La ricerca sulle cellule staminali pluripotenti indotte nell’era della medicina di precisione

Takashi HamazakiNihal El RoubyNatalie C. FredetteKatherine E. Santostefano Naohiro Terada
Pubblicato per la prima volta: 18 Gennaio 2017 https://doi.org/10.1002/stem.2570

I recenti progressi nelle tecnologie di sequenziamento del DNA stanno rivelando come le variazioni genetiche umane si associano a rischi differenziali per la salute, suscettibilità alle malattie, e risposte ai farmaci. Si prevede ora che tali informazioni contribuiscano a valutare i rischi per la salute individuale, progettare piani sanitari personalizzati e curare i pazienti con precisione. È ancora impegnativo, Tuttavia, comprendere come tali variazioni genetiche causino le alterazioni fenotipiche nelle patologie e nella risposta al trattamento. Cellula staminale pluripotente indotta dall'uomo (iPSC) Le tecnologie stanno emergendo come una strategia promettente per colmare le lacune di conoscenza tra gli studi di associazione genetica e i meccanismi molecolari sottostanti. Breakthroughs in genome editing technologies and continuous improvement in iPSC differentiation techniques are particularly making this research direction more realistic and practical. Pioneering studies have shown that iPSCs derived from a variety of monogenic diseases can faithfully recapitulate disease phenotypes in vitro when differentiated into disease‐relevant cell types. It has been shown possible to partially recapitulate disease phenotypes, even with late onset and polygenic diseases. Più recentemente, iPSCs have been shown to validate effects of disease and treatment‐related single nucleotide polymorphisms identified through genome wide association analysis. In this review, we will discuss how iPSC research will further contribute to human health in the coming era of precision medicine. Cellule staminali 2017;35:545–550

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Significance Statement
Each person has a unique set of gene variations that affect susceptibility to and protection from both common and rare disorders. Although associations between human health and individual variabilities need to be validated in principle, it is still challenging to validate effects on actual biological processes. Human induced pluripotent stem cells provide a unique opportunity to dissect the roles of genetic variants for pathogenesis. This review overviews recent developments how induced pluripotent stem cell research will further contribute to human health in the coming era of precision medicine.

Recent Advances in Human Genome Research Leading to an Era of Precision Medicine
The first human reference genome was drafted in 2001 after an international collaborative effort between academic institutions, with the goal of characterizing genetic variations across the human genome 1, 2. After completion of the human genome project, extended efforts to catalog these genetic variants have been made, the first of which was the international HapMap project, which aimed to build haplotype blocks of the most common genetic variations, namely, single nucleotide polymorphisms (SNPs) across human populations 3, 4. IL 1000 genomes project (http://www.1000genomes.org/) characterized common and rare genetic variations in 2,504 individuals from 26 different populations using next generation sequencing based methods and dense genotyping arrays 5. With the influx of information and availability of genotyping platforms, è diventato possibile interrogare simultaneamente milioni di SNP da centinaia a migliaia di individui attraverso l'analisi dell'associazione dell'intero genoma (GWAS). Questo approccio ha rivoluzionato il campo della genetica, consentendo che molte associazioni genetiche vengano effettuate attraverso un agnostico, approccio non guidato da ipotesi. Fin dalle prime ondate di pubblicazioni GWAS in 2005, 23,058 Le associazioni dei tratti SNP sono state pubblicate nel National Human Genome Research Institute - European (NHGRI–EBI) totale del catalogo 2,502 Studi GWAS 6

La farmacogenomica è un campo dedicato all’identificazione dei determinanti genetici della risposta ai farmaci o degli effetti avversi ed è fondamentale per il concetto di medicina di precisione, che implica l’utilizzo del genotipo per guidare la selezione dei farmaci. Nonostante un’era fruttuosa di scoperte GWAS nella farmacogenomica, many of these variants have not yet made it to clinical utilization. Uno dei principali ostacoli all’implementazione della farmacogenomica è l’ignoto collegamento meccanicistico sottostante(S) tra fenotipo e genotipo della risposta al farmaco. Mentre alcuni SNP si trovano in geni biologicamente rilevanti per il fenotipo studiato, la maggior parte delle varianti si trova in aree non codificanti del genoma, dove non è nota una connessione diretta al fenotipo e si presume un ruolo nella regolazione genetica. Decifrare il ruolo dei segnali genetici associati per rivelare come queste varianti funzionano a livello molecolare e cellulare è fondamentale per una chiara comprensione del processo patologico e per l’implementazione della medicina personalizzata.

