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Summary Genetic modification of human induced pluripotent stem cells (hiPSCs) is a powerful approach to measure and manipulate the cellular processes underlying differentiation and disease. Conventional genetic engineering of hiPSC lines requires a laborious process involving transfection, selection and expansion that can result in karyotypic abnormalities or transgene silencing during differentiation, limiting their applications. Self-amplifying RNA (saRNA) delivery is a potential alternative integration-free method for durable expression of transgenes. Here, we used saRNA to deliver transcription factors and functional reporters in hiPSCs and demonstrate that expression can persist for weeks. Specifically, saRNA delivery enables highly efficient forward programming to Ngn2-induced neurons and enables measurement of functional reporters over time. We show that a single transfection of saRNA encoded jRCaMP1b reporter in hiPSCs generates sustained expression throughout differentiation to 3D cardiac spheroids. The persistence of the reporter allows measurement of calcium dynamics at a single-cell and population level over weeks, allowing tracking of cardiomyocyte maturation and drug responses. Together, our systematic analysis shows that saRNA provides sustained transgene expression in hiPSCs, supporting integration- free cell-fate programming and measurement of functional reporters in clinically relevant model systems. HighlightsA single saRNA transfection generates durable transgene expressionsaRNA transfection of Ngn2 in hiPSCs results in robust neuronal differentiationsaRNA-delivery of functional reporters enables single-cell analysis of primary and hiPSC-derived cellssaRNA-based sensor allows monitoring of maturation and drug responses in 3D cardiac spheroids
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High-throughput screening of clinically approved drugs that prime nonviral gene delivery to human Mesenchymal stem cells
Abstract Background Human mesenchymal stem cells (hMSCs) are intensely researched for applications in cell therapeutics due to their unique properties, however, intrinsic therapeutic properties of hMSCs could be enhanced by genetic modification. Viral transduction is efficient, but suffers from safety issues. Conversely, nonviral gene delivery, while safer compared to viral, suffers from inefficiency and cytotoxicity, especially in hMSCs. To address the shortcomings of nonviral gene delivery to hMSCs, our lab has previously demonstrated that pharmacological ‘priming’ of hMSCs with the glucocorticoid dexamethasone can significantly increase transfection in hMSCs by modulating transfection-induced cytotoxicity. This work seeks to establish a library of transfection priming compounds for hMSCs by screening 707 FDA-approved drugs, belonging to diverse drug classes, from the NIH Clinical Collection at four concentrations for their ability to modulate nonviral gene delivery to adipose-derived hMSCs from two human donors. Results Microscope images of cells transfected with a fluorescent transgene were analyzed in order to identify compounds that significantly affected hMSC transfection without significant toxicity. Compound classes that increased transfection across both donors included glucocorticoids, antibiotics, and antihypertensives. Notably, clobetasol propionate, a glucocorticoid, increased transgene production 18-fold over unprimed transfection. Furthermore, compound classes that decreased transfection across both donors included flavonoids, antibiotics, and antihypertensives, with the flavonoid epigallocatechin gallate decreasing transgene production − 41-fold compared to unprimed transfection. Conclusions Our screen of the NCC is the first high-throughput and drug-repurposing approach to identify nonviral gene delivery priming compounds in two donors of hMSCs. Priming compounds and classes identified in this screen suggest that modulation of proliferation, mitochondrial function, and apoptosis is vital for enhancing nonviral gene delivery to hMSCs.
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- Award ID(s):
- 1659777
- PAR ID:
- 10230178
- Date Published:
- Journal Name:
- Journal of Biological Engineering
- Volume:
- 14
- Issue:
- 1
- ISSN:
- 1754-1611
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1186/s13036-020-00238-1
