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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Sasaki-Honda et al.17
Graduate School and Faculty
Experimental models: Cell lines
HC #1 hiPS cell lines
of Medicine of Kyoto University
(approval numbers #R0091)
Oligonucleotides
MscI-EagI adapter oligos (Fwd: CCACAACCATGA
This paper
N/A
This paper
N/A
PB-TA-ERN
Laboratory of Knut Woltjen (Kim et al.)11
Addgene Plasmid #80474, KW110
PB-TAC-ERN
Laboratory of Knut Woltjen (Kim et al.)11
Addgene Plasmid #80475, KW111
PB-TA-ERP2
Laboratory of Knut Woltjen (Kim et al.)
11
Addgene Plasmid #80477, KW542
PB-TAC-ERP2
Laboratory of Knut Woltjen (Kim et al.)11
Addgene Plasmid #80478, KW543
PB-TA-ERN2
This paper
KW1406
PB-TAC-ERN2
This paper
TTGAACAAGATGGATTGCACGCAGGTTCTCC)
MscI-EagI adapter oligos (Rev: GGCCGGAGAACCTG
CGTGCAATCCATCTTGTTCAATCATGGTTGTGG)
Recombinant DNA
KW1413
43
PB-CAG-rtTA-ires-Puro
Collinson et al.
pENTR-MyoD
Tanaka et al.5
EMCV-Neo-MYOD1 (PB-TA-ERN2-MyoD)
This paper
KW1410
EMCV-Neo-MYOD1-mCherry
This paper
KW1415
N/A
N/A
(PB-TAC-ERN2-MyoD)
EMCV-Puro-MYOD1-mCherry (PB-TAC-ERP2)
This paper
KW1409
FMDV-Neo-MYOD1-mCherry (PB-TAC-ERN-MyoD)
Tanaka et al.5
KW698
EMCV-Puro-MYOD1 (PB-TA-ERP2-MyoD)
Uchimura et al.12
KW879
CAG-rtTA-EGFP (PB-CAG-rtTA-ires-GFP)
This paper
KW1480
p5E-CAG
Laboratory of Knut Woltjen
KW140
pENTR-rtTA-Adv
Laboratory of Knut Woltjen
KW555
p3E-IRES-EGFPpA
Kwan et al.44
CC389
pDestPB53
Laboratory of Knut Woltjen
KW136
EMCV-Puro-mCherry (PB-TA-ERP2-MyoD)
This paper
KW1561
EMCV-Neo-mCherry (PB-TA-ERN2-mCherry)
This paper
KW1562
CAG-EGFP (PB-GFPa)
Laboratory of Knut Woltjen
KWan091
piggyBac transposase expression
Laboratory of Knut Woltjen,
KW158
vector (pCAG-PBase)
(Kim et al.)11
Software and algorithms
BD FACS Diva software
BD Biosciences
N/A
FlowJo software
Tree Star
N/A
Endmemo
http://www.endmemo.com/index.php
N/A
GraphPad Prism
GraphPad
N/A
BZ-X710
Keyence
N/A
iScience 26, 107685, October 20, 2023
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RESOURCE AVAILABILITY
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Hidetoshi Sakurai
(hsakurai@cira.kyoto-u.ac.jp).
Materials availability
All unique/stable reagents generated in this study are available from lead contact, Hidetoshi Sakurai, with a completed Material Transfer
Agreement.
Data and code availability
Data: The data generated and analyzed during the current study are available from the lead contact upon reasonable request.
Code: This study did not generate/analyze [dataset/code].
Other items: Any additional information to reanalyze the data reported in this study is available from the lead contact upon reasonable
request.
EXPERIMENTAL MODEL AND STUDY PARTICIPANT ETAILS
Human iPSC lines
HC #1 hiPS cell lines served as healthy controls. HC #1 described in key resources table. HC #1 hiPSCs line was used in following approval by
the Ethics Committee of the Graduate School and Faculty of Medicine of Kyoto University (approval numbers #R0091). Karyotype was evaluated by G-banding method in LSI Medience.
Feeder-free hiPSC culture
Tet-mCherry or Tet-MyoD hiPSCs were cultured on Easy iMatrix-511 silk-coated plates (#892024, Nippi) in StemFit medium (AK02N, Ajinomoto) containing 100 mg/mL G418 (#938044, NacalaiTesque) or 0.5 mg/mL puromycin dihydrochloride (160-23151, Wako Chemicals). Cells
were passaged every 7 days using Accutase (#12679-54, NacalaiTesque) and seeded on Easy iMatrix-511 silk-coated 6-well plates in the presence of 10 mM Y-27632 (NacalaiTesque) at a density of 1.53104 cells/well for the first 2 days after plating. At 48 h after passaging, Y-27632 was
removed and replaced with StemFit medium containing the appropriate antibiotic.
