Possiblerolesofepigeneticsinstemcell therapyforParkinson ... · As indicated above, a genetic predisposition to PD involves alterations in certain genes, such as PARK (1–15) including,

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Possible roles of epigenetics in stem celltherapy for Parkinson’s diseaseCassandra Thompson‡ ,1,2, Paulina Otero‡ ,1,2, Bhairavi Srinageshwar1,2,3, Robert B Petersen3,

Gary L Dunbar1,2,3,4,5 & Julien Rossignol*,1,2,3

1Field Neurosciences Institute laboratory for Restorative Neurology, Central Michigan University, Mt. Pleasant, MI 48859, USA2Program in Neuroscience, Central Michigan University, Mt. Pleasant, MI 48859, USA3College of Medicine, Central Michigan University, Mt. Pleasant, MI 48859, USA4Department of Psychology, Central Michigan University, Mt. Pleasant, MI 48859, USA5Field Neurosciences Institute, St. Mary’s of Michigan, Saginaw, MI 48604, USA*Author for correspondence: Tel. +1 989 774 3405; [email protected]‡Authors contributed equally

Parkinson’s disease (PD) is a neurodegenerative disease with loss of dopaminergic neurons. PD has ge-netic and epigenetic influences that determine specific changes in the brain. Epigenetic changes result indefective methylation of genes leading to differential gene-expression causing PD. This review providesan overview of stem cell transplantations as potential therapies for PD, with a focus on the epigeneticchanges, prior or following transplantation. To date, no reports have addressed epigenetic alterationsfollowing stem cell transplantation into the PD brain. Given the potential for affecting the efficacy ofstem cell therapy, increased attention needs to be given to the epigenetic processes that occur duringstem cell culture and transplantation to maximize the therapeutic potential of stem cells to PD.

Graphical abstract:

Trans-differentiation

DA neurons

DA neurons

DA neurons

Differentiation

Skin derived inducedpluripotent stem cells

Bone marrow derivedmesenchymal stem cells

Fetus derivedembryonic stem cells

Dopaminergic neuronal (DA) lineage

Differentiation

Long-term epigenetic impact on the PD brainfollowing transplantation of the DA neurons

Transplantation of the differentiated DAneurons into PD brain

Site of DA injection

?Epigenetic signatureEpigenetic signature

First draft submitted: 14 November 2019; Accepted for publication: 25 February 2020; Published online:12 May 2020

Keywords: epigenetics • methylation • Parkinson’s disease • stem cells • stem cell therapy • stem cell transplantations

Epigenomics (Epub ahead of print) ISSN 1750-191110.2217/epi-2019-0347 C© 2020 Future Medicine Ltd

https://orcid.org/0000-0002-8662-0547

Review Thompson, Otero, Srinageshwar, Petersen, Dunbar & Rossignol

Parkinson’s disease (PD) is a neurodegenerative disorder that results from the degeneration of the dopaminergic(DAergic) neurons in the substantia nigra pars compacta [1]. DAergic neurons are found in the mid-brain andproduce the neurotransmitter, dopamine (DA). DAergic neurons act to maintain voluntary movement and specificbehaviors such as mood, addiction and stress [2]. Loss of DAergic neurons leads to reduced production of DAresulting in the characteristic PD symptoms. Given that brain abnormalities increase with age in PD patients andknowing that the symptoms of PD usually manifest at the age of 60 years or older, age is the major risk factor fordeveloping PD [1]. The disease involves both motor and nonmotor symptoms that affect the quality of life. Thenonmotor symptoms of PD include dementia, insomnia, day-time sleepiness, hallucinations, excessive sweating,weight loss and impulse control disorders [3,4].

PD dementia and Lewy body dementia (LBD) have similar presentations and prognosis. Patients with LBD showmotor deficits, hallucinations and cognitive impairment similar to PD patients. However, the progression of thesesymptoms is different in PD and LBD. Patients with PD will first exhibit abnormal motor symptoms followed bycognitive decline. Patients are diagnosed with PD if they develop dementia more than 12 months after the onset ofmotor symptoms. In contrast, patients with LBD present with cognitive decline followed by motor symptoms [5].

