
After the first research declaring the generation of human induced pluripotent stem cells (hiPSCs) in 2007, several attempts have been made to model neurodegenerative disease
Parkinson’s disease (PD) is a progressive, age-related neurodegenerative disease with noteworthy motor impairments, and is the second most common neurodegenerative disease after Alzheimer’s disease (AD). PD is primarily linked with the explicit loss of midbrain dopaminergic (mDA) neurons in the substantia nigra pars compacta (SNpc), and physically displays as weakened movements in affected individuals (1, 2). The formation of unique and filamentous inclusion bodies called Lewy bodies (LBs), comprised mostly of alpha-synuclein (α-syn,
Human pluripotent stem cells (hPSCs), including embryonic stem or induced pluripotent stem cells (hESCs/hiPSCs), differentiated into specific types of neurons have emerged as a promising model for studying human neural diseases (10–14) and have the potential to be used as cell sources for transplantation (15, 16). Particularly, disease-specific hiPSCs provide us with an exceptional opportunity to recapitulate human disease phenotypes
In this review, it was investigated whether the α-syn aggregation phenomena in PD or DLB can be reproduced in hiPSC-based models, the present extent of development, and the type of further researches necessitated in future.
At the time of initial cloning, α-syn was called as the ‘precursor of non-Aβ component of AD amyloid’ (precursor of NAC, NACP) because the NAC was first detected in and isolated from AD amyloid plaques (20). α-Syn protein is a soluble protein and exists in the form of an unfolded monomer (21). Furthermore, it has been considered that α-syn undergoes a conformational change to the α-helical structure only upon binding to lipid vesicles (22). As unstructured α-syn monomers tend to eagerly undergo a conformational change to β-sheet structure and aggregate together, it has been hypothesized that binding of lipid vesicles with the α-helical conformation of α-syn monomers is a crucial intrinsic mechanism for sequestering unfolded cytosolic α-syn to prevent spontaneous α-syn aggregation. However, recent studies indicate that α-syn exists in the form of an α-helically folded tetramer in the physiological conditions and not as a natively unfolded monomer, and rarely this tetramer is converted into the pathological aggregates (23, 24). The PD missense mutations located in the lipid-binding motif of α-syn increase the transition from tetramer to monomer (25), leading to the formation of β-sheet oligomers and eventually to insoluble aggregates in pathological conditions. A growing number of evidence indicates a causative role of α-syn misfolding and aggregation in the pathogenesis of PD (2–7, 26, 27). α-Synucleinopathies (also called synucleinopathies) are neurodegenerative diseases representing the abnormal accumulation of intracellular aggregates of α-syn in neurons (LB and Lewy neurite) or glial cells (28). Among α-synucleinopathies, PD, DLB, and multiple system atrophy (MSA) are of the most common occurrence. The incident rate of α-synucleinopathies related to parkinsonism was 21.0 per 100,000 person-years (PD, 68% of α-synucleinopathies; DLB, 28%; MSA, 4%), based on the investigation of the medical records in Olmsted County, Minnesota, USA, 1991–2005 (29). Classical neurotoxin-based animal models do not model the molecular pathology of α-synucleinopathies (8, 9). However, many attempts have been made to recapitulate α-synucleinopathies in the mammalian system using α-syn transgenic mouse models, viral vector models of α-syn overexpression, and α-syn transmission models [reviewed in (9)].
Phosphorylation is the most widely and deeply studied posttranslational modification of α-syn. This modification may affect the ability of aggregate formation as well as the subcellular localization and function of α-syn. In recent years, an increasing number of studies have reported that α-syn within LBs is subjected to phosphorylation at serine 129 (S129), and it may have serious implications for α-syn-induced neurodegeneration (7, 30–34). Especially, S129-phosphorylated α-syn (pS129-α-syn) is an excellent marker for α-synucleinopathies because ~90% of α-syn in LBs is phosphorylated at S129, compared with only ~4% of α-syn under physiological conditions (7). However, the molecular and cellular mechanisms of α-syn aggregation controlled by phosphorylation and other effects of α-syn phosphorylation at S129, remain to be elucidated (35, 36), probably due to a lack of a pathophysiologically relevant model system for investigating α-syn aggregation of α-synucleinopathies. Therefore, it should be clearly addressed whether α-syn phosphorylation is a cause or a consequence of aggregation, or whether phosphorylation is neurotoxic or neuroprotective in α-synucleinopathies including PD and DLB.
