Human pluripotent stem cells (hPSCs), including human embryonic stem cells and human induced pluripotent stem cells (hiPSCs), provide promising sources for regenerative therapy, disease modeling, and drug screening. Relevant efforts have been invested in establishing robust induction protocols for PSC-derived dopaminergic (DA) neuron generation by mimicking brain development-related signaling pathways. However, these protocols require fully trained techniques and a long time to yield mature DA neurons. In this study, to accelerate the entire process, we generated a hiPSC line differentiating into DA neurons by the inducible force expression of two transcription factors ASCL1 and LMX1A. Using this hiPSC line, we established a rapid and simple induction protocol to generate mature DA neurons in 28 days. The induced DA neurons were characterized by gene expression and immunohistochemical analyses of fundamental DA neuronal markers. Moreover, the cell functional properties were analyzed by a multielectrode array system on day 28. This resource offers future applications for high-throughput screening, such as drug development and toxicology that require highly validated DA neurons.
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References
1.
VierbuchenT, OstermeierA, PangZP, KokubuY, SüdhofTC and WernigM. (2010). Direct conversion of fibroblasts to functional neurons by defined factors. Nature, 463:1035–1041.
2.
ZhouQ, BrownJ, KanarekA, RajagopalJ and MeltonDA. (2008). In vivo reprogramming of adult pancreatic exocrine cells to β-cells. Nature, 455:627–632.
3.
IedaM, FuJD, Delgado-OlguinP, VedanthamV, HayashiY, BruneauBG and SrivastavaD. (2010). Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell, 142:375–386.
4.
SzaboE, RampalliS, RisueñoRM, SchnerchA, MitchellR, Fiebig-ComynA, Levadoux-MartinM and BhatiaM. (2010). Direct conversion of human fibroblasts to multilineage blood progenitors. Nature, 468:521–526.
5.
Rivetti Di Val CervoP, RomanovRA, SpigolonG, MasiniD, Martín-MontañezE, ToledoEM, La MannoG, FeyderM, PiflC, et al. (2017). Induction of functional dopamine neurons from human astrocytes in vitro and mouse astrocytes in a Parkinson's disease model. Nat Biotechnol, 35:444–452.
6.
PfistererU, KirkebyA, TorperO, WoodJ, NelanderJ, DufourA, BjörklundA, LindvallO, JakobssonJ and ParmarM. (2011). Direct conversion of human fibroblasts to dopaminergic neurons. Proc Natl Acad Sci U S A, 108:10343–10348.
7.
NolbrantS, GiacomoniJ, HobanDB, BruzeliusA, BirteleM, Chandler-MilitelloD, PereiraM, OttossonDR, GoldmanSA and ParmarM. (2020). Direct reprogramming of human fetal- and stem cell-derived glial progenitor cells into midbrain dopaminergic neurons. Stem Cell Rep, 15:869–882.
8.
CaiazzoM, Dell'AnnoMT, DvoretskovaE, LazarevicD, TavernaS, LeoD, SotnikovaTD, MenegonA, RoncagliaP, et al. (2011). Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature, 476:224–227.
9.
KimJ, SuSC, WangH, ChengAW, CassadyJP, LodatoMA, LengnerCJ, ChungCY, DawlatyMM, TsaiLH and JaenischR. (2011). Functional integration of dopaminergic neurons directly converted from mouse fibroblasts. Cell Stem Cell, 9:413–419.
10.
ThomsonJA, Itskovitz-EldorJ, ShapiroSS, WaknitzMA, SwiergielJJ, MarshallVS and JonesJM. (1998). Embryonic stem cell lines derived from human blastocysts. Science, 282:1145–1147.
11.
TakahashiK, TanabeK, OhnukiM, NaritaM, IchisakaT, TomodaK and YamanakaS. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131:861–872.
12.
DoiD, SamataB, KatsukawaM, KikuchiT, MorizaneA, OnoY, SekiguchiK, NakagawaM, ParmarM and TakahashiJ. (2014). Isolation of human induced pluripotent stem cell-derived dopaminergic progenitors by cell sorting for successful transplantation. Stem Cell Rep, 2:337–350.
13.
KirkebyA, NolbrantS, TiklovaK, HeuerA, KeeN, CardosoT, OttossonDR, LelosMJ, RifesP, et al. (2017). Predictive markers guide differentiation to improve graft outcome in clinical translation of hESC-based therapy for Parkinson's disease. Cell Stem Cell, 20:135–148.
14.
KimTW, PiaoJ, KooSY, KriksS, ChungSY, BetelD, SocciND, ChoiSJ, ZabierowskiS, et al. (2021). Biphasic activation of WNT signaling facilitates the derivation of midbrain dopamine neurons from hESCs for translational use. Cell Stem Cell, 28:343–355.
15.
