A Combinatorial Cell and Drug Delivery Strategy for Huntington’s Disease Using Pharmacologically Active Microcarriers and RNAi Neuronally-Committed Mesenchymal Stromal Cells
Abstract
:1. Introduction
2. Materials and Methods
2.1. siRNA-LNCs
2.2. Fluorescent siRNA-LNCs-DiD
2.3. BDNF-Releasing, Laminin (LM)-Coated PAMs
2.4. LNC and PAM Characterization
2.5. MIAMI E/F Cells
2.6. MIAMI E/F Cell Transfection
2.7. LNC Cell Time Retention
2.8. MIAMI E/F Cell Neuronal Differentiation
2.9. Formation of PAMs-Cell Constructs
2.10. Preparation of Brain Organotypic Slices
2.11. Injection of the Cells-PAMs Constructs into Organotypic Slides
2.12. Reverse Transcription and Real Time Quantitative PCR
2.13. Immunocytofluorescence
2.14. Immunofluorescence
2.15. MIAMI Cell Secretome Analysis
2.16. Data Analysis
3. Results
3.1. MIAMI E/F Cell Transfection
3.2. MIAMI E/F Cells Commitment into GABA-Like Progenitors
3.3. MIAMI E/F Cell Commitment into GABAergic-Like Neurons
3.4. Adherence of MIAMI E/F SHH-siREST on PAMs
3.5. Characterization of MIAMI-SHH-siREST on PAMs
3.6. Behavior of PAMs and MIAMI-SHH-siREST in Huntington’s Disease Model
4. Discussion
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Appendix A
Heading | BDNF | PIGF-1 | b-NGF | HGF | SDF-1 | LIF | VEGF-A |
---|---|---|---|---|---|---|---|
MIAMI E/F media (Control) | 4.55 ± 0.294 | 14.36 ± 0.20 | 0.00 | 124.93 ± 1.52 | 481.95 ± 3.27 | 49.35 ± 3.43 | 34.21 ± 0.676 |
Supernatant MIAMI E/F | 1.85 ± 0.139 | 24.38 ± 1.25 | 51.07 ± 72.22 | 152.87 ± 42.87 | 385.73 ± 52.87 | 65.80 ± 2.54 | 482.53 ± 32.54 |
Step 1 Media (Control) | 0.00 | 0.00 | 0.00 | 0.00 | 13.61 ±0.65 | 0.00 | 0.00 |
supernatant MIAMI E/F SHH siREST | 5.95 ± 0.59 | 11.70 ± 0.26 | 46.68 ± 66.05 | 304.12 ± 216.21 | 1746.64 ± 63.87 | 63.78 ± 64.24 | 2205.48 ± 505.54 |
Step 2 Media without BDNF (Control) | 0.00 | 0.00 | 0.00 | 0.00 | 14.76 ± 0.01 | 0.00 | 0.00 |
supernatant MIAMI E/F SHH siREST on LM-blanks PAMS | 7.21 ± 0.19 | 14.73 ± 0.46 | 128.04 ± 12.21 | 192.08 ± 25.37 | 1567.67 ± 92.07 | 195 ± 39.81 | 11,028.92 ± 2576.19 |
supernatant MIAMI E/F SHH siREST on LM-BDNF PAMS | 13.77 ± 0.35 | 29.02 ± 0.41 | 148.30 ± 6.18 | 219.43 ± 15.76 | 1467 ± 138.48 | 180.11 ± 20.07 | 11,003.58 ± 1189.29 |
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Gene | Full name | NM number | Sequences |
---|---|---|---|
ACTB | Actin | NM_001101.3 | F: CCAGATCATGTTTGAGACCT |
R: GGCATACCCCTCGTAGAT | |||
BDNF | Brain-derived neurotrophic factor | NM_001143816 | F: CAAACATCCGAGGACAAGG |
R: TACTGAGCATCACCCTGG | |||
DARPP32 | Dopamine- and cAMP-regulated phosphoprotein | NM_181505 | F: GAGAGCCTCAGGAGAGGG |
R: CTCATTCAAATTGCTGATAGACTGC | |||
Dlx2 | Distal-less homeobox 2 | NM_004405 | F: GACCTTGAGCCTGAAATTCG |
R: ACCTGAGTCTGGGTGAGG | |||
GAD67 | Glutamic Acid Decarboxylase 67 | NM_000817 | F: GGTGGCTCCAAAAATCAAAGC |
R: CAATGTCAGACTGGGTAGCG | |||
GAPDH | glyceraldehyde-3-phosphate dehydrogenase | NM_001289745.1 | F: CAAAAGGGTCATCATCTCTGC |
R: AGTTGTCATGGATGACCTTGG | |||
GDNF | Glial cell line-derived neurotrophic factor | NM_011675.2 | Qiagen, ref #QT00001589 |
Pax6 | Paired box 6 | NM_000280 | F: TTTCAGCACCAGTGTCTACC |
R: TAGGTATCATAACTCCGCCC | |||
NGF | Nerve growth factor | NM_002506 | Qiagen, ref #QT00043330 |
REST | RE1-silencing transcription factor | NM_001193508.1 | F: ACTCATACAGGAGAACGCC |
R: GTGAACCTGTCTTGCATGG | |||
VEGFA | Vascular endothelial growth factor A | NM_001204384 | F: CAGCGCAGCTACTGCCATCCA |
R: CAGTGGGCACACACTCCAGGC | |||
ACTB | Actin | NM_001101.3 | F: CCAGATCATGTTTGAGACCT |
R: GGCATACCCCTCGTAGAT |
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André, E.M.; Delcroix, G.J.; Kandalam, S.; Sindji, L.; Montero-Menei, C.N. A Combinatorial Cell and Drug Delivery Strategy for Huntington’s Disease Using Pharmacologically Active Microcarriers and RNAi Neuronally-Committed Mesenchymal Stromal Cells. Pharmaceutics 2019, 11, 526. https://doi.org/10.3390/pharmaceutics11100526
André EM, Delcroix GJ, Kandalam S, Sindji L, Montero-Menei CN. A Combinatorial Cell and Drug Delivery Strategy for Huntington’s Disease Using Pharmacologically Active Microcarriers and RNAi Neuronally-Committed Mesenchymal Stromal Cells. Pharmaceutics. 2019; 11(10):526. https://doi.org/10.3390/pharmaceutics11100526
Chicago/Turabian StyleAndré, Emilie M., Gaëtan J. Delcroix, Saikrishna Kandalam, Laurence Sindji, and Claudia N. Montero-Menei. 2019. "A Combinatorial Cell and Drug Delivery Strategy for Huntington’s Disease Using Pharmacologically Active Microcarriers and RNAi Neuronally-Committed Mesenchymal Stromal Cells" Pharmaceutics 11, no. 10: 526. https://doi.org/10.3390/pharmaceutics11100526