Induced pluripotent stem cell models of lysosomal storage ... · Induced pluripotent stem cell...

REVIEW SPECIAL COLLECTION NEURODEGENERATION

Induced pluripotent stem cell models of lysosomal storagedisordersDaniel K Borger Benjamin McMahon Tamanna Roshan Lal Jenny Serra-Vinardell Elma Aflaki andEllen Sidransky

ABSTRACTInduced pluripotent stem cells (iPSCs) have provided newopportunities to explore the cell biology and pathophysiology ofhuman diseases and the lysosomal storage disorder researchcommunity has been quick to adopt this technology Patient-derivediPSC models have been generated for a number of lysosomalstorage disorders includingGaucher disease Pompe disease Fabrydisease metachromatic leukodystrophy the neuronal ceroidlipofuscinoses Niemann-Pick types A and C1 and several of themucopolysaccharidoses Here we review the strategies employed forreprogramming and differentiation as well as insights into diseaseetiology gleaned from the currently available models Examples areprovided to illustrate how iPSC-derived models can be employed todevelop new therapeutic strategies for these disorders We alsodiscuss how models of these rare diseases could contribute to anenhanced understanding of more common neurodegenerativedisorders such as Parkinsonrsquos disease and discuss key challengesand opportunities in this area of research

KEY WORDS Gaucher disease IPSC models Lysosomal enzymesLysosomal storage disorders Neurodegeneration

IntroductionThere are over 50 types of lysosomal storage disease a class ofinherited metabolic disease caused by the absence or deficiency of alysosomal protein Most lysosomal storage diseases (LSDs) resultfrom mutations in metabolic enzymes that are active in thelysosome although a handful are caused by defects in lysosomaltransport or vesicular trafficking (Boustany 2013 Feng et al 2002Ward et al 2000) Regardless of the function of the mutated geneLSDs are universally characterized by the intracellular accumulationof undigested storage material although the composition of thismaterial varies between the LSDs The deficiency of functionalprotein and the subsequent accumulation of storage material canimpair normal lysosomal function in affected cells and interrupt adiverse array of cellular activities (Ballabio and Gieselmann 2009)The phenotypic consequences in patients are extremely variedranging from asymptomatic or sub-clinical manifestations tochronic visceral musculoskeletal or immunological disease to

lethal acute neuronopathic disease Even within a given LSD therecan be vast phenotypic heterogeneity with many LSDs exhibitingboth early- and late-onset forms (Boustany 2013)

Individual LSDs are extremely rare disorders but taken togetherthey are thought to affect up to 1 in 4000 live births (Al-Jasmi et al2013 Applegarth et al 2000 Meikle et al 1999 Pinto et al 2004Poorthuis et al 1999 Poupe tovaacute et al 2010) Furthermore due inpart to the multi-organ nature of many of these diseases andbecause of a continuing lack of truly effective treatments for manyLSDs these diseases are often characterized by high mortalityand morbidity (Stone and Sidransky 1999) Current therapies areextremely costly and are often lifelong treatments LSDs thereforeconstitute a significant burden on affected individuals and theirfamilies and on healthcare systems as a whole and LSDs have longbeen a major focus of rare disease research Moreover a growingappreciation of the role of lysosomal dysfunction in aging and age-related neurodegenerative disorders has led to a recent surge ofinterest in LSDs These factors have combined to help fuel theapplication of a number of emerging biotechnologies towards LSDresearch However there has been mixed success in generatingsuitable animal models of LSDs for research in this field (Farfel-Becker et al 2011 Lawson andMartin 2016 Pastores et al 2013)and hence investigators have directed efforts toward developingalternative disease models

One technology in particular ndash the development of inducedpluripotent stem cells (iPSCs) ndash has been broadly adapted byinvestigators researching the LSDs and human iPSC lines are alreadycontributing significantly to our understanding and treatment of theserare diseases Here we review the use of human iPSCs in LSDresearch highlighting the strategies that have been used to generateiPSCs and iPSC-derived cell models and to evaluate their relativesuccess in accurately phenocopying the human disease The benefitsof these cells in untangling disease etiology and developing noveltherapeutics are discussed as well as the limitations We also brieflyhighlight how the insights gleaned from studying LSDs using thesecellular models could contribute to a better understanding of morecommon neurodegenerative diseases

Induced pluripotent stem cells an overviewIn 2006 Shinya Yamanaka and his colleagues at Kyoto Universityreported that by forcing expression of four genes ndash OCT34 SOX2KLF4 and MYC (collectively known as OSKM) ndash via retroviraltransduction they were able to convert murine fibroblasts into fullypluripotent stem cells The profile and potency of these murineiPSCs were similar to those in embryonic stem cells (Takahashiand Yamanaka 2006) The following year three papers ndash one byYamanakarsquos group (Takahashi et al 2007) and one by GeorgeDaleyrsquos group (Park et al 2008a) both using the OSKM cocktailand a third by James Thomsonrsquos group (Yu et al 2007) usingOCT34 SOX2 NANOG and LIN28 (OSNL) ndash showed that theReceived 27 December 2016 Accepted 28 April 2017

Medical Genetics Branch National Human Genome Research Institute NationalInstitutes of Health Bethesda MD 20892 USA

Author for correspondence (sidransemailnihgov)

ES 0000-0002-3019-8500

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (httpcreativecommonsorglicensesby30) which permits unrestricted usedistribution and reproduction in any medium provided that the original work is properly attributed

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same basic technique used in mice could also be employed togenerate iPSCs from human somatic cells Since then numerousadvances have been made in identifying new factors that inducereprogramming which now include RNAs and small moleculesnew modes of introducing the necessary factors to cells and newcell types that can be reprogrammed (Table 1) These discoverieshave done much to inform our understanding of how stem cellsachieve and maintain pluripotency Recent work clearlydemonstrates how iPSC-derived cells are a remarkable tool forresearch of human diseases (see Box 1) These advantages havemade iPSC-derived cell models a natural choice for studies of theLSDs as discussed below

iPSC models of LSDsCells of the neuronal and hematopoietic lineages are the usualdifferentiation targets for iPSC-derived models of LSDs (28 of 39studies pursuing differentiation see Table 2) because these are thecells most often affected by these diseases (Figs 1 and 2) Althoughmurine iPSC lines have been derived from five mouse models ofLSDs (Kawagoe et al 2011 Meng et al 2010 Ogawa et al 2013)human iPSCs and iPSC-derived cell models which have beengenerated for at least 11 LSDs (Table 2) have become the focus ofthe field as they more closely mimic the human disease Asdiscussed below human iPSC models of LSDs are alreadycontributing to our understanding and treatment of these rarediseases

Gaucher diseaseGaucher disease (GD) is a recessive disorder caused by mutations inGBA1 resulting in a deficiency in a lysosomal hydrolase namedglucocerebrosidase (Mistry et al 2017) Usually this deficiencyleads to glycolipid accumulation in macrophages and manifests inhematological visceral and skeletal symptoms with a variabledegree of severity GD is classified into three types non-

neuronopathic (type 1) acute lethal neuronopathic (type 2) andchronic neuronopathic (type 3) and more severe mutations areassociated with neuronopathic manifestations Furthermorepopulation studies have established that individuals with GBA1mutations both carriers and affected individuals are at anincreased risk of developing Parkinsonrsquos disease an age-relatedneurodegenerative disorder as well as other Lewy body disorders(Nalls et al 2013 Sidransky et al 2009) The discovery of this linkbetween GBA1 and Parkinsonrsquos disease has played a large part in arecent explosion of GD research

However both seasoned GD researchers and those new to thefield have been limited by a dearth of effective models for studyingboth GD and GBA1-associated Parkinsonrsquos disease Mouse modelsfor GD have been developed but none faithfully recapitulate thefeatures of the disease seen in humans (Farfel-Becker et al 2011)Human cell lines have also been of limited use Fibroblasts frompatients with GD have long been used to study the disease but thesecells do not store the implicated glycolipids and do not showobvious signs of pathology (Saito and Rosenberg 1985) Applyingconduritol β-epoxide (CBE) an irreversible inhibitor ofglucocerebrosidase to a common human monocytic cell line suchas THP-1 or to SH-SY5Y cells (a human neuroblastoma cell lineused to model neurons) leads to glycolipid accumulation in thesecell lines and this approach has been used to provide in vitromodelsfor GD (Hein et al 2007 Prence et al 1996) However it isdifficult to generalize findings in immortalized cell lines to cellsin vivo The limited options for studying GD in the laboratory hasdirected attention toward iPSCs