METHOD DETAILS
Plasmid construction
FMDV-Neo piggyBac Gateway Destination vectors PB-TA-ERN (KW110) and PB-TAC-ERN (KW111), and EMCV-Puro piggyBac Gateway
Destination vectors PB-TA-ERP2 (KW542) and PB-TAC-ERP2 (KW543) were described in key resources table. EMCV-Neo piggyBac Gateway
Destination vectors PB-TA-ERN2 (KW1406) and PB-TAC-ERN2 (KW1413) were constructed by replacement of the FMVD IRES sequence with
an FseI-MscI EMCV IRES fragment from PB-CAG-rtTA-ires-Puro (a gift from Dr. Jonathan Draper) 42 and annealed MscI-EagI adapter oligos
(Fwd: CCACAACCATGATTGAACAAGATGGATTGCACGCAGGTTCTCC, Rev: GGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCA
TGGTTGTGG). MyoD expression vectors were constructed by Gateway cloning of the MYOD1 cDNA from pENTR-MyoD 5 into the series
of piggyBac Gateway Destination vectors described above, resulting in EMCV-Neo-MYOD1 (PB-TA-ERN2-MyoD, KW1410), EMCV-NeoMYOD1-mCherry (PB-TAC-ERN2-MyoD, KW1415), and EMCV-Puro-MYOD1-mCherry (PB-TAC-ERP2, KW1409). The FMDV-Neo-mCherry
(PB-TAC-ERN-MyoD, KW698) and EMCV-Puro (PB-TA-ERP2-MyoD, KW879) MyoD expression vectors were constructed by Gateway cloning
of the MYOD1 cDNA into KW111 and KW542.
PB-CAG-rtTA-ires-GFP (KW1480) was constructed using Multisite Gateway cloning of p5E-CAG (KW140; Kim et al. in review), pENTR-rtTAAdv (KW555) and p3E-IRES-EGFPpA (CC389; Kwan et al., 200744) into pDestPB53 (KW136; Kim et al. in review).
Generating Tet-mCherry and Tet-MyoD hiPSCs with customized Tetracycline inducible vectors
To generate Tet-mCherry or Tet-MyoD hiPSCs, 5.0 mg of Tet-mCherry (EMCV-Neo/Puro-mCherry) or Tet-MyoD vectors and 5.0 mg of piggyBac transposase expression vector were electroporated into 13106 hiPSCs by using a NEPA21 electroporator (Nepagene). Briefly, hiPSCs
were treated with 10 mM Y-27632 1 day before electroporation. Cells were dissociated into single cells with accutase, and 1.0x106 cells
were resuspended in Opti-MEM. Tet-mCherry or Tet-MyoD vectors with pigguBac transposase expression vector were electroporated under
the conditions described in Table S1. The electroporated cells were plated on Easy iMatrix-511 silk-coated 6-well plates at 2.03105 cells per
well in StemFit medium containing Y-27632. Selection with 100 mg/mL G418 or 0.5 mg/mL puromycin was started 48 h after the electroporation. After 5 days of antibiotic selection, the cells were passaged with continuous antibiotic selection.
Direct skeletal muscle cell differentiation
Skeletal muscle cell differentiation of Tet-MyoD hiPSCs was performed as following. Briefly, 3.03105 cells were seeded on Matrigel-coated
(#356231, BD Biosciences) 6-well plates (1:100) in StemFit medium supplemented with 10 mM Y-27632. At 24 h after seeding, the medium was
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changed to primate embryonic stem cell medium (RCHEMD001, ReproCELL) without Y-27632. After 24 h, 1.5 mg/mL dox (LKT Laboratories)
was added to the culture medium. After an additional 24 h, the medium was changed into differentiation medium composed of a-MEM (with
L-Gln, ribonucleosides, and deoxyribonucleosides, #21444-05, NacalaiTesque) with 5% KSR (#10828028, Invitrogen), 5% Penicillin Streptomycin Mixed solution (#09367-34, NacalaiTesque), 200 mM 2-mercaptoethanol and 1.5 mg/mL dox. Culturing was continued until day 7
with daily medium changes for immunofluorescence microscopy.
Quantification of skeletal muscle cell differentiation
The efficiency of skeletal muscle cell differentiation was analyzed using Keyence software. The total number of nuclei and MHC-positive nuclei
were counted. The differentiation efficiency was then calculated by dividing the number of MHC-positive nuclei by the total number of nuclei.
