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 IDRIS1,2, FAZLINA NORDIN1,*, 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
Diserahkan: 23 Ogos 2022/Diterima: 3 Februari 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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*Pengarang untuk surat-menyurat;
email: nordinf@ppukm.ukm.edu.my
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