As macrophages are the primary cell type affected in all GDtypes they are a natural choice for studies of this disease (Fig 2) Ithas generally been accepted that glycolipid-engorged macrophagesdrive the systemic inflammation commonly seen in GD indicatedby elevated inflammatory factors in the blood of affectedindividuals (Pandey and Grabowski 2013) Differentiating iPSCs

Table 1 Factors impacting the generation of iPSCs

Variable involved iniPSC generation Options Advantages Disadvantages References

Reprogrammingfactors (RFs)

Different cocktails (OSKM vs OSNL) Both cocktails effective OSKM+NANOG and LIN28 (sixfactor reprogramming) havehighest efficiency

Liao et al 2008

Type of vector Integrative retrovirus lentivirus Effective reprogramming Insertional mutagenesis canintroduce other genomic errorsresidual expression of RFs canimpede differentiation andcause genomic instability

Okita et al 2007 Papapetrouet al 2009 Ramos-Mejiaet al 2012 2010 Stadtfeldet al 2008

Integrative and polycistronic RFs(linked by self-cleaving peptideand flanked by loxP or transposonsites)

Reduces number ofinsertion events allowsexcision of RFs

Cre excision leaves long terminalrepeat can cause mutagenesis

Chang et al 2009 Papapetrouand Sadelain 2011 Woltjenet al 2009 Yusa et al 2009

Non-integrative adenovirus orSendai RNA virus

Avoids mutagenesisNo residual RFexpression

Low reprogramming efficiency Fusaki et al 2009 Kiskinis andEggan 2010 Miyoshi et al2011 Warren et al 2010

Donor cells Fibroblasts Relatively simple to obtainvia skin punch biopsyavailable via bio-repositories

Requires invasive skin biopsyclinical data often missing inbiorepositories

Blood Blood draw is minimallyinvasive

Other drugs can interfere Staerk et al 2010

Umbilical cord blood Can be reprogrammedwith only two RFs

Seldom available for raredisorders like LSDs

Meng et al 2012

Keratinocytes Easy to obtain can bereprogrammed quickly

Not often in repositories Aasen et al 2008 Tolar et al2011

OSNL OCT34 SOX2 NANOG and LIN28 OSKM OCT34 SOX2 KLF4 and MYC

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derived from patients with type 1 GD into macrophages Panickeret al (2014) found that these GD iPSC-derived macrophages secretegreatly elevated levels of pro-inflammatory factors includinginterleukin (IL)-10 IL-6 IL-1β and tumor necrosis factor(TNF)-α when challenged with lipopolysaccharide a commonpro-inflammatory stimulus GD iPSC-derived macrophages alsoexhibit elevated secretion of chitotriosidase an antifungal factorthat is often used as a biomarker for the severity of visceral GDsymptoms (Panicker et al 2014) This pro-inflammatory profile of

GD macrophages has been further explored using primary humanmacrophages (Aflaki et al 2016b) In addition GD iPSCs werereprogrammed to macrophages which exhibited deficientglucocerebrosidase activity increased glycolipid storage andimpaired translocation of glucocerebrosidase to the lysosomeComparing these cells with primary macrophages made frommonocytes isolated from the same patients a similar phenotypeincluding impaired chemotaxis and reactive oxygen speciesproduction (ROS) was observed demonstrating the ability of theiPSC-derived cells to phenocopy the primary cells (Aflaki et al2014) GD iPSCs have also been used to explore hematopoiesis inGD leading to the conclusion that glucocerebrosidase deficiencydirectly impairs hematopoietic development (Sgambato et al2015)

In order to study the neuropathology and the role of GBA1 inparkinsonism GD iPSCs have also been differentiated into neuronsThe first such study by Mazzulli et al (2011) utilized a fibroblastcell line derived from the cells of a 20-year-old male with GD tomake dopaminergic (DA) neurons Subsequently two other groupsalso differentiated GD iPSCs to neurons that stain positive fortyrosine hydroxylase (TH) a marker for DA neurons (Panickeret al 2012 Tiscornia et al 2013) In addition these studiesdemonstrated that cells from infants with type 2 GD could besuccessfully differentiated despite their profound deficiency ofglucocerebrosidase A more recent study showed that iPSC-derivedneurons from patients with GD exhibit abnormal lysosomal functionand altered lysosomal biogenesis (Awad et al 2015)

iPSCs neuronal precursor cells (NPCs) and neurons generatedfrom an infant with type 2 GD all have similar degrees ofglucocerebrosidase deficiency compared with the original patientfibroblasts (Sun et al 2015) These cells had increased levels ofboth glucosylsphingosine and glucosylceramide ndash the enzymesubstrates Functional studies utilizing whole-cell patch-clampingof the type 2 GD iPSC-derived neurons demonstrated excitationcharacteristics of neurons and interestingly these cells showedreductions in action potential amplitudes and sodium and potassiumcurrents The authors suggest that the abnormal electrophysiologicalproperties observed in these neurons provide new clues into thepathogenesis of the neuronopathic phenotype in Gaucher disease(Sun et al 2015)

In another study Aflaki et al (2016a) examined differentiatedneurons from iPSCs from patients with type 1 GD with and withoutParkinsonrsquos disease and from a patient with GD2 The neuronshad deficient glucocerebrosidase stored glucosylceramide andglucosylsphingosine and co-localization studies revealed greatlyreduced levels of lysosomal glucocerebrosidase in the DA neuronsindicating an appropriate Gaucher phenotype (Aflaki et al 2016a)Overall these studies indicate that iPSC-based models of Gaucherdisease successfully recapitulate hallmarks of this LSD

Pompe diseasePompe disease is an autosomal recessive LSD caused by mutationsin GAA the gene coding for the glycolytic enzyme α-glucosidasewhich lead to glycogen accumulation in myocytes (Dasouki et al2014) Pompe disease is divided into two major types based on ageof onset an infantile-onset form where glycogen accumulationoccurs primarily in cardiomyocytes and a later-onset form whereglycogen accumulation is primarily restricted to skeletal muscle(Chan et al 2017) Like many other LSDs Pompe disease researchhas been constrained by a lack of physiologically relevant modelsAlthough Pompe disease mouse models exhibit cellularmanifestations similar to those in humans their overall clinical

Box 1 Advantages of iPSC technology

iPSCs offer an effective means of developing in vitro human cellularmodels for diseases which previously lacked such models

They enable the generation of certain cell types that are difficult orimpossible to obtain directly from humans

Sufficient numbers can be generated to perform cell-based experimentsand drug screens

iPSCs can be derived from a plethora of cell types and then differentiatedinto different cellular types (Fig 1)

Once generated they can be frozen thawed and expanded therebyproviding an unlimited supply of cells for research

iPSCs are free of the controversy and legal limitations facing embryonicstem cell use

Reprogram cells

iPSCs

Cardiomyocytes Hematopoieticprogenitor cells

Motor neuronsDopaminergic neurons

Neural cells

Adult fibroblast cells

Fig 1 Patient-derived fibroblasts can be reprogrammed into iPSCs andthen differentiated into different cellular lineages Adult fibroblasts arereprogrammed into iPSCs which can be differentiated into different lineagesNeural precursor cells can be further differentiated into specific types ofneurons such as dopaminerigic neurons or motor neurons