Some of the motor symptoms of PD include gait problems, bradykinesia, akinesia and rigidity [6]. PD is associatedwith both genetic and environmental factors. Familial PD is associated with mutations in the PARK gene family,which includes the SNCA. In addition, LRRK 2 and PINK1 genes are associated with both familial and sporadicPD. Additionally, constant and frequent exposure to tobacco and pesticides are two of the environmental factorsthat may lead to sporadic PD [7]; interestingly, both have been shown to result in epigenetic changes [8].

One of the potential therapies for treating the symptoms of PD is to increase the amount of DA in the brain.However, DA does not cross the blood–brain barrier and treatment has relied on precursors of DA such as levodopa(L-dopa). Although L-dopa can provide symptomatic relief, individuals using L-dopa often experience significantside effects [9]. Another approach for treating PD is boosting the level of GDNF in PD patients. Although GDNFimproves the survival of DAergic neurons, clinical trials attempting to increase GDNF levels in the brains of PDpatients have not been successful to date [10–12]. Therefore, researchers have been looking for alternative strategies toprovide long-term treatment for PD, including transplantation of different types of stem cells, such as mesenchymalstem cells (MSCs), neural stem cells (NSCs), induced pluripotent stem cells (iPSCs) and embryonic stem cells(ESCs) into the PD patient’s brain.

Epigenetic changes following stem cell transplantation may be affected by a variety of factors. Genetic andepigenetic changes that occur when NSCs are cultured and passaged prior to transplantations can affect thetherapeutic efficacy of these cells following transplantation. Loss of expression of certain genes during in vitro cellpassaging poses significant concerns about whether the transplanted NSC line will be able to differentiate intoDAergic neurons in vivo. Treating NSCs with antioxidants and vitamins, specifically Vitamin C, has resulted ina significant increase in the NURR1 and FOXA2 gene expressions compared to untreated NSCs. Expression wasmaintained both before and after differentiation and transplantation. The NSCs generated were able to alleviatePD behavioral symptoms in a rat model by modulating epigenetic changes [13].

As indicated above, a genetic predisposition to PD involves alterations in certain genes, such as PARK (1–15)including, LRRK 2 (PARK8), and SNCA (PARK1/4). In addition to the genetic alterations found in the PD brain,there are also specific epigenetic alterations that regulate the expression of certain genes in the brain of PD patients.The following sections will review the current knowledge of the epigenetics associated with PD and stem-cell-basedtherapy. Given that the objective of stem cell therapy for PD is to differentiate/trans-differentiate stem cells into aneuronal-like lineage, specifically DAergic neurons, the main population of cells that degenerate in PD [14], theseepigenetic events have particular import for application of this therapeutic strategy.

Epigenetics & PDEpigenetics plays an important role in the molecular basis of cellular function, relying on chemical modificationsthat alter the activity of DNA without changing its sequence. Epigenetic changes result from a variety of factorsthat can be intrinsic or extrinsic to the organism. Examples include hormones, metabolism, diet, temperature, light,drugs, air pollution, stress, etc. Epigenetic changes alter development, aging, adaptation, and can affect the courseof neurodegenerative diseases, such as PD [15].

10.2217/epi-2019-0347 Epigenomics (Epub ahead of print) future science group

Possible roles of epigenetics in stem cell therapy for Parkinson’s disease Review

The specific mechanisms underlying the onset and progression of PD have yet to be elucidated. However, anumber of genetic and cellular mechanisms have been implicated, as well as epigenetic modifications [8]. Theepigenetic modifications include DNA methylation, histone modifications, altered expression of miRNAs and longnoncoding RNAs (lncRNAs) that regulate expression of genes that also affect stem cells and their development.Thus, a thorough understanding of the epigenetic mechanisms mediating stem cell function and differentiationcould enhance the efficacy of stem-cell-based treatments for PD [16] as detailed in the examples below.

DNA methylationMethylation of DNA is one of the most extensively studied epigenetic mechanisms underlying many diseases. Theaddition of a methyl group to DNA generally results in inhibition of transcription. Although this can occur atmany genomic sites, the most prevalent site of methylation is in CpG islands associated with promoter regions.In PD patients, these regions become dysregulated, resulting from either excess or reduced DNA methylation [17],which in turn results in under or overexpression of specific genes.

