The Establishment of In-Vitro Human Induced Pluripotent Stem Cell-Derived Neurons

Various methods have been developed to generate and differentiate induced pluripotent stem (iPS) cells using different reprogramming strategies. Lentiviruses remain a strategic method for cell reprogramming as it is highly efficient in gene transfer. The latest lentiviral technology includes a fourth-generation packaging system that claims to be efficient and safe, both key factors in driving cell reprogramming towards clinical applications. However, modifications made to enhance safety of lentiviral vectors have been shown to affect vector performance. In this study, we established that fourth-generation lentiviral packaging system are able to produce high titre lentiviruses with high transduction efficiencies and subsequently tested the robustness and reproducibility of generating iPS cells from adult human dermal fibroblasts using these lentiviruses. The use of fourth-generation lentiviruses consistently generates iPS cells with similar efficiency and quality in different primary cell lines. We demonstrate that the iPS generated on human-derived, mitomycin-C inactivated feeder cells can support and maintain iPS cells. Derived iPS cell clones highly express key pluripotency markers and can spontaneously differentiate into cells from the three embryonic germ layers. Derived iPS cells were also able to be differentiated into neural stem cell lineage, producing cells expressing Nestin and SOX2 and can mature into neurons with more than 70% efficiency. Our data demonstrate that the use of fourth-generation lentiviral packaging to produce lentiviruses for iPS cells generation is robust and reproducible as it can generate iPS cells from different adult dermal fibroblasts with the potential to differentiate into neural stem cells and neurons. The use of safer lentiviral packaging systems in combination with established vector plasmids will help to expedite the generation of iPS cells for clinical translation.<br><br>Funding Information: This research was funded by Malaysian Ministry of Health (MOH), grant number NMRR-19- 3602-52265 and Universiti Kebangsaan Malaysia (UKM) Medical Faculty Fundamental Research Grant, grant number FF-2020-327.<br><br>Declaration of Interests: The authors declare no conflict of interest.<br><br>Ethics Approval Statement: This study protocol was checked and approved by Medical Research &amp; Ethics Committee (MREC) under project code NMRR-19-3602-52265 (ref: KKM/NIHSEC/P20-422) and Universiti Kebangsaan Malaysia Ethics Committee (ref: JEP-2020-243).<br>