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phenotype differs greatly from that encountered in patients (Limet al 2014)The development of iPSCs from patients with Pompe disease has

been met with variable results Huang et al (2011) were the first toattempt reprogramming fibroblasts from patients with Pompedisease Initially there were problems working with the enzyme-deficient cells which may have resulted from metabolicimpediments to reprogramming and differentiation in thebackground of GAA deficiency The group were eventually ableto successfully recover reprogrammed control iPSCs after restoringα-glucosidase activity via lentiviral delivery of inducible wild-typeGAA prior to OSKM transduction Notably several of the patient-derived iPSC clones subsequently recovered were not transfectedwith GAA-containing vectors with the authors hypothesizing thatexogenous enzyme from nearby α-glucosidase-expressing cells wassufficient to overcome any metabolic barrier to reprogramming(Huang et al 2011) However two subsequent studies on Pompedisease described the successful generation of patient-derived iPSCclones made in the absence of exogenous enzyme (Higuchi et al2014 Raval et al 2015)Raval et al (2015) reprogrammed fibroblasts from patients

with infantile-onset Pompe disease and differentiated them intocardiomyocytes Although these cells exhibited no α-glucosidaseactivity and the lysosomes were engorged with glycogen contractilityand autophagy in these cells were not impaired and functionallyPompe disease cells were indistinguishable from controlsNonetheless the cardiomyocytes did have aberrant glycanprocessing in some proteins suggesting that this may play a role inthe development of the cardiomyopathy characteristic of this disorderAnother study focused on iPSC-derived cardiomyocytes from

patients with late-onset Pompe disease and also confirmed theaccumulation and storage of glycogen in lysosomes (Sato et al2015) The authors then partially corrected the defect usinglentiviral GAA resulting in enhanced α-glucosidase activity anddecreased glycogen accumulation (Sato et al 2015) In an attemptto further enhance α-glucosidase activity in skeletal muscle derived

from Pompe iPSCs they introduced the gene encoding transcriptionfactor EB (TFEB) a master regulater coordinating the expression oflysosomal hydrolases membrane proteins and genes involved inautophagy GAA and TFEB together yielded further biochemicalimprovement in the form of a reduction of the glycogen stores inmuscle cells and improved enzymatic activity in cells (Sato et al2016b) This finding implicated abnormal lysosomal biogenesis inthe muscular pathology of Pompe disease The same group alsoperformed metabolomic profiling of the cells which demonstratedthat oxidative stress and mitochondrial dysfunction are associatedwith the disorder The work was then replicated in iPSCs derivedfrom a genetically engineered murine Pompe model and thisconfirmed the role of oxidative stress in skeletal and cardiacdysfunction in this disorder (Sato et al 2016a) Furthermore theauthors found that the nuclear factor erythroid 2 (NF-E2) whichplays a key role in combating oxidative stress is downregulatedin Pompe cardiomyocytes and skeletal muscle implicating animpaired anti-oxidative stress response mechanism in thepathophysiology of disease

Fabry diseaseFabry disease is an X-linked recessive LSD that results fromdeficient or absent activity of the enzyme α-galactosidase Awhich leads to the progressive lysosomal accumulation ofglobotriaosylceramide (Gb3) in a variety of cell types such ascardiomyoctes (Schiffmann and Ries 2016 Ranieri et al 2016)This systemic Gb3 accumulation eventually leads to devastatingrenal cardiac and cerebrovascular dysfunction

The first Fabry disease iPSCs that were generated exhibitedultrastructural features typically seen in Fabry disease includingmembranous cytoplasmic bodies (Kawagoe et al 2013) This ledthe authors to speculate that differentiating Fabry iPSCs into otherlineages could be challenging However iPSCs were later generatedfrom fibroblasts isolated directly from patients with Fabry disease(Itier et al 2014) These iPSCs exhibited no detectable Gb3and could differentiate into cardiomyocytes Over time Gb3

Rosettes Dopaminergicneurons

iPSCs NPCs NestinTuj1Sox1

iPSCs EBs Monocytes Macrophages

CD14

Even

ts (

of m

ax) CD68

100 101 102 103 104

Days100 20 200

Fluorescence

Fig 2 Differentiation of iPSCs to neurons and macrophages Representative fluorescent microscopy images illustrating how rosettes a distinct form ofneuronal stems cells that stain positive for Sox1 are generated from the iPSCs (top row) Rosettes are then differentiated into neuronal progenitor cells (NPCs)which stain positive for the neuronal markers Nestin and Tuj1 Further differentiation into mature dopaminergic neurons which can be visualized by stainingwith tyrosine hydroxylase can take as long as 200 days To make monocytes and macrophages (bottom row) the first stage is generation of embryoid bodies(EBs visualised here by phased light microscopy) which are spherical aggregates that recapitulate many features of early embryogenesis Monocytes whichcan be identified by the immunological marker CD14 can then be separated by a fluorescence-activated cell sorter and harvested Finally CD14-positivemonocytes can be differentiated into CD68-postive macrophages The plots show the separation of CD14- and CD68-positive cells based on fluorescenceintensity and the smaller panel provides a representative light microscopy image of macrophages

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Table 2 Summary of iPSC models of lysosomal storage diseases generated to date

DiseaseImplicatedgene(s) Reference

Reprogrammingmethod

Differentiationtarget(s) Observations

New therapeuticstested

SphingolipidosesGaucher disease GBA1 Park et al 2008b Retrovirus ndash ndash ndash

Mazzulli et al 2011 Retrovirus DA neurons GBA1 deficiencycontributes toα-synucleinaccumulation

ndash

Panicker et al 2012 Lentivirus(polycistronicCre-excised)

Macrophages DAneurons

Impaired clearance ofphagocytosed materialby macrophages

Small-moleculechaperones

Tiscornia et al 2013 Nucleofection(polycistronicCre-excised)


ndash Small-moleculechaperones

Panicker et al 2014 Sendai virus Macrophages Increased secretion of pro-inflammatory factors

ndash

Aflaki et al 2014 Lentivirus(polycistronicCre-excised)

Macrophages Impaired chemotaxisrespiratory burst


Schoumlndorf et al 2014 Retrovirus DA neurons Defects in autophagy andcalcium homeostasis

ndash

Sun et al 2015 Lentivirusnucleofection

DA neurons Aberrant electrophysiologyin neuronopathic GD

ndash

Sgambato et al 2015 Pre-existing lines Hematopoieticstem cells

Impaired erythropoiesis ndash

Awad et al 2015 Pre-existing lines Neurons Impaired lysosomalbiogenesis

ndash

Westbroek et al 2016 Lentivirus Neurons ndash ndash

Aflaki et al 2016a Lentivirus Macrophages DAneurons

Augmenting GBA activitydecreases α-synucleinaccumulation


Fabry disease GLA Kawagoe et al 2013 Retrovirus andSendai virus

ndash Cytoplasmic inclusions iniPSCs

ndash

Itier et al 2014 Lentivirus Cardiomyocytes Lysosomal GL-3accumulation

SRT withglucosylceramidesynthase inhibitor

Chou et al 2017 Sendai virus Cardiomyocytes Left ventricularhypertrophy and GB3accumulation

ndash

Metachromaticleukodystrophy

ARSA Doerr et al 2015 Retrovirus Neuronal precursorcells astrocytes

Engrafted cells reducesubstrate storage inmouse model

Ex vivo gene therapy(transplant in mice)

Meneghini et al 2016 Lentivirus(polycistronicCre-excised)

Neuronal precursorcells



MucopolysaccharidosesType I (Hurlersyndrome)

IDUA Tolar et al 2011 Retrovirus Hematopoieticstem cells

Substrate accumulation inpatient-derived iPSCs

Ex vivo gene therapy(no transplantation)

Type II (Huntersyndrome)

IDS Reboun et al 2016 Sendai virus Neuronscardiomyocytes

Skewed X-inactivation iniPSCs from heterozygousfemale patient

ndash

Varga et al 2016abcd Lentivirus ndash ndash ndash

Type IIIB(Sanfilipposyndrome)

NAGLU Lemonnier et al 2011 Retrovirus (OSK andOSKM)

Neurons Substrate accumulationand disruption ofintracellular trafficking

ndash

Type IIIC(Sanfilippo type Csyndrome)

HGSNAT Canals et al 2015 Retrovirus (OSK andOSKM)

Neurons Accumulation of GAGs ndash

Type VII (Slysyndrome)

GUSB Griffin et al 2015 Retrovirus Neuronsastrocytes

Engrafted cells reduceinflammation in mousemodel


Other LSDsNeural ceroidlipofuscinoses(Batten disease)

TPP1(infantile)

Lojewski et al 2014 Retrovirus Neuronal precursorcells neurons

Golgi endosomallysosomal andmitochondrial defects

Small-moleculeinducers

CLN3( juvenile)

Chandrachud et al 2015 Retrovirus Neuronal precursorcells

Defects in autophagy ndash

Continued

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accumulated in the lysosomes of these cardiomyocytes mimickingthe phenotypic changes found in cardiac tissue from patients withFabry disease Using the cardiomyocytes as a model it wasdemonstrated that ibiglustat a glucosylceramide synthase inhibitorbeing developed as a substrate reduction therapy for Fabry diseaseprevented Gb3 accumulation and eventually cleared lysosomal Gb3(Itier et al 2014) Thus ibiglustat could be a promising therapeuticstrategy for this lysosomal storage disease