One of the first mutations linked to the development of familial PD is a missense mutation, p.Ala53Thr, in SNCAgene. Point mutations, gene duplication, copy number variants and overexpression of SNCA have all been associatedwith the formation of Lewy bodies, a neuropathological feature of PD [18]. In addition to mutations in SNCA,altered DNA methylation in introns has also been linked to the development of PD due to the dysregulation ofSNCA gene expression in the substantia nigra and cortex [19]. Jowaed and colleagues studied epigenetic regulation ofthe SNCA gene. They found that DNA methylation in intron 1 of the SNCA gene decreased expression of SNCA.As expected, inhibiting methylation resulted in increased SNCA gene expression. These findings are supportedby the observations of fewer methylated CpG sites in the DNA samples of PD patients compared with healthyindividuals. Additionally, it is likely that the methylation state of intron 1 in SNCA gene may be affected byenvironmental factors that generate epigenetic changes providing an explanation for idiopathic PD [20].

Other genes, such as PARK16, NMB (GPNMB) and STX1B, GBA are also associated with the development ofPD and exhibit an abnormal methylation status [21,22]. Recently, haploinsufficiency of GBA was shown to acceleratealpha synuclein pathology [23].

Histone modificationsHistone modification, another type of epigenetic change, alters the assembly of chromatin structures modifyinggene expression. The modified histone proteins undergo conformational changes that can result in gene silenc-ing or gene activation. These modifications include acetylation, phosphorylation, methylation, ubiquitylation orSUMOylation [24].

The most common modifications to histones are methylation and acetylation. Histone methylation occurs oneither the arginine or lysine residues in the N-terminal of H4 histone altering transcriptional activity. Histonemethyl-transferase catalyzes this reaction. Histone acetylation occurs at the ε-amino of lysine residues in the N-terminal tails of the histones H2A, H2B, H3 and H4, causing a transcriptional change. This transfer is catalyzedby histone acetyl-transferases and reversed by histone deacetylase enzymes [25].

Nicholas and colleagues demonstrated histone modifications in an acute murine model of levodopa-induceddyskinesia following dopamine depletion and levodopa treatment [26]. The specific histone change was a reductionin H3 acetylation and trimethylation and deacetylation of histone H4.

lncRNAsAnother major epigenetic mechanism used to regulate gene expression is provided through RNA-based mechanism.lncRNAs nonprotein coding transcripts can attach to genes and alter their expression [27]. Furthermore, studies havefound that lncRNAs play an important role in regulating synaptic plasticity, cognitive function and memory [28].

SNCA is regulated by lncRNA resulting in increased expression of α-synuclein α-SYN. α-SYN contributes toDAergic neuronal differentiation and survival, as well as regulation of TH activity in the brain, TH catalyzesthe production of L-dopa from tyrosine, a DAergic precursor that is critical for preventing the events leading toPD [29]. The expression of PINK1, another gene associated with PD, may be regulated by a lncRNA. Many of thelncRNAs that are dysregulated in PD modulate expression of PD-related genes [30]. Marki and colleague showedthat polymorphisms in lncRNA genes such as PINK1, UCHL1-AS, BCYRN1, SOX2-OT, ANRIL and HAR1A werehighly associated with PD patients in Hungarian populations [31]. Upregulation of SOX2-OT and 1810014B01RiklncRNA genes are associated with PD [28].

future science group 10.2217/epi-2019-0347


Another RNA-based mechanism for regulating for gene expression involves (miRNA; containing around 22nucleotides), miRNAs are noncoding RNAs that suppress protein production through degradation of mRNA or byinhibition of mRNA translation. These miRNAs control expression of their target gene post-transcriptionally andare used to control a wide variety of physiological and pathological functions. Several miRNAs regulate expressionof α-SYN. Doxakis found that overexpression of miRNA7 and miRNA153 reduced α-SYN levels [32]. Leggioand colleagues showed that miRNAs participates in a negative feedback loop with PITX3 gene, which regulatesdifferentiation and activity of DAergic neurons [27]. Moreover, miRNAs have been shown to mediate oxidativestress, one of the main causes of PD. The miR-124 agomir increases the density of TH+ neurons and decreases theupregulation of mRNA and protein levels that resulted in a decrease in apoptosis. Moreover, a number of large-scalestudies of brain tissue samples have detected alterations in downstream targets of dysregulated miRNAs that areassociated with PD. Single miRNAs have been shown to mediate the expression of hundreds of genes. Targetingthese altered miRNAs has been shown to improve functioning in PD models [33].