Developing the most ideal protocol for human mDA neuronal differentiation from hPSCs for applications in PD modeling and/or transplantation therapy has been an intense area of research during the past decade. Arenas
The classical approach to developing a protocol for the human mDA neuronal differentiation from hPSCs was based on adaptations of mouse neural stem cell (mNSC) and mESC protocols, which required co-culture with feeder cells (37). Three groups have published the initial protocols to derive mDA neurons from hESCs co-cultured with the murine stromal cell lines (38–40). In 2007, Sonntag
For initiating spontaneous differentiation from hPSC towards specific cell types, embryonic body (EB)-formation is often considered as a basic starting method. After inducing EB-formation, putative mDA neurons are generated from hESCs (42, 43). In 2009, Swistowski
Studer group introduced a “dual-SMAD inhibition” method for differentiating hPSCs into neural cells in an exceedingly efficient manner to eliminate the influence of undefined factors, including unknown secreted molecules and unidentified effects of co-culturing with murine stromal cell lines or astrocytes, as well as to increase the efficiency and to reduce heterogenous nature on neuronal differentiation (45). Combined blockage of SMAD signaling at the beginning of the monolayer differentiation protocol using Noggin (to inhibit BMP-mediated SMAD signaling) and SB431542 (to inhibit TGF-β/nodal/activin-mediated SMAD signaling), synergistically facilitated neural induction of hPSCs and eliminated the need for feeder layers (45).
The developing midbrain co-expresses the roof plate marker LMX1A and the floor plate (FP) marker FOXA2. Administration of high levels of SHH along with the dual-SMAD inhibition during neural induction has been considered as essential for FP specification (46). By synthesizing the existing knowledge of midbrain development, Studer group succeeded in generating correctly specified hPSC-derived mDA neurons in a reliable and efficient manner (47). A follow-up study has described the use of a small molecule, LDN193189 (to inhibit BMP-mediated SMAD signaling), that can replace Noggin for neural induction of hPSCs (48). Modified dual SMAD inhibition (termed “LSB” for two inhibitors LDN193189 and SB431542) along with activation of SHH and WNT signaling, enhances the efficiency and reproducibility of the monolayer differentiation of hPSC-derived mDA neurons
Jaenisch group reported the first example of PD modeling using PD patient-derived hiPSC-based DA neurons in 2009, but the focus of the study did not cover the phenotypical differences between patient’s cells and healthy controls (49). After the pioneer study, many groups attempted to model PD
The causes of sporadic PD and DLB are largely unknown; however, the aggregation of α-syn is heavily implicated in the degeneration of neurons in sporadic PD and DLB (50). In 2016, Krainc group reported the detection of the thioflavin S-positive α-syn aggregates in the FP-derived 110 days old mDA neurons, which were differentiated from a sporadic PD hiPSC (51). The authors also showed that the level of pathogenic α-syn species detected by Syn303 antibody (52) was significantly increased in the 1% Triton X-100-insoluble-and-2% SDS-soluble fraction of the sporadic PD mDA neurons, as compared to healthy controls (51). Another report described the increased level of pS129-α-syn in the FP-derived 28–49 days old mDA neurons differentiated from a sporadic PD hiPSC, although the authors did not assess the formation of α-syn aggregates in these neurons (53).
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Groundbreaking researches suggested that α-syn exists as a helically folded tetramer in the physiological conditions and not in the form of natively unfolded monomer; the tetramer is rarely gets converted into the pathological aggregates (23, 24). A recent study has reported that the ratio of α-syn tetramers and related multimers, which resist aggregation, to monomers was reduced in the FP-derived 60–65 days old mDA neurons differentiated from a PD hiPSC with a point mutation in
It is feasible to reproduce the molecular pathology of α-synucleinopathies without using any exogenous factors and by employing only hiPSC-based PD modeling system along with the patient’s genetic background. However, only a handful of papers demonstrated success in recapitulating α-syn aggregation
Even though hiPSCs were generated by reprogramming of somatic cells from aged PD patients, their ages seemed to be reset to embryonic ages during the reprogramming process (73–76). Moreover, hiPSC-derived neurons, such as human mDA neurons, are largely regarded as embryonic stage neurons (73–76). Therefore, resolving an aging issue and/or a maturation issue is necessary to model late-onset diseases, such as PD and DLB. Apparently, the progerin-induced aging method could be used as one of the possible solutions (77).