Metachromatic leukodystrophyMetachromatic leukodystrophy (MLD) is an autosomal recessivedisorder of lipid metabolism characterized by the deficient activityof the lysosomal enzyme arylsulfatase A (ASA) resulting indeficient degradation of galactosylceramide-3-O-sulfate (sulfatide)and galactosylsphingosine-3-O-sulfate (lysosulfatide) (Gieselmann2008) At the cellular level the disease is characterized byimpaired sphingolipid metabolism and the resulting accumulationof sulfatide Progressive accumulation of sulfatide in the myelin-producing cells causes destruction of white matter in both the centraland peripheral nervous systems driving progressive deterioration ofintellectual functions and motor skills including the ability to walk

There are three clinical subtypes of this disorder late-infantilejuvenile and adult forms Symptoms seen in individuals who areaffected include peripheral neuropathy incontinence seizuresparalysis a loss of the ability to speak and visual and hearingloss Patients can eventually become unresponsive to theirsurroundings (Gieselmann 2008)

Two studies have demonstrated successful generation anddifferentiation of iPSCs for MLD In a study by Doerr et al(2015) MLD patient-derived iPSCs were differentiated into self-renewing neuroepithelial stem cells and astroglial progenitorswhich were then used to evaluate cell-based ARSA replacementTransplantation of ARSA-overexpressing precursors into ARSA-deficient mice resulted in significantly reduced sulfatide levels(Doerr et al 2015) Recently the differentiation of MLD patientfibroblasts into iPSC models was performed by Meneghini et al(2017) The patient-derived iPSCs were differentiated into neuralstem cells which shared molecular phenotypic and functionalfeatures with fetal-derived MLD neural stem cells Using lentiviralvectors MLD iPSCs were efficiently transduced achievingsupraphysiological ARSA activity which increased further afterneural differentiation A significant decrease in sulfatide storagewas

Table 2 Continued


Reprogrammingmethod



Niemann-Pickdisease type A

SMPD1 Long et al 2016 Sendai virus Neuronal precursorcells

Substrate accumulationand lysosomalenlargement

Small moleculescyclodextrins

SMPD1 Trilck et al 2013 Retrovirus Neuronal precursorcells

Cholesterol accumulation ndash

Niemann-Pickdisease type C1

NPC1 Maetzel et al 2014 Lentivirus (Cre-excised)

Hepatocytesneurons

Substrate accumulationand defects inautophagy

Cyclodextrins

Yu et al 2014 Sendai virus Neuronal precursorcells

Substrate accumulation Small moleculescyclodextrins

Lee et al 2014 Retrovirus Neurons Defects in VEGF signalingand autophagy

ndash

Soga et al 2015 Sendai virus Hepatocytesneuronalprecursor cells


Cyclodextrins

Efthymiou et al 2015 Lentivirus Neurons Dysfunction of calcium andWNTsignaling

Trilck et al 2017 Retrovirus Neurons GM2 accumulation andreducedHEX A activity

ndash

Pompe disease GAA Kawagoe et al 2011 Retrovirus Skeletal myocytes Glycogen accumulationHuang et al 2011 Retrovirus Cardiomyocytes Substrate accumulation

altered metabolic fluxand disorderedmyofibrils

ndash

Higuchi et al 2014 Retrovirus ndash Substrate accumulation iniPSCs

ndash

Raval et al 2015 Lentivirus Cardiomyocytes Defective proteinglycosylation

ndash

Sato et al 2015 Pre-existing lines Cardiomyocytes GAA overexpressionreduces glycogenstorage

Gene therapy

Sato et al 2016b Pre-existing lines Skeletal myocytes TFEB supplements GAAoverexpression innormalizing glycogenlevels

Gene therapy

Sato et al 2016a Pre-existing lines Cardiomyocytes Metabolic dysfunctionoxidative stress

ndash

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also observed when ARSA-overexpressing cells were used(Meneghini et al 2017) This study enhances our understandingof the CNS pathology in MLD and suggests that ultimately celltransplantation might provide both enzymatic reconstitution andreplacement of damaged or lost cells

Neuronal ceroid lipofuscinosesNeuronal ceroid lipofuscinoses (NCLs) also referred to collectivelyas Batten disease are a group of extremely rare and fatalneurodegenerative LSDs These diseases are characterized byintracellular accumulation of autofluorescent lipofuscin a fattylipopigment in both neurons and peripheral tissues (Mole andCotman 2015) To date mutations in 14 genes have been identifiedas being potentially causative for NCLs and there are several NCLsubtypes based on the mutated gene age of onset and the severityof neurological defects such as progressive dementia seizures andvisual failure (Mole and Cotman 2015)A study by Lojewski and co-workers in 2014 generated the first

NCL iPSCs using fibroblasts derived from two patients with late-infantile NCL linked to mutations in TPP1 (tripeptidyl peptidase 1)and four patients with juvenile NCL and mutations in CLN3 TPP1encodes a member of the sedolisin family of serine proteases andCLN3 encodes a protein involved in lysosomal function Thesepatient-derived iPSCs were differentiated into neuronal tissue Asexpected abnormalities in the endosomal-lysosomal system weredetected in the patient iPSCs but the authors noted that disease-subtype-specific lysosomal storage was only evident in theirdifferentiated neuronal derivatives They were able to correct theabnormalities in these cells by overexpressing adenovirus vector-delivered wild-type TPP1 or CLN3 These iPSC-derived neuralprogenitor cells were also used to screen potential pharmacologicalmodulators of the CLN2 encoded protein The screen demonstratedthe utility of patient-derived iPSCs as a platform for testing newtherapeutic candidates Two lipid-lowering drugs were identified ndashfenofibrate and gemfibrozil The patient with the NCL-linked TPP1mutation was treated with these compounds resulting in a smallincrease in both TPP1 levels and enzymatic activity This workfurther illustrates the value of iPSC-derived human neuronal modelsfor NCL drug discovery and evaluation

Niemann-Pick type C diseaseNiemann-Pick type C disease (NP-C) is an autosomal recessiveneurovisceral atypical LSD Mutations in NPC1 and NPC2 lead toimpaired intracellular transport of cholesterol and glycolipidswhich ultimately causes accumulation of these lipids in cells(Vance 2006) Both NPC1 and NPC2 proteins are catalysts thatmobilize the cholesterol within the multivesicular environment ofthe late endosome Children affected by NP-C present primarilywith visceral symptoms such as hepatosplenomegaly (enlargementof the liver and spleen) followed by progressive intellectual andneurological deterioration Those who present in adulthood oftendevelop psychiatric problems including depression and psychosis(Evans and Hendriksz 2017)Hepatocyte-like cells and neural progenitors derived from the

iPSC lines generated from patient-derived fibroblasts displayedcholesterol accumulation and impairment of autophagy and ATPproduction (Soga et al 2015) indicating that these cells dophenocopy the human disease Soga et al (2015) also showed thata new compound 2-hydroxypropyl-γ-cyclodextrin reducedcholesterol accumulation and restored the observed abnormalitiesin the patient-derived NPC iPSCs demonstrating the utility of thismodel for evaluating new candidate drugs

In another study patient-derived NP-C iPSC neurons were foundto have abnormal vascular endothelial growth factor (VEGF) levelsand altered sphingolipid metabolism thus recapitulating features ofthe disease in vivo (Lee et al 2014) The neurons also demonstratedinhibition of autophagosome-lysosome fusion when compared withwild-type neurons Treatment with VEGF appeared to amelioratethis defect in autophagy by correcting the sphingolipidabnormalities indicating that VEGF could be a therapeuticcandidate for Niemann-Pick type C disease

Bergamin et al (2013) successfully generated a human neuronalmodel of NP-C by inducing neuronal differentiation of multipotentadult stem cells (MASCs) isolated from patients with NP-C andcontrols In the MASCs massive lysosomal accumulation ofcholesterol was observed only in those isolated from patients withNP-C Upon neural differentiation intracellular accumulation ofunesterified cholesterol and GM2 ganglioside were observed in theNP-C neurons resulting in morphological differences thatdistinguished the diseased cells from those derived from healthydonors It is likely that these promising iPSC models will soon beused to explore the pathophysiology of NP-C