Following the elucidation of the epigenetic mechanisms associated with the development of PD, expression ofsome important proteins and factors have been found to be altered by such epigenetic changes, including:

• NURR1 is a member of nuclear receptor subfamily 4 that is essential for the proper formation and maintenanceof DAergic neurons from their precursors, the mesodiencephalic dopaminergic neurons. NURR1 is also crucialfor the migration and survival of dopaminergic neurons. Additionally, NURR1 may also be involved in regulatingthe expression of some of the enzymes and transporters, such as TH and DAT), which play roles in the productionand storage of DA [34].

• PITX3 is expressed by a subpopulation of DAergic neurons presented specifically in the substantia nigra andis required for DA neuronal differentiation, with its expression being associated with the age of onset ofPD [35]. Synergistic expression of NURR1 and pituitary homeobox 3 is necessary for ESCs to differentiate intodopaminergic neurons in the mid brain, which are the neurons responsible for the production of TH and DATin the brain [15].

• TH is an enzyme that is present in mature DAergic neurons and catalyzes the production of L-DOPA, themetabolic precursor of DA [36].

• Shh, a secreted glycoprotein, helps with the development of DAergic neurons in the neuroepithelial floorplate from the midbrain and hindbrain border. Shh also appears to increase TH gene expression in cells withdopaminergic phenotypes. Additionally, Shh increases resistance of mesencephalic DAergic neurons to MPTPand 6-OHDA insult to the brain [37].

• FGF8 and FGF2 are involved in the migration, differentiation and survival of DAergic neurons to establish themidbrain and hindbrain border [38,39].

• BDNF and GDNF promote the growth and survival of DAergic neurons. BDNF confers neurotrophic andneuroprotective effects as well as increasing dopamine reuptake and TH activity. Additionally, BDNF is thoughtto have autocrine, paracrine and retrograde effects on DAergic neurons. Similarly, GDNF enhances the survivalof DAergic neurons and increases high affinity dopamine uptake, but is more effective than BDNF [40].

• Retinoic acid, a by-product of vitamin A, determines the maturation level of DAergic neurons from its precur-sor [41].

• ASCL1 is transcription factor that activates transcription of genes leading to differentiation of DAergic neurons.Additionally, ASCL1 is also involved in the initiation of oligodendrogenesis and myelination in the postnatalcortex [42].

• LMX1a and LMX1b are also involved in the growth and maintenance of dopamine-producing neurons duringembryogenesis. Downregulation of these genes results in impaired mitochondrial function and formation ofα-SYN aggregates, thereby leading to the loss of DAergic neurons [43].

Epigenetic variations are now considered a major focus for understanding the changes that occur in the brain ofPD patients. Thus, defining the epigenetic changes that occur after transplantation of stem cells in PD therapy alsomerits study.

Stem cell transplantation in the PD brainDifferent types of stem cells have been utilized for treatment of PD. In vitro work with mesenchymal stem cells(MSCs), induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) has led to transplantation studies



in both animal models of PD and in humans [44–48]. Transplantation studies provide further insight into whetherand how the stem cells arrive at affected brain locations, whether and how each of the different types of stem cellssuccessfully differentiate into a DAergic neuronal lineage and whether the differentiated DAergic cells can integrateinto the brain circuit and reduce the behavioral symptoms of PD. To date, the epigenetic status of the transplantedstem cells are rarely assessed.

Dezawa and colleagues described induction of neuronal cells from bone marrow (BM)-derived stromal cellsusing trophic factors and gene transfer. These cells were then transplanted into animal models of neurodegenerativediseases such as PD. The study showed that the cells were able to successfully integrate into the neuronal circuitand provide therapeutic effects as evidenced by improved function [49].

