Generating a Cell Model to Study ER Stress in iPSC-Derived Medium Spiny Neurons from a Patient with Huntington’s Disease
Abstract
1. Introduction
2. Results
2.1. Generation and Characterization of iPSC Lines of a Patient with HD
2.2. Generation of Transgenic iPSC Lines Expressing XBP1-TagRFP Biosensor
2.3. Testing Capability of Obtained iPSC Lines to Differentiate into Neural Derivatives
2.4. Detection of XBP1-Mediated ER Stress in MSNs
3. Discussion
4. Materials and Methods
4.1. Ethical Statement
4.2. Generation of iPSC Lines by Reprogramming PBMCs of an HD Patient
4.3. Assessment of CAG Repeat Tract Length in HTT Gene
4.4. Generation of Transgenic iPSC Lines with XBP1-TagRFP Biosensor
4.5. Spontaneous Differentiation
4.6. Immunofluorescence Staining
4.7. Directed Differentiation iPSC into Medium Spiny Neurons
4.7.1. Protocol No. 1 [27]
4.7.2. Protocol No. 2 [26]
4.7.3. Protocol No. 3 [25]
4.8. Analysis of MSN and ER Stress Marker Genes Expression by RT-qPCR
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
iPSC | Induced pluripotent stem cell |
HD | Huntington’s disease |
ER | Endoplasmic reticulum |
ERAD | Endoplasmic reticulum-associated protein degradation system |
UPR | Unfolded protein response |
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Protocol | Transgenic iPSC Line | MSN Culture Name |
---|---|---|
No. 1 | iHD46Q-7.1-XBP-1 | XBP-1 |
No. 2 | iHD46Q-7.1-XBP-6 | XBP-6F |
No. 3 | XBP-6ST | |
No. 1 | iHD46Q-7.1-XBP-7 | XBP-7 |
No. 1 | iHD46Q-7.1-XBP-8 | XBP-8 |
No. 2 | XBP-8F | |
No. 3 | XBP-8ST | |
No. 1 | iHD46Q-7.1-XBP-16 | XBP-16 |
No. 1 | K6-4f (healthy donor) | K6-4 |
Neuronal Culture | ATF4 | sXBP1 | CHOP | BIP |
---|---|---|---|---|
XBP-7 | up | up | up | |
XBP-8 | up | up | up | up |
XBP-16 | up | up | up | up |
XBP-6F | up | up | ||
XBP-8F | down | up | up | up |
XBP-6ST | down | up | up | up |
XBP-8ST | down | up |
Gene | Sequence | |
---|---|---|
Markers of ER stress | ATF4 | TTCTCCAGCGACAAGGCTAAGG/CTCCAACATCCAATCTGTCCCG |
CHOP | GGTATGAGGACCTGCAAGAGGT/CTTGTGACCTCTGCTGGTTCTG | |
Bip (GRP78) | CTGTCCAGGCTGGTGTGCTCT/CTTGGTAGGCACCACTGTGTTC | |
sXBP1 | TCTGCTGAGTCCGCAGCAG/GAAAAGGGAGGCTGGTAAGGAAC | |
Markers of pluripotency | NANOG | CAGCCCCGATTCTTCCACCAGTCCC/CGGAAGATTCCCAGTCGGGTTCACC |
SOX2 | GCTTAGCCTCGTCGATGAAC/AACCCCAAGATGCACAACTC | |
OCT4 | CTTCTGCTTCAGGAGCTTGG/GAAGGAGAAGCTGGAGCAAA | |
Markers of MSN | GABRA2 | CCCAATGCACTTGGAGGATTTCC/AGAGCCATCAGGAGCAACCTGT |
ARPP21 | CCTACCTCAACCACGCAACAGT/CCTGTTGACCAGACAAGACTGG | |
CTIP2 (BCL11B) | CTCCCTTTGGATGCCAGTGTCA/GGCTCCAGGTAGATGCGGAAG | |
DRD1 | TGGTCTGTGCTGCCGTTATCAG/CAATCTCAGCCACTGCCTTCCA | |
DRD2 | CAATACGCGCTACAGCTCCAAG/GGCAATGATGCACTCGTTCTGG | |
CALB1 | TTTCCTGCTGCTCTTCCGATGC/GCTCCTCAGTTTCTATGAAGCCA | |
Detection of transgene insertions | XBP1-TagRFP | CCGGACCACTTTGAGCTCTAC/AGGCGCACCGTGGGCTTGTAC |
M2rtTA | CAGCCGGAACACGGCGGCATC/ACACCGTGCGTTTTATTCTGTC | |