The mucopolysaccharidosesThe mucopolysaccharidoses (MPSs) are a heterogeneous group ofLSDs that are clinically characterized by progressive dysfunction inmultiple organ systems and reduced life expectancy (Coutinho et al2012) Apart fromMPS II (also known as Hunter Syndrome) whichis inherited in an X-linked manner the MPSs are autosomalrecessive diseases Individuals with MPSs are typically healthy atbirth but during early childhood they experience onset of symptomsthat include deterioration of skeletal joint airway and cardiactissue impaired hearing and vision and in some MPSs cognitiveimpairment There are nine subtypes of MPS described to date eachcaused by a deficiency in a lysosomal enzyme required forglycosaminoglycan (GAG) degradation The result of thisdeficiency is accumulation of partially degraded GAG withinlysosomes and elevated levels of GAG fragments in the urine bloodand cerebral spinal fluid (Coutinho et al 2012)

IPSCs have been generated from patients with MPS IH (Hurlersyndrome) which is caused by the deficiency of α-L-iduronidaseThe study indicated that the deficient enzyme is not required forstem cell renewal (Tolar et al 2011) The iPSCs showed lysosomalstorage defects characteristic of MPS IH and could be differentiatedto both hematopoietic and non-hematopoietic cells The authorsdemonstrated that when the differentiated cells were gene-correctedwith virally delivered α-L-iduronidase the specific epigeneticprofile associated with de-differentiation of MPS IH fibroblasts intoMPS-iPSCs was maintained highlighting the potential of thesecells to generate autologous hematopoietic grafts devoid ofimmunologic complications (Tolar et al 2011) Hematopoieticcell transplantation is currently being performed as a life-savingtreatment for MPS IH However a suitable hematopoietic donor isnot found for all affected individuals and the therapy is associatedwith significant morbidity as well as mortality (Aldenhoven et al2008) The potential to generate gene-corrected autologous stemcells could potentially provide a more optimal graft fortransplantation avoiding current complications

As it is an X-linked disorder MPS II manifests almostexclusively in males however an iPSC model has been generatedfrom a symptomatic female with a heterozygous mutation in the IDS(iduronate 2-sulfatase) gene (Reboun et al 2016) This geneencodes a member of the sulfatase family of proteins which isinvolved in the lysosomal degradation of heparan sulfate and

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isms

dermatan sulfate iPSCs generated from the patientrsquos peripheralblood demonstrated characteristic pluripotency markers anddeficient iduronate 2-sulfatase activity This study reported thatX-inactivation analyzed at three X-chromosome loci showedextreme skewing in two of the patientrsquos cell types favoringexclusive expression of the mutated allele iPSCs derivedprincipally from males affected by MPSII have also beensuccessfully generated by Varga et al (2016abcd)In their initial attempt at generating iPSCs for MPS IIIB

(Sanfillipo syndrome type B) Lemonnier et al (2011) wereunsuccessful and the authors speculated that accumulation ofimproperly metabolized GAG in patient-derived iPSCs interferedwith growth factor signaling Co-culture of the patient-derivediPSCs with feeder cells secreting α-N-acetylglucosaminidaseshowed that the deficient enzyme in MPS IIIB was necessary toexpand the resulting iPSCs (Lemonnier et al 2011)iPSC lines have also generated from two patients with MPS IIIC

(Sanfilippo syndrome type C) (Canals et al 2015) Neurons derivedfrom these lines recapitulated features of the disease includinglow acetyl-CoA α-glucosaminide N-acetyltransferase activityaccumulation of GAG and an increase in lysosome size andnumber which was not seen in genetically corrected patient-specific iPSC-derived cultures Furthermore the authors observedearly defects in neuronal activity neuronal-wide degradation andaltered effective connectivity in the patient-derived cells Since themechanism underlying the brain dysfunction and behavioralphenotype in this disorder are poorly understood theidentification of these early functional phenotypes provide newinsight into disease pathogenesis Furthermore the model has utilityfor drug development (Canals et al 2015)Another study of mucopolysaccharidoses used human iPSCs

generated from patients with MPS VII (Sly syndrome) MPS VIIiPSCs were differentiated into neuronal precursor cells and thentransplanted into a well-characterized mouse model of the disease(Griffin et al 2015) The patient-derived neural stem cells engraftedalong the rostrocaudal axis of the CNS primarily within white mattertracts surviving around four months Genetically corrected iPSC-derived neural stem cells were transplanted into the striatum ofadult post-symptomatic MPSVII mice resulting in a reversal ofneuropathology in a zone surrounding the grafts (Griffin et al2015) This study suggested the potential of ex vivo gene therapy inthe brain for LSDs discussed further below

A therapeutic revolution for the LSDsUntil relatively recently therapeutic options for LSDs have beenlargely limited to palliative care and physical therapy Bone marrowtransplant has been attempted as a means to treat a handful of theLSDs but transplant-associated morbidity and mortality and thefailure of this procedure to alleviate neurological manifestations insome LSDs have limited its wider application (Rovelli 2008) Thismade the development of enzyme replacement therapy (ERT)which is currently available or in clinical trials for eight LSDs (Ries2017) a revolution in the field of LSDs ERT involves intravenousinfusion of the deficient enzyme with the aim of clearing storedmaterial and restoring normal lysosomal function in affected cellsERT is effective in preventing or reversing visceral cardiovascularmusculoskeletal and even peripheral neurological manifestations ofthose diseases for which it is available (Barton et al 1991Schiffmann et al 2003 2001 Winkel et al 2004) However theinfused enzymes are unable to cross the blood-brain barrier andthus have little impact on brain phenotypes in neuronopathic LSDsFurthermore it is an inconvenient and extremely expensive

treatment requiring infusions at regular intervals for theremainder of the patientrsquos life at a cost upwards of US$200000per year (Kanters et al 2014 van Dussen et al 2014)

Another therapeutic approach substrate reduction therapy (SRT)involves the administration of small-molecule inhibitors aimed atreducing the synthesis of storage material To date SRT hasdemonstrated only mixed success in managing neurologicalsymptoms of LSDs One SRT drug miglustat has shown somepromise in slowing neurological decline in Niemann-Pick type Cdisease but the same drug (and a second SRT elglucerase) showedno impact on the neurological symptoms in GD (Patterson et al2007 Schiffmann et al 2008 Poole 2014 Shayman 2010) OtherSRT drugs are currently in clinical trials for Pompe diseaseGaucher disease and Niemann-Pick C (Parenti et al 2015)

These realities paired with recent technological developmentshave pushed the development of new and improved treatmentmodalities to the forefront of LSD research Modifications are beingdeveloped to allow enzymes infused intravenously to cross theblood-brain barrier and enter neurons and glial cells (Grubb et al2008 Sorrentino et al 2013) Gene therapy and corrective stemcell therapies are also being investigated in animal models aspotential treatments for severe LSDs with a particular focus onlethal neuropathic LSDs (Sands and Haskins 2008) Alongsidethese developments new approaches using small-moleculepharmacological chaperones have attracted much attention as apotential therapy (Parenti et al 2015)

iPSC-based therapiesOne exciting development in iPSC research for LSDs is thepossibility of ex vivo gene therapy especially as a means oftreating neuronal manifestations of these diseases This processinvolves developing patient-derived iPSCs transducing thesecells with wild-type forms of the mutant gene differentiating thesegene-corrected cells into neuronal precursors and transplantingthem back into the patientrsquos central nervous system (Griffin et al2015) This process attempts to achieve the same aim as in vivogene therapy by establishing a long-term source of wild-typeenzyme within the brain but without injection of adenovirus intopatients

Recent studies have assessed the efficacy of human iPSC-derivedcell transplants into mouse models of two LSDs metachromaticleukodystrophy (MLD) and Sly disease (MPS VII) Beforetransplant these LSD mice lines were crossed withimmunodeficient mice to avoid immune rejection As discussedearlier Doerr et al (2015) generated neuroepithelial stem cells andastroglial progenitors fromMLD patient iPSCs that were transducedwith a vector containing the wild-type ARSA and transplanted intothe brains of MLD mice This did result in a significant reduction ofsulfatide in the vicinity of transplanted cells Griffin et al (2015)similarly transplanted neural stem cells differentiated from iPSCsfrom patients with Sly disease and noted GUSB activity along withcorrection of disease-associated microglial pathology These studiesillustrate the success of correcting brain pathology using geneticallyreprogrammed iPSCs and the survival of neural stem cells andastroglial progenitors after several months However noexperiments were performed to assess whether disease symptomsin the mice were reduced

iPSCs as a platform for drug screeningSmall-molecule chaperones are another strategy that could beappropriate for the treatment of LSDs Such drugs would functionby binding endogenous mutant enzyme stabilizing the protein and