We are constantly attempting to improve mDA neuronal differentiation protocols for modeling PD more complete, beyond EB-derived, feeder-dependent, neural rosette-derived, and FP-derived methods (Fig. 1). Very recently, the protocols to differentiate hPSCs or neuroepithelial stem cells into human midbrain organoids have been developed for the generation of more ideal human mDA neuronal models (78–80). These midbrain organoids will serve as a completer and more important biologically relevant cell sources for PD modeling.
Environmental factors may cause PD along with genetic factors. Peng
This work was supported by the National Research Foundation of Korea (NRF) grants funded by different governments of Korea (Ministry of Education, NRF-2017R1A6A3A03010524; Ministry of Science and ICT, NRF-2018R1C1B5045395) and by the research fund of Hanyang University (HY-2018). I sincerely apologize to colleagues whose work has not been cited in this review due to space limitations.
The author has no conflicting interests.
Selected reports showing the endogenous α-synuclein aggregation using PD hiPSC-derived cell models
Genetic mutation | Tested model | Differentiation protocol (ref.) | Differentiation marker | Stimulus | Aggregate | Cell death | Note | Ref. | |
---|---|---|---|---|---|---|---|---|---|
WB | ICC | ||||||||
Unknown | mDA | 2D-based (45, 47) | n.d. | No | Yes | Yes | n.d. | Increased detergent-insoluble α-syn aggregates at day 110; thioflavin S-positive α-syn aggregates formation at day 110; lysosomal defects at day 90 | (51) |
p.[Ala53Thr] |
mDA GABAergic GLUergic | EB-based (43, 45, 49) ~20% TH+; ~25% GABA+; ~20% VGLUT1+ | No | n.d. | Yes | Yes | Detection of thioflavin S-positive and proteinase K-resistant α-syn aggregates at day 50; increased pS129-α-synat day 50; decreased neurite length at day 50 | (54) | |
p.[Ala53Thr] |
mDA | 2D-based (45, 47) ~73% TH+/GIRK2+ | OTX2, LMX1A, FOXA2, NURR1, TH, GIRK2 (ICC) | No | n.d. | Yes | No | Increased thioflavin T-positive or S129-phosphorylated α-syn aggregates formation and ROS production than genetically corrected controls at day 35 | (55) |
paraquat | n.d. | n.d. | Yes | Increased apoptosis than genetically corrected controls at day 35 | |||||
maneb | n.d. | n.d. | Yes | Increased apoptosis than genetically corrected controls at day 35 | |||||
rotenone | n.d. | n.d. | Yes | Increased apoptosis than genetically corrected controls at day 35 | |||||
p.[Ala53Thr] |
mDA | 2D-based (45, 47) | n.d. | No | n.d. | Yes | n.d. | Detection of thioflavin S-positive α-syn aggregates at day 90; reduced aggregates formation by 758 treatment at day 90 | (56) |
Duplication | Neuron | EB-based (82) | n.d. | No | Yes | n.d. | n.d. | Neural rosette-derived neurons co-cultured with astrocytes; increased detergent-insoluble α-syn aggregates; increased presence of α-syn aggregation intermediates | (57) |
Triplication | Cortical | 2D-based (45) ~70% neurons activated by glutamate | Glutamate response (Ca2+ imaging) | No | n.d. | Yes | Yes | Increased filamentous α-syn aggregates formation and cell death than genetically corrected controls at day 70–90; increased NADH redox index than control at day 70–90 | (58) |
Triplication | mDA | EB-based (53) ~28% TH+/TUBB3+ (83) | No | n.d. | Yes | n.d. | Neural rosette-derived mDA; increased number and size of punctate α-syn aggregates at day > 71 than at day 45–50 | (59) | |