Another study by Funk and Alexanian described a method for generating mature neural-like cells from BM-derived MSCs (BM-MSCs) by exposing them to epigenetic modifiers. This changed their innate characteristicsand produced cells in a primitive pluripotent-like stage. Next, these pluripotent-like cells were exposed to anNSC environment, which resulted in their differentiation into mature neural-like cells. The re-programmed cellswere then analyzed, and these cells were found to release neurotrophic factors, such as NGF and BDNF, whichhave shown potential for treatment based on their promoting the survival and growth of neurons in brain underneurodegenerative conditions, including PD [50].

ESCs in PDESCs have been studied extensively as a potential therapy for PD. An early study by Bjorklund and colleaguesreported on the effects of ESC transplantation following lesions of the medial forebrain in female Sprague Dawley(SD) rats. The ESCs were derived from mouse blastocysts and injected into the right striatum of the PD rat.Following the ESCs injection, PET and MRI scans showed that ESCs readily develop along a DAergic neuronallineage within the brain, exhibiting changes in the structure of the striatum following transplantation. In addition,behavioral improvement was also noted, indicating that these neurons successfully restored function in thesemodels [51].

In order to assess the importance of gene alterations, Brederlau and colleagues conducted a study investigatingthe effect of predifferentiation time of human ESCs grafted into a female SD rats of PD. The ESC from humanorigin (hESC) cells were predifferentiated for 16, 20 and 23 days. SD rats were lesioned in the right nigra-striatalpathway and then received human embryonic stem cells (hESC) injections in anterior, lateral and ventral locationsof the striatum. Grafted predifferentiated cells did not express DA phenotypes and no motor improvement wasobserved. In contrast, the cells that were allowed to predifferentiate for 16 days prior to grafting exhibited tumorgrowth in vivo, but those that predifferentiated for approximately 20 days or more did not exhibit this adverseeffect [52]. This confirms the importance of the differentiation state of transplanted neurons and the need forcharacterizing epigenetic markers that indicate a propensity for cancer development or the ability to differentiateinto DA producing cells.

Clinical trials for human transplantation of embryonic DAergic neurons have provided mixed results. In an earlydouble-blind study, 40 randomly assigned PD patients with severe symptoms were either surgically injected withembryonic DAergic neurons bilaterally into the putamen or underwent a sham surgery that entailed drilling intothe skull without disruption of the dura. The results of this trial indicated that the transplanted cells provided morerelief for younger individuals with PD as opposed to older individuals [53].

However, hESC transplantation has provoked considerable controversy surrounding the ethical use of fetal tissueas well as other issues, such as transplant-induced dyskinesia, that has dampened the enthusiasm for pursuingthis line of research [54]. Nonetheless, hESC transplantation has significant therapeutic potential for treatingneurodegenerative diseases, like PD, and outcomes from this type of therapy could be improved if further insightsinto epigenetic alterations were understood well enough to optimize their effectiveness.

iPSCs in PDiPSCs for treatment of PD are an alternative to hESC transplantation, where the patient’s own cells are differentiatedinto DAergic neurons. Ethical concerns have been raised about the potential for undesired genomic alteration viaiPSC transplantations that may result in cancer. However, more recent research indicates that the reprogrammingprocess for iPSCs is not excessively mutagenic, suggesting that iPSCs can be used safely for stem-cell-basedtherapy [55]. These cells are usually derived from either skin or blood and are re-programmed to a state similar to



that of ESCs. These iPSCs can be differentiated into a variety of lineages, including motor neurons, gametes andblood cells [56–58].

Transplantation of iPSCs have been performed in cynomolgus monkey (CM) models of PD that were generatedby treating CMs with MPTP. The efficacy of the iPSCs treatment was evaluated following transplantation of thecells into the putamen. The iPSCs were derived from MF25-04 nonhuman primate models and trans-differentiatedinto a DAergic neuronal lineage prior to transplantation. The graft resulted in reduced PD symptoms followingtransplantation into CM models. Histological analysis showed that there was increased microglial density, withouta concomitant increase in the immune response within the putamen [59].