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Disea

seModelsampMechan

isms

thereby increasing enzymatic activity Like SRT drugs these smallmolecules would be able to enter the brain but unlike SRT drugsthey would act by directly addressing the underlying enzymedeficiency Currently high-throughput drug screens are commonlyused Different small-molecule libraries have been assembledcontaining a hundred thousand to a million compounds that canbe tested simultaneously (Inglese et al 2006 Zheng et al 2007)Other libraries containing FDA-approved compounds are alsoavailable for such screens Chaperones for different LSDs have beenidentified by employing assays that screen for compounds thatimpact enzymatic activity (Motabar et al 2010) These assays wereinitially utilized to identify enzyme inhibitors that bind to the activesite (Zheng et al 2007) Subsequently tissue extracts were used toidentify non-inhibitory chaperones that are now being developedfurther (Jung et al 2016)iPSC-derived cell models can play a role in identification of

small-molecule drugs as well as providing a new platform for testingnew drugs Although it is currently difficult to generate a largeenough number of cells to use in high-throughput screening theycan still serve as a valuable validation tool for candidate drugsIn the case of GD two different groups examined specificsmall-molecule inhibitors of glucocerebrosidase that act aspharmacological chaperones and both observed improvement inthe clearance of erythrocytes and reduction in the secretion of pro-inflammatory factors in iPSC-derived macrophages (Panicker et al2014 Tiscornia et al 2013) Furthermore Aflaki et al (2014)demonstrated correction of glucocerebrosidase activity lipidstorage chemotaxis and reactive oxygen species (ROS)production in iPSC-derived macrophages treated with a novelnon-inhibitory chaperone These results demonstrate that iPSC-derived cells provide opportunities for both the identification anddocumentation of responses to new therapiesA different strategy to improve the folding of mutant lysosomal

enzymes exploits proteostasis regulators In GD calcium channelblockers were shown to partially restore enzymatic activity inpatient fibroblasts rescue of activity was thought to involveupregulation of the intrinsic molecular chaperones ofglucocerebrosidase (Wang et al 2011) IPSC-derived modelsmight prove useful in the identification and testing of suchregulators as well as enabling a better understanding of theirmechanism of action Therapies based on heat shock proteins arealso under consideration for several LSDs (Kirkegaard et al 2016)Theoretically therapies combining chaperones and proteostasisregulators could enhance efficacy and iPSCs are also an effectiveplatform for testing and optimizing such combinatorial therapies

Insights into common neurodegenerative diseasesOne of the most profound benefits of iPSC models is the ability torecapitulate the hallmark characteristics of cells affected bycommon neurodegenerative disorders In particular thedifferentiation of iPSCs into DA neurons has provided the abilityto investigate the previously unattainable diseased neuronsimplicated in neuronopathic GD and Parkinsonrsquos disease Acomplete understanding of the basis of the relationship betweenglucocerebrosidase and parkinsonism is still lacking (Aflaki et al2017) augmenting the need for new tools and models A recentstudy by Woodard et al generated iPSC-derived neuronal modelsfrom a set of monozygotic twins discordant for PD both of whomcarried an N370S mutation in GBA1 (Woodard et al 2014) Thestudy revealed increased α-synuclein levels in DA neurons in thetwin with Parkinsonrsquos disease Such investigations provide aplatform upon which the complex association between GBA1 and

Parkinsonrsquos disorder can be further elucidated and ultimatelycharacterized In another study Aflaki et al examined differentiatedDA neurons from patients with GD1 GD1-with Parkinsonrsquos diseaseand GD2 (Aflaki et al 2016a) These cells were then used to testnon-inhibitory compounds that could be potential leads for drugdevelopment Ultimately such studies have shown that iPSC-derived neurons can circumnavigate the difficulties in studyinghuman tissue in neurodegenerative disorders

Caveats and limitations of iPSC-based models for LSDsDespite the advantages provided by iPSCs for modeling differentLSDs there are some issues that are important to take intoconsideration Some of the limitations of this technology are listedin Box 2 and discussed below

Metabolic impediments to reprogramming and differentiationThe process of reprogramming is energetically demanding andcells must undergo extensive metabolic remodeling in order tosuccessfully transition to pluripotency (Choi et al 2015Panopoulos et al 2012) When generating iPSC-derived cellmodels from patients with LSDs there is the possibility that themetabolic disruption accumulation of storage material andsubsequent cellular dysfunction seen in LSDs could negativelyimpact the reprogramming process iPSC lines for several LSDshave exhibited extensive disease-related pathology Although mostiPSC models of LSDs have been developed without the rescue ofthe deficient enzyme difficulties in reprogramming of patient cellshave been reported as highlighted in specific sections above(Huang et al 2011 Lemonnier et al 2011 Tiscornia et al 2013)

Phenocopying do these cells provide a faithful model of diseaseTwo universal metrics for assessing the effectiveness of an iPSC-derivedmodel of LSDs are enzyme deficiency and substrate storageMost but not all LSD iPSC lines have exhibited these featuresbefore differentiation however the presence of these defects indifferentiated cells is required for them to be considered a potentialmodel of disease When evaluating other observed cellularphenomena researchers generally aim to compare their findingsto established pathologies in human patients or animal modelswhen available Perhaps the strongest support for the effectivenessof the ability of iPSC-derived cells to phenocopy their in vivocounterparts was provided by the observation that macrophagesdifferentiated from both Gaucher iPSCs and peripheral blood

Box 2 Limitations of iPSC-based disease models oflysosomal storage disorders

Developing iPSC-based disease models is expensive labor-intensiveand requires time

Reprogramming is energetically demanding and can be affected bymetabolic defects intrinsic to the disease being modeled

The donor cells must be carefully and completely phenotyped

Controls are needed with an appropriate genetic background

The model may not reflect later-onset disease phenotypes

iPSC-derived differentiated cells might not retain aging-associated genesignatures and cellular properties

699


Disea

seModelsampMechan

isms

monocytes derived from the same patients exhibited similar cellularphenotypes (Aflaki et al 2014)However in many cases pluripotent stem cell (both ESCs and

iPSC)-derived differentiated cells often best resemble cells of theearly embryo (lt6 weeks of development) rather than cells fromadult tissues (Keller 2005 Patterson et al 2012) Owing to theirimmature state the functionality of such cells could be differentfrom their adult counterparts For this reason maturation of cells cansometimes be required and this is achieved by supplementation ofchemical compounds that promote more rapid maturation(Chambers et al 2012) Another strategy used to generate moremature and functional pluripotent stem cell-derived cells is to try toreproduce the in vivo conditions by co-culturing with other celltypes from the native tissue environment such as glia cells in thecase of neurons Furthermore three-dimensional approaches such asthe generation of organoids that reproduce the organ architecturein vitro or by microfluidics systems (organ-on-a-chip) that are ableto recreate dynamic multi-tissue structures have been considered(Cornacchia and Studer 2017) Another limitation is that iPSC-derived differentiated cells might not retain aging-associated genesignatures and cellular properties such as senescence andproliferation mitochondrial metabolism and related oxidativestress (Lapasset et al 2011 Marion et al 2009 Prigione et al2010 Suhr et al 2009) This could pose a problem when studyingaging-related disease pathophysiology in vitro such as bonepathology in Gaucher disease Attempting to control the cellularage of differentiated cell linages has become a major challengeparticularly when developing models of neurodegenerativediseases For this reason strategies aimed at modeling the effectof aging such as treatment with ROS or the manipulation ofparticular transcriptional regulators signaling pathways andepigenetic markers are being considered (Cornacchia and Studer2017 Miller et al 2013)

