Sains Malaysiana 52(3)(2023): 863-876

http://doi.org/10.17576/jsm-2023-5203-14

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

(Penubuhan Neuron Berpunca Sel Stem Pluripoten In Vitro Manusia)

IZYAN MOHD IDRIS^1,2, FAZLINA NORDIN^1,*, NUR JANNAIM MUHAMAD², JULAINA ABDUL JALIL², FATIMAH DIANA AMIN NORDIN², ROSNANI MOHAMED², ADIRATNA MAT RIPEN², GEE JUN TYE³, WAN SAFWANI WAN KAMARUL ZAMAN⁴, MUHAMMAD DAIN YAZID¹ & MIN HWEI NG¹

¹Centre for Tissue Engineering and Regenerative Medicine (CTERM), Universiti Kebangsaan Malaysia Medical Centre (UKMMC), 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

²Institute for Medical Research (IMR), National Institutes of Health (NIH), Ministry of Health Malaysia, Jalan Setia Murni U13/52, Seksyen U13 Setia Alam, 40170 Shah Alam, Selangor Darul Ehsan, Malaysia

³Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia (USM), 11800 USM, Penang, Malaysia

⁴Department of Biomedical Engineering, Faculty of Engineering, Universiti Malaya, 50603, Kuala Lumpur, Federal Territory, Malaysia

Received: 23 August 2022/Accepted: 3 February 2023

Abstract

Induced pluripotent stem cells (iPSCs) have been generated using different reprogramming strategies. Lentiviruses remain a strategic method for cell reprogramming as it is highly efficient in gene transfer. The latest fourth-generation lentiviral packaging systems claimed to be efficient and safe. However, modifications made to enhance safety of lentiviral vectors have been shown to affect vector performance. In this study, we established that the fourth-generation lentiviral packaging system can produce high-titre lentiviruses with high-transduction efficiencies. Subsequently, the robustness and reproducibility of generating iPSCs from adult human dermal fibroblasts were tested using these lentiviruses. The use of fourth-generation lentiviruses consistently generates iPSCs with similar efficiency and quality in different primary cell lines. This study demonstrated that the human-derived iPSCs can be maintained using mitomycin-C inactivated feeder cells. The iPSC clones highly expressed key pluripotency markers and can spontaneously differentiate into cells from the three embryonic germ layers. The iPSCs generated were able to differentiate into neural stem cell lineages, producing cells expressing Nestin and Sox2 as well as able to further differentiate into neurons with more than 70% efficiency. The data demonstrated that the use of the fourth-generation lentiviral packaging to produce lentiviruses for iPSCs generation is robust and reproducible as it can generate iPSCs from different adult dermal fibroblasts with the potential to differentiate into neural stem cells and neurons. The use of safer lentiviral packaging systems combined with established vector plasmids will help to expedite the generation of iPSCs for clinical applications.

Keywords: Induced pluripotent stem cells; lentivirus; neural stem cells; neurons; reprogramming

Abstrak

Sel induk pluripoten teraruh (iPS) telah dihasilkan menggunakan strategi pengaturcaraan semula yang berbeza. Lentivirus kekal sebagai kaedah strategik untuk pengaturcaraan semula sel kerana ia sangat cekap dalam pemindahan gen. Sistem pembungkusan lentivirus generasi keempat terkini dikatakan lebih cekap dan selamat. Walau bagaimanapun, pengubahsuaian yang dibuat untuk meningkatkan keselamatan vektor lentivirus telah ditunjukkan boleh menjejaskan prestasi vektor. Dalam kajian ini, kami mendapati bahawa sistem pembungkusan lentivirus generasi keempat boleh menghasilkan lentivirus dengan titer tinggi serta kecekapan transduksi yang tinggi dan seterusnya menguji keteguhan dan kebolehulangan penjaanan sel iPS daripada fibroblas kulit manusia menggunakan lentivirus ini. Penggunaan lentivirus generasi keempat secara tekal menjana sel iPS dengan kecekapan dan kualiti yang sama dalam talian sel primer yang berbeza. Kami menunjukkan bahawa iPS yang dihasilkan di atas sel penyuap yang dinyahaktifkan menggunakan mitomisin-C yang berasal daripada manusia boleh menyokong dan mengekalkan sel iPS. Klon sel iPS yang diperoleh mengekspresikan penanda pluripotensi utama dan boleh secara spontan membezakan menjadi sel daripada tiga lapisan sel embrio. Sel iPS yang diperoleh dapat dibezakan kepada keturunan sel induk saraf yang mengekspresikan Nestin dan Sox2 dan boleh matang menjadi neuron dengan kecekapan lebih daripada 70%. Data kami menunjukkan bahawa penggunaan pembungkusan lentivirus generasi keempat untuk menghasilkan lentivirus untuk penjanaan sel iPS adalah teguh dan boleh dihasilkan semula kerana ia boleh menjana sel iPS daripada fibroblas kulit dewasa yang berbeza dengan potensi untuk membeza menjadi sel stem saraf dan neuron. Penggunaan sistem pembungkusan lentivirus yang lebih selamat dalam gabungan dengan plasmid vektor yang mantap akan membantu mempercepatkan penjanaan sel iPS untuk terjemahan klinikal.

Kata kunci: Lentivirus; neuron; pengaturcaraan semula; sel induk pluripoten terjana; sel induk saraf

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^*Corresponding author; email: nordinf@ppukm.ukm.edu.my