Further primate studies indicate significant epigenetic effects on transplanted cells that subsequently differen-tiate into neuronal-like cells. Kikuchi and colleagues analyzed genetic factors affected by iPSCs following theirtransplantations into CM models. Lines from PD patients and healthy individuals have been established. Thesecell lines have been analyzed for FOXA2, TUJ1, NURR1, and multiple tumorigenic markers in in vitro. Followinganalysis, the cells were transplanted into the putamen of MPTP PD monkeys. The study showed that irrespectiveof the source of iPSCs (PD patient vs healthy individuals), the iPSCs were able to differentiate into DAergicneurons in vivo and the PD monkeys recovered from motor deficits compared with controls. Post-transplantation,histological analysis revealed that the characteristics and the morphology of the TH+ cells were similar to that ofthe graft recipient’s cells, further showing that these cells were able to produce DAT in the mid-brain as well asGIRK2, which is produced by the DAergic neurons in the nigrostriatal pathway. Following this, the PD monkeyswere randomly divided into two groups based on whether they had excellent or poor TH+ cell re-innervation.The study also found that the PD monkeys that had excellent innervation showed upregulation of 11 specificgenes. Interestingly, the most prominent of these genes is Dlk1, which was previously shown to have a role inmid-brain facilitating migration of DAergic neurons, thereby improving the innervation of TH+ cells. This studydemonstrates the efficacy of iPSC transplantations and the significance of the epigenetic changes that occur in themleading to upregulation of genes specific to DAergic neuron migration, location and dopamine release [60].

Reprogrammed iPSCs from fibroblast also effectively integrate into the embryonic mouse brain. Wernig andcolleagues transplanted fibroblast-derived iPSCs into the brains of embryonic mice in utero and reported thatthey differentiated into a variety of neuronal and glial lineages. They then transplanted iPSCs into rats that weregiven the neurotoxin, 6-OHDA, which kills DAergic neurons, causes of symptoms of PD. When iPSCs that weredifferentiated toward a DAergic neuronal lineage were grafted into the striatum of these 6-OHDA-treated rats,the researchers observed the presence of more TH+ cells in their brain. However, they also noted the formation oftumors occurred frequently in the rats given iPSC transplantations, underscoring serious safety concerns that needto be addressed before grafting of iPSCs can be considered for clinical use [61]. Clearly, the epigenetic alterationsof the iPSCs need to be studied thoroughly before any hope of them being used for clinical purposes can beseriously entertained. As a first step, Roessler and colleagues analyzed the epigenetic signatures of iPSC-derivedDAergic neurons and confirmed that these markers are important for assessing the long-term functionality of thetransplanted cells [35].

MSCs in PDMSCs are adult multipotent stem cells that are used extensively in stem cell therapy studies for a variety ofneurodegenerative diseases, including PD. MSCs can differentiate into a variety of lineages, including neuronal,adipocytes, chondrocytes and osteoblasts, as well as ectodermal neurocyte [62]. Transplantation of MSCs into thebrains of rodents that are manipulated to model PD has often yielded improvements in motor function andincreased TH and DA levels, demonstrating the promise of this approach. For example, MSC transplantation intothe MPTP-treated mice showed reduced motor dysfunction, as well as trans-differentiation of BM-MSCs intoa DAergic neuronal lineage. In addition, TH+ cells in the substantia nigra were increased, relative to what wasobserved in control mice [45].

PD research is predominantly conducted in rat models. However, some studies have investigated stem-cell-basedtherapy in murine PD models. BM-MSCs were extracted from male C57BL/6 mouse that were subsequently,treated with MPTP, and followed by implantation of the MSCs into the right striatum. As with the rat model,the mice showed improved rotational behaviors when subjected to motor coordination tasks following MSCtransplantation [63].

Work in our lab indicates that the efficacy of transplanting BM-MSCs is dependent on the type of dopaminergicinduction method and the number of passages the MSCs undergo prior to transplantation [39,64]. Shall and



colleagues analyzed changes in mRNA expression in BM-MSCs isolated from male SD rats at four or 40 passagesfollowing either direct or indirect induction of dopaminergic differentiation. The MSCs differentiated into aDAergic lineage (expressing TH, DAT and MAP2 genes) following either direct or indirect induction, althoughdirect dopaminergic induction of early passaged cells (P4) was more efficient than indirect dopaminergic inductionof late passaged cells [64].

