Generation and characterization of two human induced pluripotent stem cell lines from myotonic dystrophy type 1 patients

Abstract

The neuromuscular disorder myotonic dystrophy Type 1 (DM1) is brought on by CTG trinucleotide repeat expansions in the dystrophia myotonica-protein kinase (DMPK) gene, which leads to progressive myotonia and muscle weakness. We used Sendai virus reprogramming to generate two induced pluripotent stem cell (iPSC) lines (SCVIi134-A and SCVIi137-A) from peripheral blood mononuclear cells (PBMCs) of female DM1 patients carrying CTG repeat expansion. Both lines have normal karyotypes, show expression of undifferentiated human iPSC state markers, and differentiate into all three germ layers. These iPSC lines provide a platform for studying RNA toxicity at the molecular level and for drug development.

1. Resource table:

Unique stem cell line identifier	1) SCVIi134-A 2) SCVIi137-A
Alternative name(s) of stem cell lines	1) SCVI 692 (SCVIi134-A) 2) SCVI 770 (SCVIi137-A)
Institution	Stanford Cardiovascular Institute
Contact information of the distributor	Joseph C. Wu
Type of cell lines	iPSCs
Origin	Human
Additional origin info is required for human ESC or iPSC	Age: 60 (SCVIi134-A), 64 (SCVIi137-A), Sex: Female (both lines), Ethnicity if known: White (both lines)
Cell Source	PBMC
Clonality	Clonal
Method of reprogramming	Sendai virus vectors
Genetic Modification	Yes
Type of Genetic Modification	Spontaneous mutation
Evidence of the reprogramming transgene loss (including genomic copy if applicable)	RT-qPCR
Associated disease	Myotonic Dystrophy Type 1 (DM1, OMIM ID: 160900)
Gene/locus	DMPK, 19q13.32, CTG repeat expansion
Date archived/stock date	1) 11/18/2022 (SCVIi134-A) 2) 01/06/2021 (SCVIi137-A)
Cell line repository/bank	SCVi134-A: https://hpscreg.eu/cell-line/SCVIi134-A SCVi137-A: https://hpscreg.eu/cell-line/SCVIi137-A
Ethical approval	The Administrative Panel on Human Subjects Research (IRB) granted approval for the lines’ production under IRB #29904, “Derivation of Human-Induced Pluripotent Stem Cells (Biorepository).

2. Resource utility

Current treatment options for DM1 patients are limited to symptom management and supportive care without addressing the underlying disease mechanism. These two iPSC lines derived from female DM1 carriers provide valuable tools for studying DM1 pathophysiology, RNA toxicity mechanisms, somatic instability of CTG repeats, and screening potential therapeutic approaches in a patient-specific manner.

3. Resource details

DM1 is the most prevalent multisystemic genetic disorder marked by progressive muscle weakness, myotonia, and dysfunction across multiple organs such as the heart, brain, and endocrine system (Wenninger, 2018). It follows an autosomal dominant inheritance pattern and results from expanded CTG trinucleotide repeats located in the 3′ untranslated region of the DMPK gene on chromosome 19q13.32. Whereas unaffected individuals carry 5–35 CTG repeats, affected DM1 patients have expansions ranging from 50 to several thousand repeats, with disease severity generally correlating with repeat length (Liao, 2022). Additionally, the somatic instability of the expanded repeats results in an increasing number of repeats with each generation, a phenomenon termed anticipation. The expanded CUG-containing RNA transcripts sequester muscleblind-like (MBNL) RNA-binding proteins, leading to misregulated alternative splicing of numerous transcripts, which is considered the primary pathogenic mechanism in DM1 (Hale, 2023).

Two iPSC lines (SCVIi134-A and SCVIi137-A) were generated and characterized from female DM1 carriers’ peripheral blood mononuclear cells (PBMCs): SCVIi134-A (200–700 CTG repeats) and SCVIi137-A (850–1300 CTG repeats) (Table 1). Sendai virus vectors containing the four Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc) were used to reprogram PBMCs into iPSCs. The resulting iPSC lines showed typical colony morphology with defined borders and compact cells with high nucleus-to-cytoplasm ratios (Fig. 1A, Scale bar: 130 μm). The undifferentiated human iPSC state markers of both iPSC lines were confirmed through multiple approaches: 1) Using iPSC-derived cardiomyocytes (iPSC-CMs) as the negative control and a wild-type (WT) iPSC line as the positive control. RT-qPCR analysis demonstrated that our two iPSC lines expressed undifferentiated human iPSC state markers (Fig. 1B). 2) Immunofluorescence staining demonstrated robust expression of key undifferentiated human iPSC state markers, including Oct3/4, Nanog, and Sox2 (Fig. 1C, Scale bar: 130 μm). Both iPSC lines successfully differentiated into derivatives of all three germ layers, as evidenced by the expression of lineage-specific markers: Otx2 and Pax6 for ectoderm, Sox17 and Foxa2 for endoderm, and Brachyury and Tbx6 for mesoderm (Fig. 1D, Scale bar: 130 μm). Karyotype analysis demonstrated normal chromosomal integrity in both cell lines, and no chromosomal aberration were found when compared against the reference dataset (Fig. 1E). Both cell lines tested negative for mycoplasma contamination (Fig. 1F). STR analysis verified the origin of the iPSC lines from their respective donors’ PBMCs.