Selecting donor cellsDetermining which donor cells to use to model the LSDs can beimpacted by the paucity of available patient samples As a result ofthe rarity of these diseases biorepositories are often the only sourceof fibroblasts from patients with LSDs However informationregarding disease phenotypes can be lost when patient cells areentered into biorepositories In the worst cases iPSC lines can becompletely misidentified In fact the first two GD iPSC lines weregenerated using the same fibroblast line from the samebiorepository but the publications disagreed over the diseasephenotype of the donor (Mazzulli et al 2011 Park et al 2008b)More generally a major asset of patient-derived iPSCs lies in thecorrelation of the iPSC phenotype with the patient phenotypewhich is particularly important when considering the vastphenotypic heterogeneity that characterizes the LSDs Studies thatsource fibroblasts directly from well-characterized patients aretherefore particularly valuable

CostPerhaps the largest impediment to the development and use of iPSC-derived models is the cost Firstly reagents media consumables andgrowth factors are quite expensive Furthermore modeling anydisease using iPSCs is labor-intensive and requires a great investmentin human resources This is compounded by the long periods of timerequired for the reprogramming process iPSC validation anddifferentiation to relevant cell types This is particularly true whenattempting to establish adult-differentiated cells andor to recapitulatelater-onset disease phenotypes Moreover because these are rare

diseases it is difficult to generate a large number of LSD iPSCmodels with different genotypes in order to perform studies withadequately high statistical power

Identifying the appropriate controlsIdentifying and generating appropriate controls with the samegenetic background of the disease model can also be challengingTo overcome the differences in genetic background and also clonalvariability which can occur during reprogramming (Gore et al2011 Hussein et al 2011) generating isogenic lines usinggenome-editing systems [such as transcriptional activator-likeeffector nucleases (TALENs) or clustered regulatory interspacedshort palindromic repeat (CRISPR)Cas-based systems] is desirable(Gaj et al 2013) These technologies can also be used to introducedisease-specific mutations in wild-type cells in order to generate aphenotype However these endeavors are likely to also bechallenging and labor intensive

ConclusionThe ability to generate iPSC models of different LSDs is markedlychanging the approach to modeling these disorders In particularthese new methods of generating diseased macrophages neuronsand cardiomyocytes closely resembling the primary diseasephenotypes provide new tools to probe disease pathogenesis andto test therapeutic strategies One issue that has remained unresolvedis to what extent the phenomena observed in the disease models arephysiologically relevant as opposed to being a result of thereprogramming or differentiation process New advances in geneediting could help to answer these questions To confirm thatchanges observed in the cell models are a result of the specificmutation TALENs andor CRISPR-Cas strategies can now be usedto correct diseased iPSCs by editing out the disease-causingmutations (Kim et al 2017) This technology while requiringextensive optimization will enable researchers to ascertain whatfeatures of the cellular models are a direct functional consequence ofthe LSD-associated mutation

Differentiating the iPSCs into different neuronal lineages willhelp to elucidate the cause of neuronopathic forms of LSDsIn addition although a link between Gaucher disease and thesynucleinopathies is clearly established it has not been definitivelyascertained whether mutations in other LSD genes are similarlyrelated to more common neurodegenerative disorders iPSCmodels of these rare often lethal disorders could provide uniqueopportunities to phenotype neurons expressing the mutantlysosomal genes

The generation of organoid disease models from iPSCs is arapidly growing field developed to bridge the gap between studiesin cell lines and in vivo modeling Such research has beensupported by progress in stem cell work and in new biomaterialsThis has enabled researchers to develop 3D culture systemsmimicking conditions found in human tissues Developingorganoids to model the different lysosomal storage disorders isclearly of great interest and likely to be an expanding field in thefuture

This article is part of a special subject collection lsquoNeurodegeneration fromModels toMechanisms to Therapiesrsquo which was launched in a dedicated issue guest edited byAaron Gitler and James Shorter See related articles in this collection at httpdmmbiologistsorgcollectionneurodegenerative-disorders

AcknowledgementsThe authors acknowledge the assistance of Julia Fekecs with preparation of thefigures

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seModelsampMechan

isms

Competing interestsThe authors declare no competing or financial interests

FundingThis work was supported by the Intramural Research Programs of the NationalHuman Genome Research Institute and the National Institutes of Health

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Lim J A Li L and Raben N (2014) Pompe disease from pathophysiology totherapy and back again Front Aging Neurosci 6 177

Lojewski X Staropoli J F Biswas-Legrand S Simas A M Haliw L SeligM K Coppel S H Goss K A Petcherski A Chandrachud U et al (2014)Human iPSC models of neuronal ceroid lipofuscinosis capture distinct effects ofTPP1 and CLN3 mutations on the endocytic pathway Hum Mol Genet 232005-2022

Long Y Xu M Li R Dai S Beers J Chen G Soheilian F Baxa UWangM Marugan J J et al (2016) Induced pluripotent stem cells for diseasemodeling and evaluation of therapeutics for Niemann-Pick disease type A StemCells Transl Med 5 1644-1655

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same basic technique used in mice could also be employed togenerate iPSCs from human somatic cells Since then numerousadvances have been made in identifying new factors that inducereprogramming which now include RNAs and small moleculesnew modes of introducing the necessary factors to cells and newcell types that can be reprogrammed (Table 1) These discoverieshave done much to inform our understanding of how stem cellsachieve and maintain pluripotency Recent work clearlydemonstrates how iPSC-derived cells are a remarkable tool forresearch of human diseases (see Box 1) These advantages havemade iPSC-derived cell models a natural choice for studies of theLSDs as discussed below

iPSC models of LSDsCells of the neuronal and hematopoietic lineages are the usualdifferentiation targets for iPSC-derived models of LSDs (28 of 39studies pursuing differentiation see Table 2) because these are thecells most often affected by these diseases (Figs 1 and 2) Althoughmurine iPSC lines have been derived from five mouse models ofLSDs (Kawagoe et al 2011 Meng et al 2010 Ogawa et al 2013)human iPSCs and iPSC-derived cell models which have beengenerated for at least 11 LSDs (Table 2) have become the focus ofthe field as they more closely mimic the human disease Asdiscussed below human iPSC models of LSDs are alreadycontributing to our understanding and treatment of these rarediseases

Gaucher diseaseGaucher disease (GD) is a recessive disorder caused by mutations inGBA1 resulting in a deficiency in a lysosomal hydrolase namedglucocerebrosidase (Mistry et al 2017) Usually this deficiencyleads to glycolipid accumulation in macrophages and manifests inhematological visceral and skeletal symptoms with a variabledegree of severity GD is classified into three types non-

neuronopathic (type 1) acute lethal neuronopathic (type 2) andchronic neuronopathic (type 3) and more severe mutations areassociated with neuronopathic manifestations Furthermorepopulation studies have established that individuals with GBA1mutations both carriers and affected individuals are at anincreased risk of developing Parkinsonrsquos disease an age-relatedneurodegenerative disorder as well as other Lewy body disorders(Nalls et al 2013 Sidransky et al 2009) The discovery of this linkbetween GBA1 and Parkinsonrsquos disease has played a large part in arecent explosion of GD research

However both seasoned GD researchers and those new to thefield have been limited by a dearth of effective models for studyingboth GD and GBA1-associated Parkinsonrsquos disease Mouse modelsfor GD have been developed but none faithfully recapitulate thefeatures of the disease seen in humans (Farfel-Becker et al 2011)Human cell lines have also been of limited use Fibroblasts frompatients with GD have long been used to study the disease but thesecells do not store the implicated glycolipids and do not showobvious signs of pathology (Saito and Rosenberg 1985) Applyingconduritol β-epoxide (CBE) an irreversible inhibitor ofglucocerebrosidase to a common human monocytic cell line suchas THP-1 or to SH-SY5Y cells (a human neuroblastoma cell lineused to model neurons) leads to glycolipid accumulation in thesecell lines and this approach has been used to provide in vitromodelsfor GD (Hein et al 2007 Prence et al 1996) However it isdifficult to generalize findings in immortalized cell lines to cellsin vivo The limited options for studying GD in the laboratory hasdirected attention toward iPSCs

As macrophages are the primary cell type affected in all GDtypes they are a natural choice for studies of this disease (Fig 2) Ithas generally been accepted that glycolipid-engorged macrophagesdrive the systemic inflammation commonly seen in GD indicatedby elevated inflammatory factors in the blood of affectedindividuals (Pandey and Grabowski 2013) Differentiating iPSCs

