any of GFP+/DAPI + cells (Fig. 3B, C, Fig. 4B, and C), we analyzed data statistically using the Student-t-test and ANOVA depending on the experimental design. Data are presented in the text and figures as means ± standard deviation (SD). The statistical significance level was set at p < 0.05.
Animals at P 27 - P 30 (D 26–28) were anesthetized by intraperitoneal administration of xylazine (12.5 mg/kg) and ketamine-HCl (87.5 mg/kg) in saline. One-third dose of the anesthetic mix was administered as boosters every 30 minutes. Stimuli presentation and acquisition of evoked potentials were performed via an RME (Haimhausen, Germany) Hammerfall DSP Multiface II sound card (44.1 kHz sampling rate). Monaural auditory thresholds were assessed in response to 50 ms tone pips at frequencies of 4, 8, 16 and 22 kHz at stimulus levels from 20 to 95 dB SPL at 5-dB steps. Five hundred repetitions were presented for each ear, all conditions were randomized. Sound stimuli were delivered via custom insert earpieces attached to TDT (Tucker Davis Technologies, Inc., Alachua, FL) FF1 magnetic speakers. Evoked auditory potentials were recorded with platinum subdermal needle electrodes placed at the apex (active), and nape of the neck (reference) with a ground electrode placed in the hind leg. The threshold was defined as the lowest level at which any wave in the ABR could be clearly detected by visual inspection.