BFCN | EB-based (84, 85) ~36% VCHT+/TUBB3+ (83) | No | n.d. | Yes | n.d. | Neural rosette-derived BFCN; increased number and size of diffused α-syn aggregates at day > 71 than at day 45–50 | |||
Triplication | mDA | 2D-based (45, 47) ~70% TH+/FOXA2+ | LMX1A, FOXA2, TH (ICC) | No | Yes | Yes | n.d. | Increased detergent-insoluble α-syn aggregates at day 55–330; thioflavin S-positive α-syn aggregates formation at day 100 or 120; increased low expressing TH cells at day 330; lysosomal defects at day 180 or 330; reduced aggregates formation by 758 treatment at day 120 | (51, 56) |
p.[Gly2019Ser] |
Astrocyte | 3D-based (86) ~95% GFAP+ | No | n.d. | Yes | n.d. | Increased α-syn puncta area at day 28; transmitting α-syn to mDA neurons during 28 days co-culture | (61) | |
p.[Arg42Pro] |
mDA | EB-based (44) ~7% TH+ | FOXA2, TH (ICC) | No | u.c. | u.c. | n.d. | Decreased mDA neuronal differentiation than healthy control (~22% TH+) at day 28 | (62) |
p.[Arg275Trp] |
mDA | EB-based (44) ~15% TH+ | FOXA2, TH (ICC) | No | u.c. | u.c. | n.d. | Decreased mDA neuronal differentiation than healthy control (~22% TH+) at day 28 | (62) |
p.[Arg42Pro] |
mDA | EB-based (44) ~7% TH+ | FOXA2, TH (ICC) | No | u.c. | u.c. | n.d. | Decreased mDA neuronal differentiation than healthy control (~22% TH+) at day 28 | (62) |
p.[Asn52fs] |
mDA | EB-based (44) ~7% TH+ | FOXA2, TH (ICC) | No | u.c. | u.c. | n.d. | Decreased mDA neuronal differentiation than healthy control (~22% TH+) at day 28 | (62) |
p.[Val324fs] |
mDA | 2D-based (45, 47) ~70% TH+ | LMX1A, FOXA2, NURR1, TH (ICC) | No | Yes | u.c. | n.d. | Increased Triton X-100-insoluble α-syn aggregates at day 60; increased intracellular dopamine level; increased susceptibility to mitochondria toxin at day 60 | (63) |
p.[Gln456Ter] |
mDA | 2D-based (45, 47) ~75% TH+ | LMX1A, FOXA2, NURR1, TH (ICC) | No | Yes | u.c. | n.d. | Increased Triton X-100-insoluble α-syn aggregates at day 60; increased susceptibility to mitochondria toxin at day 60 | (63) |
p.[Leu1059Arg] |
mDA | 2D-based (45, 47) ~60% TH+/FOXA2+ | FOXA2, TH (ICC) | No | Yes | n.d. | n.d. | Increased detergent-insoluble α-syn aggregates at day 90; lysosomal defects at day 90 | (56) |
p.[Asn409Ser] |
mDA | 2D-based (45, 47) ~60% TH+/FOXA2+ | LMX1A, FOXA2, TH (ICC) | No | Yes | Yes | n.d. | Increased detergent-insoluble α-syn aggregates at day 90; thioflavin S-positive α-syn aggregates formation at day 100 or 120; increased low expressing TH cells at day 330; lysosomal defects at day 110, 120 or 180; reduced aggregates formation by 758 treatment at day 120 | (51, 56) |
p.[Asn409Ser] |
mDA | 2D-based (45, 47) ~75% TH+ | No | u.c. | n.d. | No | Reduced ratio of tetramers and related multimers, which resist aggregation, to monomers at day 60–65 | (71) | |
α-PFF | Yes | n.d. | Yes | Increased detergent-insoluble α-syn aggregates at day 73; increased pS129-α-syn at day 73; increased cell death at day 73 or 79 |
2D, monolayer differentiation. 3D, neurosphere differentiation. 758, NCGC00188758. α-PFF, α-syn pre-formed fibril (72). BFCN, basal forebrain cholinergic neuron. cortical, cortical neuron. del, deletion. EB, embryonic body differentiation. EX, exon. fs, frameshift. GABAergic, GABAergic neuron. GLUergic, glutamatergic neuron. ICC, confirmed by immunocytochemistry. mDA, midbrain dopaminergic neuron. n.d., not determined. NPC, neuronal precursor cell. p., protein sequence. qRT-PCR, quantitative real-time PCR. u.c., unclear. WB, confirmed by western blotting.
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