It is well known that sporadic PD occupies > 90% of total PD cases, but genetic factors still play an important role in understanding PD etiology. The use of genetic PD iPSCs offers the promise of addressing the contribution of individual genetic factors and functional relevance of underlying molecular pathways in the development of PD. Currently, 16 familial PD-related genes have been identified. Among them, SNCA, LRRK2, Parkin, PINK1 and GBA mutations have mostly been studied in genetic PD iPSCs[50] (Table 1). Researchers have made efforts to illustrate the pathological features of DA neurons or other neuronal cells derived from these genetic PD iPSCs. It is generally considered that the fate of these mutated genes is often loss of function that induces aberrant accumulation of inactive proteins[51].

Accumulation of α-synuclein is a major feature of PD, which is encoded by SNCA gene. SNCA A53T mutant and triplication SNCA are familial PD SNCA mutants. In these patients, α-synuclein level in the midbrain region is three times that in somatic cells, revealing that it is easy to cause accumulation of α-synuclein in DA neurons. After Soldner et al[52] successfully generated the first DA neurons from SNCA mutant iPSCs, a number of SNCA A53T mutant or triplication SNCA mutant iPSC lines were generated, and elevated α-synuclein levels were found in these iPSC-derived DA neurons[40,53-57]. Other cellular types such as forebrain cortical neurons, neural precursor cells, and GABAergic neurons were further induced from SNCA mutant iPSCs, and endoplasmic reticulum (ER) stress, oxidative stress, or maturation inability but no significant alteration of α-synuclein protein or mRNA level were detected in these iPSC-derived cells[56-59]. These studies show that SNCA gene mutations may have great influence on a variety of neuronal subtypes and not just DA neurons.

N1437H, R1441C and G2019S mutations of LRRK2 are known to cause PD. Among them, G2019S is the most common familial PD mutation in LRRK2[60]. Many studies have pointed out the crucial role of the loss-of-function of LRRK2 mutant in reducing neurite outgrowth and numbers, and process complexity[61-65]. Oxidative stress, mitophagic dysfunction, and DNA damage have been observed in LRRK2 G2019S iPSC-derived DA neurons. In addition, self-renewal and neuronal differentiation of LRRK2 G2019 mutant neural stem cells are reported to be impaired, providing evidence on the crucial role of LRRK2 in neural development[62].

Synergistic effect of PINK1 and Parkin are important to maintain cellular homeostasis such as mitochondrial quality in DA neurons[66,67]. It is suggested that overexpression of Parkin can largely rescue the defects in PINK1 mutants through mitochondrial translocation[66,68]. Parkin improves the uptake of dopamine through enhancing the expression of DAT as well[69]. PINK1 or Parkin mutant iPSC-derived DA neurons display abnormal phenotypes such as mitophagy and autophagy impairment, vulnerability to various stresses, and accumulation of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α), which are consistent with previous studies[63,70-75]. These findings support the synergistic effect of PINK1 and Parkin, providing an inspiration for developing therapeutic strategies for PD.

GBA1 gene mutations are reported to be associated with an increased risk of sporadic PD[76]. A number of studies on patient-specific iPSC-derived DA neurons harboring GBA1 mutations have indicated that β-glucocerebrosidase (GBA1) has high correlation with elevated α-synuclein levels as well as autophagic and lysosomal defects[77-79]. Furthermore, calcium homeostasis imbalance, and reduced dopamine storage and uptake are also found in GBA1 mutant DA neurons[77-79]. However, how these above-mentioned phenotypes are triggered by GBA1 mutant remains to be explored.

Sporadic PD-derived iPSCs lack known PD mutations. The first case of generation of sporadic PD patient iPSCs was reported by Soldner et al[80]. They generated iPSCs from skin biopsies obtained from sporadic PD patients by application of modified lentiviruses carrying loxP sites flanking the integrated provirus for improving the efficiency of reprogramming. The advantage of this method was the use of inducible excisable lentivirus, rendering the iPSCs free of reprogramming transgenes. However, the phenotypic analysis of these iPSCs was not performed in that study. Sanchez-Danes et al[71] generated healthy and sporadic PD iPSCs via retroviral delivery of OCT4, KLF4 and SOX2, and then differentiated them into DA neurons. By comparing with the healthy control group, they found that DA neurons differentiated from sporadic PD-patient-derived iPSCs after long-term culture displayed increased expression of cleaved caspase 3, shortened neurite length, and defective autophagosome clearance[81]. Fernández-Santiago et al[65] reported genome-wide DNA methylation of DA neurons derived from LRRK2-mutant and sporadic PD patients to explore the relationship between DNA methylation and alteration of gene expression and enhancer elements. They found that alterations of epigenetic signature significantly affected DNA methylation in sporadic PD patients. This study provides evidence that it is a common phenomenon that aberrant protein turnover and altered morphology and methylation patterns occur in sporadic PD-patient-derived DA neurons. Piwi-interacting RNA (piRNA) seems to have relevance to aberrant protein turnover and altered morphology and methylation patterns in sporadic PD-patient-derived DA neurons. piRNA is a complex of piwi protein and RNA, and is a large class of small noncoding RNA molecules expressed in animal cells that are involved in the epigenetic and post-transcriptional silencing of transposons. Schulze et al[82] have shown that the specific alteration of sin- and line-derived piRNA in DA neurons could be a new mechanism for the causation of PD[82-84].