PiaoJ, ZabierowskiS, DuboseBN, HillEJ, NavareM, ClarosN, RosenS, RamnarineK, HornC, et al. (2021). Preclinical efficacy and safety of a human embryonic stem cell-derived midbrain dopamine progenitor product, MSK-DA01. Cell Stem Cell, 28:217–229.
16.
DoiD, MagotaniH, KikuchiT, IkedaM, HiramatsuS, YoshidaK, AmanoN, NomuraM, UmekageM, MorizaneA and TakahashiJ. (2020). Pre-clinical study of induced pluripotent stem cell-derived dopaminergic progenitor cells for Parkinson's disease. Nat Commun, 11:3369.
17.
NgYH, ChandaS, JanasJA, YangN, KokubuY, SüdhofTC and WernigM. (2021). Efficient generation of dopaminergic induced neuronal cells with midbrain characteristics. Stem Cell Rep, 16:1763–1776.
18.
ThekaI, CaiazzoM, DvoretskovaE, LeoD, UngaroF, CurreliS, ManagòF, Dell'AnnoMT, PezzoliG, et al. (2013). Rapid generation of functional dopaminergic neurons from human induced pluripotent stem cells through a single-step procedure using cell lineage transcription factors. Stem Cells Transl Med, 2:473–479.
19.
FaedoA, LaportaA, SegnaliA, GalimbertiM, BesussoD, CesanaE, BelloliS, MorescoRM, TropianoM, et al. (2017). Differentiation of human telencephalic progenitor cells into MSNs by inducible expression of Gsx2 and Ebf1. Proc Natl Acad Sci U S A, 114:E1234–E1242.
20.
ZhangY, PakCH, HanY, AhleniusH, ZhangZ, ChandaS, MarroS, PatzkeC, AcunaC, et al. (2013). Rapid single-step induction of functional neurons from human pluripotent stem cells. Neuron, 78:785–798.
21.
ChenM, MaimaitiliM, HabekostM, GillKP, Mermet-JoretN, NabaviS, FebbraroF and DenhamM. (2020). Rapid generation of regionally specified CNS neurons by sequential patterning and conversion of human induced pluripotent stem cells. Stem Cell Res, 48:101945.
22.
ChenSW, HungYS, FuhJL, ChenNJ, ChuYS, ChenSC, FannMJ and WongYH. (2021). Efficient conversion of human induced pluripotent stem cells into microglia by defined transcription factors. Stem Cell Rep, 16:1363–1380.
ChandaS, AngCE, DavilaJ, PakC, MallM, LeeQY, AhleniusH, JungSW, SüdhofTC and WernigM (2014). Generation of induced neuronal cells by the single reprogramming factor ASCL1. Stem Cell Rep, 3:282–296.
25.
HerdyJ, SchaferS, KimY, AnsariZ, ZangwillD, KuM, PaquolaA, LeeH, MertensJ and GageFH (2019). Chemical modulation of transcriptionally .enriched signaling pathways to optimize the conversion of fibroblasts into neurons. Elife, 8:e41356.
26.
AnderssonE, TryggvasonU, DengQ, FrilingS, AlekseenkoZ, RobertB, PerlmannT and EricsonJ. (2006). Identification of intrinsic determinants of midbrain dopamine neurons. Cell, 124:393–405.
27.
Doucet-BeaupréH, GilbertC, ProfesMS, ChabratA, PacelliC, GiguèreN, RiouxV, CharestJ, DengQ, et al. (2016). Lmx1a and Lmx1b regulate mitochondrial functions and survival of adult midbrain dopaminergic neurons. Proc Natl Acad Sci U S A, 113:E4387–E4396.
28.
NakagawaM, TaniguchiY, SendaS, TakizawaN, IchisakaT, AsanoK, MorizaneA, DoiD, TakahashiJ, et al. (2014). A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells. Sci Rep, 4:3594.
29.
AmimotoN, NishimuraK, ShimohamaS and TakataK. (2021). Generation of striatal neurons from human induced pluripotent stem cells by controlling extrinsic signals with small molecules. Stem Cell Res, 55:102486.
30.
KimS-I, Oceguera-YanezF, SakuraiC, NakagawaM, YamanakaS and WoltjenK (2016). Inducible transgene expression in human iPS cells using versatile all-in-one piggyBac transposons. Methods Mol Biol, 1357:111–131.
31.
NelanderJ, HebsgaardJB and ParmarM (2009). Organization of the human embryonic ventral mesencephalon. Gene Expr Patterns, 9:555–561.
32.
PowellSK, O'SheaC, TownsleyK, PrytkovaI, DobrindtK, ElahiR, IskhakovaM, LambertT, ValadaA, et al (2021). Induction of dopaminergic neurons for neuronal subtype-specific modeling of psychiatric disease risk. Mol Psychiatry. DOI: 10.1038/s41380-021-01273-0.
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