Table 1.

Characterization and validation.

Classification	Test	Result	Data
Morphology	Photography	Normal iPSC-like morphology	Fig. 1 Panel A
Phenotype	Quantitative analysis: RT-qPCR	Positive expression of undifferentiated iPSC state markers Nanog, Sox2, and Oct3/4 in the two DM1 lines and absent in differentiated CMs	Fig. 1 Panel B
	Qualitative analysis: Immunofluorescence staining	Positive expression of Nanog, Sox2, and Oct3/4 in both lines	Fig. 1 Panel C
Genotype	Whole genome array (KaryoStat™ Assay) Resolution 1–2 Mb]	Normal karyotype: 46, XX for both lines	Fig. 1 Panel E
Identity	Microsatellite PCR (mPCR) or	N/A	N/A
	STR analysis	Exact match	Submitted to an archive with a journal
Mutation analysis (IF APPLICABLE)	Southern blot or WGS	N/A	N/A
	Southern blot or WGS	N/A	N/A
Microbiology and virology	Mycoplasma	Mycoplasma testing by luminescence: Negative	Fig. 1 Panel F
Differentiation potential	Directed differentiation	Positive IF staining of three germ layer markers	Fig. 1 Panel D
List of recommended germ layer markers	Expression of these markers has to be demonstrated at mRNA(RT-qPCR) or protein (IF) levels, at least 2 markers need to be shown per germ layer	Ectoderm: Pax6, Otx2 Endoderm: Sox17, Foxa2 Mesoderm: Brachyury, Tbx6	Fig. 1 Panel D
Donor screening (OPTIONAL)	HIV 1 + 2, Hepatitis B, Hepatitis C	N/A	N/A
Genotype additional info (OPTIONAL)	Blood group genotyping	N/A	N/A
	HLA tissue typing	N/A	N/A

Fig. 1.

4. Materials and Methods

4.1. Reprogramming

The methodology was outlined in the prior study by (Liu, 2023). PBMCs were extracted from whole blood using the Ficoll-Paque gradient (GE Healthcare #17–5442–02) and washed with DPBS (ThermoFisher #14190144). Cells were cultured in StemPro-34 SFM (ThermoFisher #10639011) supplemented with SCF (100 ng/mL, Peprotech #300–07), FLT3 (100 ng/mL, ThermoFisher #PHC9414), IL-3 (20 ng/mL, Peprotech #200–3), IL-6 (20 ng/mL, ThermoFisher #PHC0063), and EPO (20 ng/mL, ThermoFisher #PHC9631). CytoTune-iPSC 2.0 Sendai Kit (ThermoFisher #A16517) was used for reprogramming. Transduced cells were seeded on Matrigel-coated plates (1:500, Corning #356231) and cultured in StemPro-34 medium. On day 7, the medium was changed to StemMACS iPS-Brew XF (Miltenyi #130–104–368). The cultures were maintained until colonies of iPSCs appeared, usually 10–15 days after transduction. Colonies were manually selected, grown, and then cryopreserved for use in later experiments.

4.2. Cell culture

Human iPSCs were maintained on Matrigel-coated plates (1:500, Corning #356231) in StemMACS iPS-Brew XF medium with supplements (Miltenyi #130–104–368, #130–104–370) at 37°C with 5% CO₂ and non-regulated (ambient) O₂. Cells were passaged at 70–80% confluency using 0.5 mM EDTA (Invitrogen #15575–038) with 10 μM ROCK inhibitor Y-27632 (Selleck #S1049) added for 24 h. The medium was replaced every 2 days.

4.3. Trilineage differentiation

Passage 23 iPSCs were used to verify differentiation potential of iPSCs into endoderm, mesoderm, and ectoderm lineages. Ectoderm was generated using the Human Pluripotent Stem Cell Functional Identification Kit (R&D Systems #SC027B). Endoderm was produced using StemDiff^™ Definitive Endoderm Differentiation Kit (STEMCELL^™ #05110). Mesoderm was derived using 6 μM CHIR99021 (Selleck #S2924) in RPMI medium with B27-Minus Insulin supplements, Minus Insulin (ThermoFisher #A1895601) for 48 h.