Astrocytes are the major group of cells in the central nervous system, with a range of functions that provide both structural and metabolic support for neurons. Accumulating evidence suggests that astrocyte dysfunction leads to the pathogenesis of PD, especially familial PD[85-88]. As summarized by Booth et al[85], mutations in DJ-1, SNCA, PLA2G6, LRRK2 and GBA lead to abnormal glutamate uptake, mitochondrial dysfunction, inflammatory response, water transport defect, and autophagy impairment. Gunhanlar et al[86] found that in a coculture system of astrocytes and neurons at a consistent ratio (60:40), neuron maturation was distinctly upregulated according to electrophysiological maturity. Accordingly, astrocytes are widely used in implementing cellular modeling approaches to the study of neurodegenerative disorders[87]. Similarly, astrocytes could help DA neurons defend against the neurotoxins and attenuate the mitochondrial dysfunction, as observed by Feng et al[88]. Remarkably, the astrocytes and neuron co-culture system improved the outgrowth of neuron markers, and stabilized the mitochondrial function through downregulation of ROS and increased mitochondrial function[88]. On the contrary, astrocytes were also used in inducing PD degeneration phenotypes by Santos et al[89] and di Domenico et al[90]. In Santos et al’s research, astrocytes were activated and became inflammatory, and were co-cultured with DA neurons[89], while Domenico et al[90] co-cultured PD-patient-derived astrocytes with normal DA neurons. Normal DA neurons were induced to display apoptosis and multiple system dysfunction after co-culture with dysfunctional astrocytes[89,90]. These compelling findings emphasized that astrocytes may substantially participate in PD pathogenesis (Table 2).

DA neurons modulate several brain functions such as motor control, reward behavior, and cognition[99]. Recapitulation of the in vivo developmental profile of a specific cell type provides a powerful strategy for manipulating cell-fate choice during the process of human iPSC differentiation. In vitro generation of functional DA neurons is critical in pluripotent cell biology for both experimental and clinical applications. The neural stem/progenitor cell (NSPC) strategy and the floor-plate cell strategy are two useful protocols for generating DA neurons. The NSPC strategy is widely used in neuronal differentiation, in which NSPCs are isolated from rosettes[100,101] or induced by defined medium with many supplements[102]. The floor-plate strategy was proposed by Hynes et al and is based on the fact that the floor plate is a critical signaling center during neural development located along the ventral midline of the embryo[103]. Lorens et al[100] derived the floor-plate cells from human embryonic stem cells (hESCs) using a modified protocol by dual Smad inhibition. The floor-plate cells are predisposed to differentiate into mature ventral midbrain DA neurons with a higher efficiency than rosette-based neurons[101]. The floor-plate-derived midbrain DA neurons are able to control dopamine release and selective dopamine reuptake, as well as other features such as synaptic transmission. Importantly, PD patient iPSC-derived DA neurons show cellular PD phenotypes such as increased accumulation of mitochondrial ROS and cytoplasmic α-synuclein, mitochondrial DNA damage, shortened neurites, and impaired autophagy[80,104,105].

Aging is a crucial risk factor for all late-onset neurodegenerative diseases. One important challenge found in iPSC-based PD models is to appropriately reproduce the late-onset characteristics. Reprogramming somatic cells to iPSCs resets their identity back to an embryonic state. The ability of iPSCs to undergo unlimited division while maintaining genomic integrity provides a way to overcome the senescence barrier. Even if these iPSCs differentiate into mature DA neurons, it still needs a lot of time to culture for mimicking aging DA neurons, presenting a significant hurdle for modeling PD. Thus, how to induce aging in iPSC-derived DA neurons is an important issue. Justine et al[72] compared young and aged human fibroblasts; they found a predominant difference in progerin level between adolescent and aged cells. They further demonstrated that overexpression of progerin in PD iPSC-derived DA neurons in vitro or in vivo promoted cell aging for modeling late-onset PD features such as pronounced dendrite degeneration, progressive loss of tyrosine hydroxylase expression, and enlarged mitochondria or Lewy body inclusions[72]. Thus, progerin expression could accelerate aging in iPSC-derived DA neurons for inducing PD pathogenic phenotypes, and introducing progerin expression could be a useful strategy to manifest disease phenotypes in iPSC-based late-onset PD models. However, it should be noted that neurons are fragile for undertaking the delivery of exogenous genes. The low efficiency of transfection in neurons remains a big challenge for widespread and convenient application of progerin expression to model iPSC-based late-onset neurodegenerative diseases.

