Urinary Cells Collection and Expansion
Zhou et al. detailed the procedure for urinary cells isolation and expansion in 2012 [13]. Typically, approximately 200 mL of urine were collected into sterile 50 mL tubes and centrifuged at 800g for 5 min at room temperature, followed by gently and quickly pouring out the supernatant, so as not to pour out the precipitate. About 5 mL of each tube is left and mixed into a centrifugal tube. Then, 20 mL of washing buffer was added and centrifuged at 800g for 5 min at room temperature. The supernatant was carefully removed, leaving ~0.2 mL plus the pellet. Afterward, 1 mL of the primary culture medium was added to re-suspend the cell pellet, and then transfered the volume into a single well of a 12-well plate (coated beforehand with 0.1% gelatin). Next, 1 mL of the primary culture medium for the first 3 days was added, however without removing any medium. Approximately 4 days after plating, most of the medium was discarded, and 1 mL of REGM medium (LONZA, CC-3190, Switzerland) was subsequently added. Later, half of the medium could be changed every day to monitor the cell growth with a microscope until the density reached 80-90%. Finally, we split the obtained urinary cells (UCs) into ratios of 1:3 or 1:4 to a 6-well plate to allow them to expand quickly.
hiPSCs derivation and maintenance
The iPSCs were acquired by reprogramming the UCs as described in the previous work [13]. The iPSCs were reprogrammed from the UCs of a healthy 23-year-old male using the same method. For the following experiments, all iPSCs were cultured on 5μL/mL Matrigel (Corning, 354277, USA) in mTeSRTM1 (Stemcell Technologies, 5872, Canada).
Generation and differentiation of iPSC-derived NSCs through TGF-β/Smad and BMP inhibition
hiPSCs were expanded for 4 days in mTeSRTM 1 medium. Undifferentiated hiPSC colonies were broken into fragments using a P1000 pipette and re-plated onto 5μL/mL Matrigel-coated dishes in embryoid bodies (EBs) + 2i medium (DMEM/F12, 20% KSR, NEAA (1x), GlutaMax (1x), 0.1% beta-Mecaptoenthano, 5μM SB431542 and 5μM dorsomorphin) to generate EBs for 4 days. The EBs were re-plated onto Matrigel-coated dishes in N2B27 medium (1:1 mixture of N2 and B27.N2 medium consists of 1×N2, DMEM/F-12, NEAA (1x), GlutaMAX (1x), 5μg/mL insulin, 1mM L-glutamine, 100 μM 2-mercaptoethanol. B27 medium consists of Neurobasal, 1×B27). Neural rosettes were visible and matured within 14 days. Rosettes were picked and then dissociated into single cells in accutase for 2 minutes (Sigma, A6964, USA) that are suspended in culture. After 7-10 days, the single cells produced round neurospheres in N2B27 medium.Neurospheres were collected in a 15 mL tube with N2B27 medium and could be stored at AT for a week. To further acquire neurons, neurospheres were dissociated into single cell using accutase and transferred onto Matrigel-coated 24-well plate at the density of 2×104 cells/well in N2B27 medium and then change the culture medium every other day. Cells were cultured for 1months and obtained a mixture of neural cells containing astrocytes and neurons was obtained.
Karyotype analysis
Karyotype analysis was performed in iPSCs at passage 15 and in iNSCs at passage 5. When the cells had reached the logarithmic phase, Colcemid was added to a final concentration of 20 μg/mL for 2 h. The Supernatant was removed, and the pellet was resuspended in 8 mL of 0.075 M KCl and incubated at 37 °C for 20 min. The cells were fixed with fresh Carnoy's Fixative (3:1 ratio of methanol: glacial acetic acid). Twenty metaphases were analyzed at 450–500 band resolution using Ikaros (MetaStstems, Germany) on an Olympus BX51 microscope.
Teratoma formation
Human iPSC cells were harvested in 1.5 mL tube and two million cells were injected into the flank subcutaneously and Lower limb intramuscularly of NOD/SCID mice. After 8–10 weeks, euthanasia of mice with rising CO2 levels was performed. Tumors were collected and embedded in paraffin, stained with hematoxilin/eosin and then histologically analyzed.
Immunofluorescence staining
Cells were briefly fixed in 4% paraformaldehyde for 15 min at room temperature. After permeabilization with 0.5% Triton X-100 in PBS for 5 min, the cells were blocked with 0.5% Triton X-100 and 10% goat serum for 30 min. Next, the cells were incubated with primary antibodies in 10% goat serum at 4 °C overnight and then incubated with secondary antibodies (Supplementary Table.2) diluted in 10% goat serum for 1 h at room temperature. Nuclei were counterstained with DAPI (Beyotime Biotechnology, C1005, China) for 5 min. Images were acquired with an inverted fluorescence microscope (Olympus, IX73, Japan).
Quantitative Real-time PCR (qRT-PCR)
Total RNA was extracted using TRIzol reagent (Invitrogen, 15596026, USA). Total cDNA was prepared with HiScript II Q RT SuperMix for RT-qPCR (Vazyme, R223-01, China). qRT-PCR was then performed using specific primers in a CFX96 Real-Time System (Bio-Rad, USA). Primers are listed in Supplementary Table 2.
Flow cytometry
Cells were collected and fixed in 4% paraformaldehyde for 30 min at 37 °C. After washing with PBS, the cells were resuspended in FACS buffer. Then, the cells were incubated with the primary antibodies overnight at 4 °C, and secondary antibodies were carried out at room temperature for 30min (Table 2). After washing, the cells were resuspended in 200–300 μl of the FACS buffer and proceeded for analysis on BD Accuri C6 Plus. The data were analyzed with FlowJo V10 (FlowJo LLC, BD) and BD Accuri C6 Plus software.
Electrophysiology
Electrophysiological recordings were performed at using a whole-cell, voltage- or current-clamp technique. Whole-cell recordings were made with 6 - 9-MΩ borosilicate glass electrodes and specific protocols were depicted in each figure. An Axopatch 200B amplifier (Axon Instruments, USA) was used to record the electrophysiological signals. The data were acquired and analyzed using Clampfit 10.2 software (Molecular Devices, USA). Borosilicate glass pipettes had resistances of 4–8 MV when filled with a solution containing the following (mM): 140 potassium methanesulfonate, 10 HEPES, 5 NaCl, 1 CaCl2, 0.2 EGTA, 3 ATP-Na2, 0.4 GTP-Na2, pH 7.2 (adjusted with KOH). The bath solution contained the following (mM): 127 NaCl, 3 KCl, 1 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, 10 D-glucose, 2CaCl2, pH7.4 (adjusted with NaOH). Cells plated on coverslips were placed in a submerged recording chamber and were continually perfused with the bath solution equilibrated with 95% O2 and 5% CO2. All electrophysiological experiments were performed at room temperature.
Mycoplasma test
The Lonza MycoAlertTM mycoplasma detection kit was used to estimate the mycoplasma according to the instruction.
Short tandem repeat (STR) analysis
STR analysis was performed on the urine cells and established iPSCs with detection of 21 loci (Amelogenin, D3S1358, vWA, D7S820, CSF1PO, PentaE, D8S1179, D21S11, D16S539, D2S1338, PentaD, D19S433, TH01, D13S317, THOX, D18S51, D6S1043, D1S1656, D5S818, D12S391, FGA) by GUANGZHOU IGE BIOTECHNOLOGY LTD, China.
Data analysis
Statistical analysis for all the experimental data was performed using GraphPad Prism 7 and Microsoft Excel. The data were presented as mean ± SD. Statistical significance was determined using paired T-test.