4.4. Immunofluorescent staining

Passage 22 iPSCs were fixed in 4% paraformaldehyde (Fisher #50980487) for 15 min and permeabilized with 0.1% PBST (Sigma-Aldrich #9036–19–5) for 10 min at room temperature. Cells were blocked for 30 min in 0.1% PBST with 5% Donkey Serum (Sigma-Aldrich #D9663) or Goat serum (Sigma #G9023), depending on secondary antibody origin. Primary antibodies diluted in 0.1% PBST were applied overnight at 4°C (Table 2). After washing, cells were incubated with secondary antibodies for 30 min at room temperature (Table 2). Nuclei were stained with NucBlue Fixed Cell ReadyProbes Reagent (ThermoFisher #R37606). Images were acquired using an ECHO Revolve microscope.

Table 2.

Reagents details.

Antibodies and Primers for Immunocytochemistry, Flow Cytometry, and PCR
Marker	Antibody	Dilution	Company (Cat #)	RRID
Undifferentiated iPSC state marker	Rabbit Anti-Nanog	1:200	Proteintech (142951–1-AP)	AB_1607719
Undifferentiated iPSC state marker	Mouse IgG2bκ Anti-Oct-3/4	1:200	Santa Cruz Biotechnology (Sc-5279)	AB_628051
Undifferentiated iPSC state marker	Mouse IgG1κ Anti-Sox2	1:200	Santa Cruz Biotechnology (Sc-365823)	AB_10842165
Differentiation marker: Ectoderm	Goat Anti-Otx2	1:200	R&D Systems (963273)	AB_2157172
Differentiation marker: Ectoderm	Rabbit Anti-Pax6	1:100	Thermo Fisher Scientific (426600)	AB_2533534
Differentiation marker: Endoderm	Goat Anti-Sox17	1:200	R&D Systems (963121)	AB_355060
Differentiation marker: Endoderm	Rabbit Anti-Foxa2	1:250	Thermo Fisher Scientific (701698)	AB_2576439
Differentiation marker: Mesoderm	Goat Anti-Brachyury	1:200	R&D Systems (963427)	AB_2200235
Differentiation marker: Mesoderm	Rabbit Anti-Tbx6	1:200	Thermo Fisher Scientific (PA5-35102)	AB_2552412
Secondary	Alexa Fluor 488 Goat Anti-Mouse IgG1	1:1000	Thermo Fisher Scientific (A-21121)	AB_2535764
Secondary	Alexa Fluor 488 Donkey Anti-Goat IgG (H + L)	1:1000	Thermo Fisher Scientific (A-11055)	AB_2534102
Secondary	Alexa Fluor 555 GoatAnti-Rabbit IgG (H + L)	1:500	Thermo Fisher Scientific (A-21428)	AB_2535849
Secondary	Alexa Fluor 555 Donkey Anti-Rabbit IgG (H + L)	1:500	Thermo Fisher Scientific (A-31572)	AB_162543
Secondary	Alexa Fluor 647 Goat Anti-Mouse IgG2b	1:250	Thermo Fisher Scientific (A-21242)	AB_2535811
Primers for PCR and qPCR
Target	Size of Band	Forward/Reverse primer (5′–3′)
Sendai virus genome (qPCR)	181 bp	Mr04269880_mr
GAPDH (qPCR)	471 bp	Hs02786624_g1
SOX2 (qPCR)	258 bp	Hs04234836_s1
NANOG (qPCR)	327 bp	Hs02387400_g1

4.5. Mycoplasma detection

Passage number 21 iPSCs were used for mycoplasma detection. MycoAlertTM Detection Kit (Lonza #LT07–318) was used according to manufacturer guidelines.

4.6. RNA extraction and RT-qPCR

RNA extraction and RT-qPCR analysis at passages number 21 and 24 were performed to evaluate the expression of markers and determine Sendai virus clearance. Total RNA was isolated from passages 21 and 24 DM1 iPSC lines, control iPSCs, and iPSC-CMs using Trizol reagent (Thermo Fisher Scientific), followed by purification with the Direct-zol RNA Microprep Kit (Zymo Research #R2062) according to the manufacturer’s protocol. Following extraction, RNA was reverse transcribed into cDNA using the iScript cDNA Synthesis Kit (BioRad #1708891). Quantitative PCR was subsequently conducted to assess expression levels of undifferentiated human iPSC state markers Nanog and Sox2, as well as to confirm the absence of Sendai viral genome, using commercially available TaqMan Fast Advanced Gene Expression Assay (Applied Biosystems, Cat #4444557) with the corresponding primers as detailed in Table 2.

4.7. Karyotyping

At passage 21, 2 × 10⁶ cells from each line were analyzed using the KaryoStat^™ assay (ThermoFisher #905403).

4.8. STR analysis

Genomic DNA was isolated from iPSCs at passage 21 and corresponding PBMCs using the DNeasy Blood & Tissue Kit (Qiagen, Cat. #69504). STR profiling was performed with the CLA Identifiler^™ Direct PCR Amplification Kit (Thermo Fisher Scientific, Cat. #A44661) according to the manufacturer’s instructions. Capillary electrophoresis was carried out on an ABI 3130xl Genetic Analyzer at the Stanford Protein and Nucleic Acid (PAN) Facility.

Data availability

Data will be made available on request.