Reference | B2M | TAGCTGTGCTCGCGCTACT/ TCTCTGCTGGATGACGTGAG |
sgRNA for AAVS1 | AAVS1 | CACCGGTCACCAATCCTGTCCCTAG/ AAACCTAGGGACAGGATTGGTGACC |
Marker | Antibody | Dilution | Company |
---|---|---|---|
Markers of ectoderm | Rabbit IgG anti-NF200 | 1:1000 | Sigma-Aldrich Cat. # N4142 |
Mouse IgG2a anti-Tubulin β 3 (TUBB3)/Clone: TUJ1 | 1:1000 | BioLegend Cat. # 801201 | |
Markers of mesoderm | Mouse IgG2a anti-αSMA | 1:100 | Dako Cat. # M0851 |
Mouse IgG1 anti-Collagen IV | 1:100 | LifeSpan Biosciences Cat # LS-C79603 | |
Markers of endoderm | Mouse IgG1 anti-CK18 | 1:200 | Abcam, ab668 |
Markers of pluripotency | Mouse IgGγ3 anti-SSEA4 | 1:200 | Abcam, ab16287 |
Rabbit IgG anti-SOX2 | 1:500 | Cell Signaling, 3579 | |
Mouse IgGγ2b anti-OCT4 | 1:50 | Santa Cruz, sc-5279 | |
Mouse IgGγ1 anti-NANOG | 1:50 | Santa Cruz, sc-293121 | |
Rabbit IgG anti-NANOG | 1:500 | REPROCELL, RCAB004P-F | |
Secondary antibodies | Goat anti-Mouse IgG (H + L) Secondary Antibody, Alexa Fluor 568 | 1:400 | Thermo Fisher Scientific, A11031 |
Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 | 1:400 | Thermo Fisher Scientific, A11008 | |
Goat anti-Mouse IgG2b CrossAdsorbed Secondary Antibody, Alexa Fluor 568 | 1:400 | Thermo Fisher Scientific, A-21141 | |
Goat anti-Mouse IgG1 Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 | 1:400 | Thermo Fisher Scientific, A-21121 | |
Goat anti-Mouse IgG3 CrossAdsorbed Secondary Antibody, Alexa Fluor 488 | 1:400 | Thermo Fisher Scientific, A21151 |
Name of Primer | Sequence |
---|---|
Forward (FAM-labeled) | [FAM-AH]TGGCGACCCTGGAAAAGCTGAT |
Reverse | GGTGGCGGCTGTTGCTGCTGCTG |
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Makeeva, V.S.; Sivkov, A.Y.; Zakian, S.M.; Malakhova, A.A. Generating a Cell Model to Study ER Stress in iPSC-Derived Medium Spiny Neurons from a Patient with Huntington’s Disease. Int. J. Mol. Sci. 2025, 26, 8930. https://doi.org/10.3390/ijms26188930
Makeeva VS, Sivkov AY, Zakian SM, Malakhova AA. Generating a Cell Model to Study ER Stress in iPSC-Derived Medium Spiny Neurons from a Patient with Huntington’s Disease. International Journal of Molecular Sciences. 2025; 26(18):8930. https://doi.org/10.3390/ijms26188930
Chicago/Turabian StyleMakeeva, Vladlena S., Anton Yu. Sivkov, Suren M. Zakian, and Anastasia A. Malakhova. 2025. "Generating a Cell Model to Study ER Stress in iPSC-Derived Medium Spiny Neurons from a Patient with Huntington’s Disease" International Journal of Molecular Sciences 26, no. 18: 8930. https://doi.org/10.3390/ijms26188930
APA StyleMakeeva, V. S., Sivkov, A. Y., Zakian, S. M., & Malakhova, A. A. (2025). Generating a Cell Model to Study ER Stress in iPSC-Derived Medium Spiny Neurons from a Patient with Huntington’s Disease. International Journal of Molecular Sciences, 26(18), 8930. https://doi.org/10.3390/ijms26188930