Immunofluorescence microscopy
To identify differentiated cells, myogenic markers were used for immunostaining. Differentiated cells were fixed with 4% or 2% PFA/DPBS (-) at
4 C for 10 min. After being washed in DPBS (-), the cells were treated with methanol:H2O2 (100:1) at 4 C for 15 min (in the case of d7 differentiated cells only) and subsequently blocked with Blocking One (#03953-95, NacalaiTesque) at 4 C for 45 min. The cells were then incubated
with primary antibody in 10% Blocking One in DPBS (-) with 0.2% Triton X-100 (Santa Cruz Biotechnology) at 4 C overnight. Next, the samples
were washed three times with DPBS (-) containing 0.2% Triton X-100. The cells were then incubated at room temperature for 1 h with secondary antibody in 10% Blocking One in DPBS (-) containing 0.2% Triton X-100. DAPI (1:5000) was used to counter-stain the nuclei. The samples
were observed under a BZ-X710 fluorescence microscope (Keyence).
For pluripotent marker immunostaining, the hiPSCs were fixed with 4% PFA/DPBS (-) at 4 C for 10 min. After being washed in DPBS (-), the
cells were blocked with Blocking One at 4 C for 45 min. The cells were then incubated at 4 C overnight with primary antibody in 10% Blocking
One in DPBS (-) containing 0.2% Triton X-100. Next, the samples were washed three times with DPBS (-) containing 0.2% Triton X-100. The cells
were then incubated at room temperature for 1 h with secondary antibody in 10% Blocking One in DPBS (-) and 0.2% Triton X-100. DAPI
(1:5000) was used to counter-stain the nuclei. The samples were observed under a BZ-X710 microscope at 2003 magnification. The primary
and secondary antibodies used in this study are listed in Table S2.
FACS
To isolate mCherry-positive and mCherry-negative populations of Tet-mCherry or Tet-MyoD hiPSCs, the cells were harvested as a single cell
suspension in HBSS buffer using Accutase and filtered through a cell strainer. Cells were analyzed and sorted on a FACS Aria II (BD
Biosciences).
Co-transfection of Tet-MyoD vector with additional rtTA or EGFP expression vector
For co-transfection, 5.0 mg Tet-MyoD vectors and piggyBac transposase expression vector with CAG-rtTA-EGFP or CAG-EGFP were electroporated simultaneously into hiPSCs using a NEPA21 electroporator. In total, 15 mg of plasmid vectors were electroporated into hiPSCs.
Flow cytometry
To measure mCherry or GFP reporter fluorescence, Tet-mCherry or Tet-MyoD hiPSCs were suspended in HBSS buffer and analyzed using a
BD LSR FortessaTM Cell Analyzer (BD Biosciences) with BD FACS Diva software (BD Biosciences). Hoechst staining (1:2000) was used to
exclude dead cells. Data were analyzed and generated by FlowJo software (Tree Star).
RNA isolation and reverse transcription
Total RNA was isolated using the ReliaPrep RNA cell Miniprep Kit System (Z6012, Promega) according to the manufacturer’s instructions. Residual genomic DNA was digested and removed using DNase I (Promega) treatment. First-strand cDNA was generated from extracted total
RNA using ReverTra Ace qPCR RT Master Mix with gDNA remover (FSQ301, TOYOBO). The qPCR was performed using Power SYBR Green
(#4368708, Applied Biosystems) and the Step One Plus thermal cycler (Applied Biosystems). All samples were normalized to the house-keeping gene Porphobilinogen Deaminase 1 (PBGD) 6. For absolute quantification of transgene expression levels, the plasmid backbone of the
EMCV-Neo/Puro-MYOD1-mCherry vectors were used to construct a standard curve with serial dilutions of 1/10 for qPCR analysis and estimation of the plasmid copy number was calculated using the web server Endmemo (http://www.endmemo.com/index.php). Each transgene’s
copy number was estimated by annotating its cycle threshold (Ct) value and comparing it to the standard curve of the plasmid copy number.
The primer sets used in this study are listed in Table S3.
Genomic DNA isolation and copy number integration analysis
Genomic DNA was extracted from mCherry-positive and mCherry-negative populations of Tet-mCherry or Tet-MyoD hiPSCs after sorting.
Genomic DNA was extracted using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich) according to the manufacturer’s
instructions. Copy number analysis was performed by qPCR as specified above, using Power SYBR Green and the Step One Plus thermal
cycler. Approximately, 8 ng of extracted genomic DNA was used for each qPCR. rtTA primer was used for detecting the Tet-mCherry or
Tet-MyoD vector and DLX5 was used as internal control. A single knocked-in Tet-MyoD vector cell line served as a single copy number control.
iScience 26, 107685, October 20, 2023
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QUANTIFICATION AND STATISTICAL ANALYSIS
For all experiments, data are reported as the mean G SD. For comparison of two samples, p value were analyzed using either an unpaired
Student’s t-test or Paired t-test. For comparison of multiple samples, p value were analyzed using one-way ANOVA followed by Tukey’s test or
Dunnett’s test. The above p value were analyzed using GraphPad Prism.
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iScience 26, 107685, October 20, 2023
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