Combinatorial programming of human neuronal progenitors using magnetically-guided stoichiometric mRNA delivery
- Massachusetts Institute of Technology, United States
- Massachusetts General Hospital, Harvard Medical School, United States
- Swiss federal Institute of Technology Zurich (ETH), Switzerland
Figures
Figure 1
Magnetically-guided spotting platform enables localized reconfigurable stoichiometrically-defined mRNA transfection.
(a) Schematic of the template used for synthesis of transcription factor mRNAs. (b) Magnetic spotting configurations with single versus dual magnets. (c) Transfection of cultured neural progenitor …
Figure 2
Interleaved transfection reduces fluctuations in delivered mRNA copy numbers and stoichiometries.
(a) Schematic illustration of multiple interleaved transfections using GFP and mCherry mRNAs. Each mRNA is delivered in multiple interleaved rounds of transfection, as opposed to being delivered all …
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Figure 2—source data 1
Ratio of red (mCherry) to green (GFP) fluorescence intensity per cell for both single and interleaved transfection protocols.
- https://doi.org/10.7554/eLife.31922.004
Figure 3
Magnetically reconfigurable spotting platform can screen and identify key transcription factors promoting dopaminergic cell fate.
(a) Timeline of the mRNA-induced differentiation process (proliferative NPC stage from day −2 to 0; early induction stage after day 0). Red arrows indicate time points of transfection before mitogen …
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Figure 3—source data 1
Quantitative TH gene expression analysis at day 17 comparing factors delivered at NPC (superscript N) and early induction (superscript I) stages.
- https://doi.org/10.7554/eLife.31922.006
Figure 4
Immunocytochemical and quantitative analysis confirms midbrain identity dopaminergic neurons generated from screen.
(a) Immunocytochemistry at day 17 for TH (green), MAP2 (red), FOXA2 (red) and DAPI (blue) performed in microwell plates; comparing triple staining DAPI+/MAP2+/TH+ and DAPI+/FOXA2+/TH+ between …
Author response image 1
Source: Courtesy of Inglese, J. & Jang, S.-W. from NIH Screening Center.
https://doi.org/10.7554/eLife.31922.012
Author response image 2
Western blots showing expression levels and lifetimes of Oct4, Sox2, Nanog, Lin28, and MyoD1 proteins in MRC-5 human fetal lung fibroblasts transfected with protein- encoding RNA, relative to levels in hES (H9) and rhabdomyosarcoma (Rh30) cells.
b-actin was used as a loading control. Left panels: The amount of RNA per 50 mL electroporation volume was varied as indicated. Cells were lysed 6 hours after transfection. Right panels: Cells were …
Author response image 3
Author response image 4
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers |
|---|---|---|---|
| Gene (Homo sapiens) | OTX2 | NA | HGNC:8522 |
| Gene (H. sapiens) | LMX1A | NA | HGNC:6653 |
| Gene (H. sapiens) | FOXA2 | NA | HGNC:5022 |
| Gene (H. sapiens) | ASCL1 | NA | HGNC:738 |
| Gene (H. sapiens) | NGN2 | NA | HGNC:13805 |
| Gene (H. sapiens) | NURR1 | NA | HGNC:7981 |
| Gene (H. sapiens) | PITX3 | NA | HGNC:9006 |
| Cell line (H. sapiens) | hNP1 | EMD Millipore | EMD Millipore:SCR055; RRID:CVCL_GS51 |
| Antibody | anti-FOXA2 (mouse monoclonal) | Abcam | Abcam:ab60721 |
| Antibody | anti-MAP2 (chicken polyclonal) | Abcam | Abcam:ab5392 |
| Antibody | anti-Tyrosine Hydroxylase (rabbit polyclonal) | Abcam | Abcam:ab112 |
| Sequence-based reagent | VMAT2 (TaqMan assay) | Thermo Fisher Scientific | Thermo Fisher Scientific:Hs00996835_m1 |
| Sequence-based reagent | GAPDH (TaqMan assay) | Thermo Fisher Scientific | Thermo Fisher Scientific:Hs02758991_g1 |
| Sequence-based reagent | OTX2 (TaqMan assay) | Thermo Fisher Scientific | Thermo Fisher Scientific:Hs00222238_m1 |
| Sequence-based reagent | PITX3 (TaqMan assay) | Thermo Fisher Scientific | Thermo Fisher Scientific:Hs01013935_g1 |
| Sequence-based reagent | FOXA2 (TaqMan assay) | Thermo Fisher Scientific | Thermo Fisher Scientific:HS00232764_m1 |
| Sequence-based reagent | LMX1A (TaqMan assay) | Thermo Fisher Scientific | Thermo Fisher Scientific:Hs00892663_m1 |
| Sequence-based reagent | TH (TaqMan assay) | Thermo Fisher Scientific | Thermo Fisher Scientific:Hs0016594_m1 |
| Sequence-based reagent | MAP2 (TaqMan assay) | Thermo Fisher Scientific | Thermo Fisher Scientific:Hs00258900_m1 |
| Sequence-based reagent | NURR1 (TaqMan assay) | Thermo Fisher Scientific | Thermo Fisher Scientific:Hs00443062_g1 |
| Other | DAPI stain | Thermo Fisher Scientific |
Additional files
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Supplementary file 1
List and details of top and bottom magnets.
- https://doi.org/10.7554/eLife.31922.008
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Supplementary file 2
List of TaqMan qRT-PCR assays used in this research.
- https://doi.org/10.7554/eLife.31922.009
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Transparent reporting form
- https://doi.org/10.7554/eLife.31922.010