Table 1 Factors impacting the generation of iPSCs

Variable involved iniPSC generation Options Advantages Disadvantages References

Reprogrammingfactors (RFs)

Different cocktails (OSKM vs OSNL) Both cocktails effective OSKM+NANOG and LIN28 (sixfactor reprogramming) havehighest efficiency

Liao et al 2008

Type of vector Integrative retrovirus lentivirus Effective reprogramming Insertional mutagenesis canintroduce other genomic errorsresidual expression of RFs canimpede differentiation andcause genomic instability

Okita et al 2007 Papapetrouet al 2009 Ramos-Mejiaet al 2012 2010 Stadtfeldet al 2008

Integrative and polycistronic RFs(linked by self-cleaving peptideand flanked by loxP or transposonsites)

Reduces number ofinsertion events allowsexcision of RFs

Cre excision leaves long terminalrepeat can cause mutagenesis

Chang et al 2009 Papapetrouand Sadelain 2011 Woltjenet al 2009 Yusa et al 2009

Non-integrative adenovirus orSendai RNA virus

Avoids mutagenesisNo residual RFexpression

Low reprogramming efficiency Fusaki et al 2009 Kiskinis andEggan 2010 Miyoshi et al2011 Warren et al 2010

Donor cells Fibroblasts Relatively simple to obtainvia skin punch biopsyavailable via bio-repositories

Requires invasive skin biopsyclinical data often missing inbiorepositories

Blood Blood draw is minimallyinvasive

Other drugs can interfere Staerk et al 2010

Umbilical cord blood Can be reprogrammedwith only two RFs

Seldom available for raredisorders like LSDs

Meng et al 2012

Keratinocytes Easy to obtain can bereprogrammed quickly

Not often in repositories Aasen et al 2008 Tolar et al2011

OSNL OCT34 SOX2 NANOG and LIN28 OSKM OCT34 SOX2 KLF4 and MYC

692


Disea

seModelsampMechan

isms














Reprogram cells

iPSCs



Neural cells



693


Disea

seModelsampMechan

isms











CD14

Even

ts (

of m

ax) CD68

100 101 102 103 104

Days100 20 200

Fluorescence


694


Disea

seModelsampMechan

isms



Reprogrammingmethod





ndash









ndash





ndash



ndash




ndash







ndash




ndash
















ndash





ndash









TPP1(infantile)




CLN3( juvenile)



Continued

695


Disea

seModelsampMechan

isms





Table 2 Continued


Reprogrammingmethod











Hepatocytesneurons


Cyclodextrins




ndash



Cyclodextrins



ndash



ndash


ndash


ndash


Gene therapy


Gene therapy


ndash

696


Disea

seModelsampMechan

isms











697


Disea

seModelsampMechan

isms












698


Disea

seModelsampMechan

isms
















699


Disea

seModelsampMechan

isms












700


Disea

seModelsampMechan

isms














































701


Disea

seModelsampMechan

isms


















































702


Disea

seModelsampMechan

isms














































703


Disea

seModelsampMechan

isms







704


Disea

seModelsampMechan

isms














Reprogram cells

iPSCs



Neural cells



693


Disea

seModelsampMechan

isms











CD14

Even

ts (

of m

ax) CD68

100 101 102 103 104

Days100 20 200

Fluorescence


694


Disea

seModelsampMechan

isms



Reprogrammingmethod





ndash









ndash





ndash



ndash




ndash







ndash




ndash
















ndash





ndash









TPP1(infantile)




CLN3( juvenile)



Continued

695


Disea

seModelsampMechan

isms





Table 2 Continued


Reprogrammingmethod











Hepatocytesneurons


Cyclodextrins




ndash



Cyclodextrins



ndash



ndash


ndash


ndash


Gene therapy


Gene therapy


ndash

696


Disea

seModelsampMechan

isms











697


Disea

seModelsampMechan

isms












698


Disea

seModelsampMechan

isms
















699


Disea

seModelsampMechan

isms












700


Disea

seModelsampMechan

isms














































701


Disea

seModelsampMechan

isms


















































702


Disea

seModelsampMechan

isms














































703


Disea

seModelsampMechan

isms







704


Disea

seModelsampMechan

isms











CD14

Even

ts (

of m

ax) CD68

100 101 102 103 104

Days100 20 200

Fluorescence


694


Disea

seModelsampMechan

isms



Reprogrammingmethod





ndash









ndash





ndash



ndash




ndash







ndash




ndash
















ndash





ndash









TPP1(infantile)




CLN3( juvenile)



Continued

695


Disea

seModelsampMechan

isms





Table 2 Continued


Reprogrammingmethod











Hepatocytesneurons


Cyclodextrins




ndash



Cyclodextrins



ndash



ndash


ndash


ndash


Gene therapy


Gene therapy


ndash

696


Disea

seModelsampMechan

isms











697


Disea

seModelsampMechan

isms












698


Disea

seModelsampMechan

isms
















699


Disea

seModelsampMechan

isms












700


Disea

seModelsampMechan

isms














































701


Disea

seModelsampMechan

isms


















































702


Disea

seModelsampMechan

isms














































703


Disea

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704


Disea

seModelsampMechan

isms



Reprogrammingmethod





ndash









ndash





ndash



ndash




ndash







ndash




ndash
















ndash





ndash









TPP1(infantile)




CLN3( juvenile)



Continued

695


Disea

seModelsampMechan

isms





Table 2 Continued


Reprogrammingmethod











Hepatocytesneurons


Cyclodextrins




ndash



Cyclodextrins



ndash



ndash


ndash


ndash


Gene therapy


Gene therapy


ndash

696


Disea

seModelsampMechan

isms











697


Disea

seModelsampMechan

isms












698


Disea

seModelsampMechan

isms
















699


Disea

seModelsampMechan

isms












700


Disea

seModelsampMechan

isms














































701


Disea

seModelsampMechan

isms


















































702


Disea

seModelsampMechan

isms














































703


Disea

seModelsampMechan

isms







704


Disea

seModelsampMechan

isms





Table 2 Continued


Reprogrammingmethod











Hepatocytesneurons


Cyclodextrins




ndash



Cyclodextrins



ndash



ndash


ndash


ndash


Gene therapy


Gene therapy


ndash

696


Disea

seModelsampMechan

isms











697


Disea

seModelsampMechan

isms












698


Disea

seModelsampMechan

isms
















699


Disea

seModelsampMechan

isms












700


Disea

seModelsampMechan

isms














































701


Disea

seModelsampMechan

isms


















































702


Disea

seModelsampMechan

isms














































703


Disea

seModelsampMechan

isms







704


Disea

seModelsampMechan

isms











697


Disea

seModelsampMechan

isms












698


Disea

seModelsampMechan

isms
















699


Disea

seModelsampMechan

isms












700


Disea

seModelsampMechan

isms














































701


Disea

seModelsampMechan

isms


















































702


Disea

seModelsampMechan

isms














































703


Disea

seModelsampMechan

isms







704


Disea

seModelsampMechan

isms












698


Disea

seModelsampMechan

isms
















699


Disea

seModelsampMechan

isms












700


Disea

seModelsampMechan

isms














































701


Disea

seModelsampMechan

isms


















































702


Disea

seModelsampMechan

isms














































703


Disea

seModelsampMechan

isms







704


Disea

seModelsampMechan

isms
















699


Disea

seModelsampMechan

isms












700


Disea

seModelsampMechan

isms














































701


Disea

seModelsampMechan

isms


















































702


Disea

seModelsampMechan

isms














































703


Disea

seModelsampMechan

isms







704


Disea

seModelsampMechan

isms












700


Disea

seModelsampMechan

isms














































701


Disea

seModelsampMechan

isms


















































702


Disea

seModelsampMechan

isms














































703


Disea

seModelsampMechan

isms







704


Disea

seModelsampMechan

isms














































701


Disea

seModelsampMechan

isms


















































702


Disea

seModelsampMechan

isms














































703


Disea

seModelsampMechan

isms







704


Disea

seModelsampMechan

isms


















































702


Disea

seModelsampMechan

isms














































703


Disea

seModelsampMechan

isms







704


Disea

seModelsampMechan

isms














































703


Disea

seModelsampMechan

isms







704


Disea

seModelsampMechan

isms







704


Disea

seModelsampMechan

isms

Induced pluripotent stem cell models of lysosomal storage ... · Induced pluripotent stem cell...

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