Calcium-Enhanced Medium-Based Delivery of Splice Modulating Antisense Oligonucleotides in 2D and 3D hiPSC-Derived Neuronal Models
Abstract
:1. Introduction
2. Materials and Methods
2.1. Neural Progenitor Cell Cultures
2.2. Neural Differentiation and Maturation
2.3. Organoid Preparation
2.4. Calcium Chloride-Enhanced Medium (CEM), Gymnotic Uptake, and Lipofectamine-Mediated ASO Uptake in 2D Neuronal Cell Culture
2.5. Antisense Oligonucleotides
2.6. NEPA 3D Electroporation
2.7. Calcium Chloride-Enhanced Media (CEM)-Mediated ASO Uptake in 3D Neuronal Cell Culture
2.8. RNA Isolation, cDNA Synthesis, and RT-PCR
2.9. Immunocytochemistry
2.10. Organoid Embedding
2.11. Immunohistochemistry on Organoid Sections
2.12. Transfection Efficiency of Antisense Oligonucleotide
3. Results
3.1. CEM Improves Survival in hiPSC-Derived Neuronal Cultures
3.2. CEM Delivery Is More Efficient in Neurons Compared to Astrocytes
3.3. Dose-Dependent CEM-Mediated ASO Uptake in D15 Brain Organoids
3.4. Long-Term On-Target Effect of CEM-Mediated ASO Uptake in D35 Brain Organoids
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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ASO | Chemistry | Sequence | |
---|---|---|---|
1 | H40 (FAM-labeled) | 2′-O-methoxyethyl-modified phosphorothioate | 5′-UCC UUU CAU CUC UGG GCU C-3′ |
2 | HTT-AON12.1 | 2′-O-methoxyethyl-modified phosphorothioate | 5′-GUC CCA UCA UUC AGG UCC AU-3′ |
3 | APP-AT040 | 2′-O-methoxyethyl-modified phosphorothioate | 5′-CUU CUG CAA AGA ACA CCU UG-3′ |
Set Parameters | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Poring Pulse | Transfer Pulse | ||||||||||
V | Length (ms) | Interval (ms) | No. | D. Rate (%) | Polarity | V | Length (ms) | Interval (ms) | No. | D. Rate (%) | Polarity |
225 | 1.5 | 50 | 4 | 10 | + | 20 | 50 | 50 | 5 | 40 | +/− |
Target | Forward/Reverse primer (5′-3′) | |
---|---|---|
RT-PCR | HTT APP | CATCACACACAGCACCAAGA/ TCTCACATCTCTGTCTGGAACC CAAGACGGAGGAGATCTCTGA/ TTGGATTTTCGTAGCCGTTC |
Manufacturer | Catalogue Number | Dilution | |
---|---|---|---|
Primary antibodies | |||
Mouse-a-MAP2 | Sigma-Aldrich, Burlington, MA, USA | MAB364 | 1:500 |
Rabbit-a-GFAP | Sigma-Aldrich Burlington, MA, USA | Z0334 | 1:500 |
Mouse-a-Nestin | Stemcell Technologies Vancouver, BC, Canada | 60091 | 1:250 |
Rabbit-a-PAX6 | Biolegend, San Diego, CA, USA | 901301 | 1:250 |
Secondary antibodies | |||
DAPI | Sigma Aldrich, Burlington, MA, USA | D9542 | 1:1000 |
Goat-a-Mouse-Alexa594 | Invitrogen, Waltham, MA, USA | A11005 | 1:500 |
Goat-a-Rabbit-Alexa594 | Invitrogen, Waltham, MA, USA | A11012 | 1:500 |
Goat-a-Mouse-Alexa488 | Invitrogen, Waltham, MA, USA | A11001 | 1:500 |
Goat-a-Rabbit-Alexa750 | Abcam, Cambridge, UK | ab175732 | 1:500 |
Channel | Label | Color | Wavelength | Target% | Exposure Type | Exposure Time (s) |
---|---|---|---|---|---|---|
Ch1 | DAPI | Blue | 386 | 25 | Fixed | 0.00612 |
Ch2 | ASO | Green | 485 | 25 | Fixed | 0.03745 |
Ch3 | MAP2 | Red | 560 | 25 | Fixed | 0.02687 |
Ch4 | GFAP | Yellow | 740 | 25 | Fixed | 0.05654 |
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Buijsen, R.A.M.; van der Graaf, L.M.; Kuijper, E.C.; Pepers, B.A.; Daoutsali, E.; Weel, L.; Raz, V.; Parfitt, D.A.; van Roon-Mom, W.M.C. Calcium-Enhanced Medium-Based Delivery of Splice Modulating Antisense Oligonucleotides in 2D and 3D hiPSC-Derived Neuronal Models. Biomedicines 2024, 12, 1933. https://doi.org/10.3390/biomedicines12091933
Buijsen RAM, van der Graaf LM, Kuijper EC, Pepers BA, Daoutsali E, Weel L, Raz V, Parfitt DA, van Roon-Mom WMC. Calcium-Enhanced Medium-Based Delivery of Splice Modulating Antisense Oligonucleotides in 2D and 3D hiPSC-Derived Neuronal Models. Biomedicines. 2024; 12(9):1933. https://doi.org/10.3390/biomedicines12091933
Chicago/Turabian StyleBuijsen, Ronald A. M., Linda M. van der Graaf, Elsa C. Kuijper, Barry A. Pepers, Elena Daoutsali, Lotte Weel, Vered Raz, David A. Parfitt, and Willeke M. C. van Roon-Mom. 2024. "Calcium-Enhanced Medium-Based Delivery of Splice Modulating Antisense Oligonucleotides in 2D and 3D hiPSC-Derived Neuronal Models" Biomedicines 12, no. 9: 1933. https://doi.org/10.3390/biomedicines12091933