Similarly, Welchko and colleagues genetically modified BM-MSCs using a virus carrying three different genes:ASCL1, LMX1A and NURR1, some of the factors important for inducing differentiation of DAergic neurons. Adetailed description of each of these genes is given above. The results from this study showed that the BM-MSCswere successfully transduced with the virus and that the cells expressed TH and DAT [39]. Transplantation of thesecells into 6-OHDA-treated rats reduced the motor deficits found in this PD model.

The studies from our lab indicate that BM-MSCs can be steered into a DAergic lineage by modifying themeither by incubating the stem cells in specific media or by introducing genes that drive differentiation. Thoughthe outcome of these studies was the production of DAergic neurons, the epigenetic makeup of the cells may bedifferent following each type of DAergic induction. Future studies are required to define the epigenetic changesoccurring under each of the conditions used.

Overall, these studies show promising therapeutic results for stem-cell-based transplantation in the treatmentof PD. However, the genetic and epigenetic changes that occur in the PD brain can readily lead to increased ordecreased expression of genes that contribute to the disease symptoms and conditions. As discussed above epigeneticchanges are associated with the development of PD, but how epigenetics impact transplantation for PD is a majorfocus of future research.

ConclusionEpigenetic mechanisms regulate genes resulting in the increase/decrease of protein levels. The epigeneticstate of the different types of stem cells and/or DAergic neuronal lineage of cells obtained by the trans-differentiation/differentiation of stem cells prior to transplantation into the PD brain may be different, affectingthe outcome of the treatment. This may, in part, help to explain why the stem cell therapies for PD exhibit varyinglevels of efficacy. Results could be affected by unknown epigenetic factors that impact the progression of PD or theviability of the transplanted cells. As studies involving iPSCs, hESCs and MSCs indicate the emerging impact ofgenomic properties on treatment efficacy, more attention must be paid to stem cell epigenetics.

Importantly, more studies involving the epigenetic changes before and after of in vivo transplantation in PDare needed, especially to evaluate specific stem cell epigenetic markers. The critical data needed are the similaritiesand differences between the epigenetic signatures of the host DAergic neurons and the DAergic neurons that aretransplanted following trans-differentiation of various types of stem cells. This important variable needs to becharacterized in order to determine which approach to stem cell therapy will produce the cells that are most likelyto eliminate the symptoms of PD.

Future perspectiveFuture transplantation studies should incorporate in their analysis information about the sources of the transplantedcells for PD. The potential epigenetic characteristics of these cell populations might play a critical role in renderingtheir therapeutic effects. It would also be beneficial for research in the future to record the different epigeneticchanges that stem cells undergo during differentiation and transdifferentiating prior to transplantation. This mightlead in choosing a specific stem cell type having epigenetic changes that may provide the most effective stem cellbased therapy for PD.

Financial & competing interest disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or finan-

cial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria,

stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.



Executive summary

• Parkinson’s disease (PD) is a neurodegenerative disorder that results from the degeneration of the dopaminergicneurons in the substantia nigra pars compacta.

• The objective of stem cell therapy for PD is to differentiate/trans-differentiate stem cells into a neuronal-likelineage, specifically dopaminergic neurons, the main population of cells that degenerate in PD, the differentepigenetic events have particular import for application of this therapeutic strategy.

Epigenetics & PD• Epigenetic changes alter development, aging, adaptation, and can affect the course of neurodegenerative

diseases, such as PD.• There are many genes that exhibit mutations leading to PD with abnormal methylation status.• There are various long noncoding RNAs and miRNA that are important for gene regulations are altered in PD.• Some of the important proteins and factors that are altered in PD include Nurr1, pituitary homeobox 3, tyrosine

hydroxylase, sonic hedgehog, FGF2, LIM homeobox transcription factor, brain-derived neurotrophic factor andGDNF.

Stem cell transplantation in PD brain• Stem cell transplantation of differentiated/transdifferentiated stem cells into dopamine neurons and cell

re-programming (induced pluripotent stem cells) are some of the strategies used as a potential therapy for PD.• Transplantation of dopaminergic neurons obtained from various stem cell sources may exhibit different

epigenetic signature that could be same/different than the host dopaminergic neurons leading to a differentialtherapy for PD.

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