Previous studies have indicated that chronic treatment with anticancer drug hydroxyurea (HU) could induce cellular senescence in human fibroblasts[106] and mouse neural stem cells[107] via induction of genes related to DNA damage and repair, mitochondrial dysfunction, and ROS increase. In our recent study[108], we found that HU induced disease phenotypes of sporadic PD-patient-specific iPSC-derived DA neurons. HU treatment significantly reduced neurite outgrowth, expression of p-Akt and its downstream targets (p-4EBP1 and p-ULK1), as well as increased the level of cleaved caspase 3 in iPSC-derived DA neurons from sporadic PD patients. Transcriptome analysis and Western blotting indicate that HU alters the expression of genes and proteins related to the ER stress pathway in healthy iPSC-derived DA neurons. It reveals that ER stress might contribute to HU-induced aging in iPSC-derived DA neurons from sporadic PD patients. Our study also found that the midbrain characteristics decline after iPSC-derived DA neurons are treated with HU, which is similar to the characterization of PD. Thus, increasing chemically induced ER stress could be an alternative approach to accelerating aging of iPSC-derived DA neurons from PD patients for manifestation of PD cellular phenotypes.

Due to the ability of human iPSCs to differentiate into human DA neurons and astrocytes, human iPSCs are a promising model for studying the pathogenesis of PD. Compared with neurotoxin-induced injuries, human iPSC-derived DA neurons from sporadic or familiar PD patients could give help us to understand the progressive changes of PD neuronal phenotypes as culture time increases[50,109]. Through this PD iPSCs model, we can verify the possible mechanisms of pathogenesis suggested in previous studies in other cell or animal models. Most importantly, studying these PD-iPSC-derived DA neurons could explain how the clinical degenerative features of human DA neurons occur[110,111]. At least the changes in some human familial- PD-iPSC-derived neurons can represent the middle or final stage of PD because this iPSC-derived DA neuronal death occurs with α-synuclein accumulation, which is consistent with the observation in PD patients. Compared with familial PD, the etiology of sporadic PD is still a major challenge because of the multifactorial etiopathogenesis of sporadic PD. Since sporadic PD is complicated, the changes in neuronal phenotype cannot reflect the pathogenic alteration in the whole brain or other systems of sporadic PD patients[112,113].

Current PD therapies help patients relieve motor symptoms, but do not effectively prevent, slow or halt the progression of PD, particularly in the loss of DA neurons[114]. Neurotoxin-based neurons or animal models are commonly used for anti-PD drug screening. Many drugs based on these artificial models have been developed but do not significantly prevent PD progression[115]. One reasonable explanation is that these neurotoxins that usually cause strong injuries in DA neurons cannot mimic the progressive death of human DA neurons in PD. In addition, DA neurons from animals are distinct from human DA neurons. In PD-iPSC-derived DA neurons, the typical PD features such as accumulation of α-synuclein, progressive degeneration, and death could be observed. These robust and reproducible PD phenotypes are amenable to screening potential compounds. Thus, PD-iPSC-derived DA neurons are more suitable for screening anti-PD drugs than artificial models are.

The inspiring success in PD treatment was achieved through allograft of human fetal midbrain cell suspensions in 1980[116]. In addition, in MPTP-treated monkey brains, monkey ESC-derived neural progenitor cells differentiated into DA neurons and cells integrated well in the striatum, thereby PD-related motor symptoms improved[117]. Compared with ESCs, iPSCs have more potential in cell replacement therapies for PD because they can be generated from patients’ own cells and differentiate into DA neural progenitor cells that specifically develop into DA neurons[118]. iPSCs have the advantage of eliminating immune rejection concerns as they are obtained from the host. The generation of iPSCs from a patient’s own somatic cells would potentially allow for a plentiful source of cells for autotransplantation. In addition, using iPSCs rather than ESCs means that this treatment would be potentially available in some countries that ban the application of ESCs, including Italy, Ireland, and most African and South American countries[119,120]. For familial PD patients, the corrected iPSCs are also a reasonable source for the transplantation of normal DA neurons to reduce motor symptoms. Recently, scientists from Japan have started a clinical trial[121] (ClinicalTrials.gov NCT02452723) to treat PD with human iPSCs. All these suggest that the transplant of human iPSCs-derived DA neurons will be a promising therapeutic strategy and customized treatment is practical due to individual differences.