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- Title
Derivation Of Human Midbrain Dopaminergic Neurons From Cord Blood-Derived Human Induced-Pluripotent Stem Cells In Vitro.
- Authors
Meng Meng Wang; King-Hwa Ling; Cheng-Biao Lu; Jun Jie Tan
- Abstract
Introduction: Cell-replacement therapies may offer promising prospect for treating Parkinson’s disease (PD). Human induced pluripotent stem cells (hiPSCs) offer possibility to generate functional midbrain dopaminergic (mDA) neurons. However, differentiation efficiency and expansion remain the challenges for subsequent scale-up and clinical applications. Methods: The episomal hiPSCs lines generated from cord blood CD34+ cells were obtained from Gibco. hiPSCs were cultured and maintained in mTesR medium. hiPSCs were seeded at 10,000/cm2 prior to differentiaton. Cell identity was assessed using immunocytochemistry and flow cytometric analysis. Results: Early neural induction was achieved via dual SMAD inhibition using SB431542 and Noggin. Co-stimulation of Wnt and sonic hedgehog signaling pathways, using glycogen synthase kinase-3β inhibitor (CHIR99021) at 0.75 μM and Shh-C24II at 200 ng/mL for day 9 showed greater ventral-midbrain patterning efficiency. FGF8b (100 ng/mL) stimulation between day 9 and day 16 was found to promote caudalization of ventral-midbrain progenitors, evident by the formation of FOXA2+/LMX1A+/OTX2+ cells by immunostaining at day 16 (n=7). The differentiation efficiency of FOXA2+/ LMX1A+ was 93.3% and LMX1A+/OTX2+ was 94.7% (n=1). Further maintenance of the ventral-midbrain progenitors in the defined maturation medium induced the formation of early neuronal phenotype with distinct projection was observed at day 24. At day 37, the cells further matured into βIII-tubulin+ neurons which also expressed tyrosine hydrolase, the rate limiting enzyme involved in dopamine biosynthesis. Conclusion: mDA neurons can be efficiently and effectively derived from hiPSCs.
- Subjects
PLURIPOTENT stem cells; DOPAMINERGIC neurons; HUMAN stem cells; INDUCED pluripotent stem cells; MESENCEPHALON; PARKINSON'S disease
- Publication
Malaysian Journal of Medicine & Health Sciences, 2022, Vol 18, p85
- ISSN
1675-8544
- Publication type
Abstract