In 2015, l’amministrazione Obama ha annunciato il lancio di un’iniziativa di medicina di precisione da parte del National Institutes of Health 7, 8 (https://www.nih.gov/precision-medicine-initiative-cohort-program) In questa iniziativa, saranno condotti studi di coorte su larga scala per integrare lo stile di vita individuale, ambiente, e informazioni genomiche, costruire una base di conoscenze completa in grado di prevedere il rischio di malattia individuale e la risposta ai trattamenti. Il sequenziamento del genoma e la caratterizzazione della variabilità genetica sono stati i primi passi verso gli obiettivi della medicina di precisione che prevedevano l'utilizzo di dati individuali per la diagnosi, trattare, e prevedere la risposta ai trattamenti medici. Con l’avvento di tecnologie di sequenziamento di prossima generazione ad alto rendimento e costi in rapida diminuzione, è possibile eseguire l'intero genoma, intero esoma (codifica proteica), e trascrittoma (Trascrizione dell'RNA) sequenziamento per sondare le associazioni con tratti fenotipici/condizioni di malattia. Inoltre, integrazione di dati omici multidimensionali (per esempio., genomica, trascrittomica, epigenomica, proteomica, e metabolomica) holds promise to elucidate biological interactions involved in complex diseases and to shed light on important genetic variants that may be missed with genetic approaches due to lack of strict statistical significance.

Although associations between human health and individual variabilities need to be validated in principle, it is still challenging to validate effects on actual biological processes. The use of induced pluripotent stem cells (iPSC) is an attractive system for modeling genetic variants to study molecular consequences in a relevant cell type. iPSC technology reprograms a fully mature somatic cell into a pluripotent stem cell that retains all the genetic characteristics of an individual patient. These iPSCs can then be differentiated into multiple different tissue types (for a growing list of validated tissue differentiation milestones, see Cell Stem Cell 18, Marzo 2016) 9, 10. I sistemi di editing genetico come CRISPR-CAS9 o TALEN amplieranno gli studi che mirano a svelare i meccanismi e le conseguenze funzionali delle variazioni genetiche 11, 12. Questo può essere fatto modificando singoli nucleotidi, introducendo o invertendo mutazioni nelle iPSC e osservando i cambiamenti fenotipici nelle cellule terminalmente differenziate.

IPSC per trovare cure per i disturbi monogenici
Le malattie possono avere eziologie monogeniche o poligeniche. Malattie monogeniche, causate dall'ereditarietà di un singolo gene difettoso sono considerate rare perché la prevalenza di ciascuna malattia è piuttosto bassa, solitamente inferiore a 1/10,000 alla nascita. Negli ultimi anni il numero di malattie con loci genetici causali noti è raddoppiato 10 anni come visto in Online Mendelian Inheritance in Man (Oh mio Dio) statistiche di ingresso. Miglioramento della diagnostica genetica e implementazione di programmi di screening (per esempio., screening neonatale e screening ad alto rischio) consentire di identificare le persone affette da questa malattia rara. Di conseguenza, colpiscono malattie genetiche rare 350 milioni di persone in tutto il mondo e la prevalenza globale di tutte le malattie monogeniche alla nascita è di circa 1/100. Dalla creazione delle iPSC umane in 2007 13, 14, c'è stata una straordinaria aspettativa di utilizzare le cellule per modellare le "malattie umane in un piatto" 15, 16. Studi pionieristici hanno dimostrato che le iPSC derivate da una varietà di disturbi monogenici possono ricapitolare fedelmente i fenotipi della malattia in vitro quando differenziate in tipi cellulari rilevanti per la malattia 17, 18. La generazione di linee iPSC da pazienti affetti da queste malattie monogeniche è un approccio utile per stabilire un modello umano in vitro duraturo ed è stato dimostrato in numerosi studi pubblicati 19-22. Gli sforzi di collaborazione tra le comunità di ricerca hanno prodotto una varietà di linee iPSC specifiche per la malattia facilmente disponibili attraverso le banche iPSC 23 i ricercatori potrebbero essere in grado di trovare linee di cellule staminali di interesse per condurre ulteriori studi meccanicistici o applicare direttamente le cellule per lo screening farmacologico.

Sebbene siano state fatte molte scoperte importanti per le malattie monogeniche attraverso la ricerca iPSC, uno degli studi più interessanti è un recente rapporto sull'acondroplasia di Yamashita et al. 24. È importante sottolineare che, gli autori hanno stabilito attentamente un metodo per differenziare le iPSC in condrociti per formare tessuto cartilagineo. This was a critical step for Yamashita et al., because developing appropriate differentiation protocols for disease‐relevant cell types can still be a limiting factor for iPSC research. They were able to successfully recapitulate abnormal cartilage formation during in vitro differentiation of iPSCs derived from patients with achondroplasia when compared to those from healthy controls. Furthermore, upon compound screening, they showed that statins, widely used lipid lowering medications, unexpectedly corrected the degraded cartilage in the iPSC model. This exemplary work clearly recapitulates disease processes in a dish and demonstrates the utility and promise of iPSC models to discover novel treatments for rare monogenic disorders.

Oltre alla differenziazione monostrato bidimensionale o tridimensionale di base (3D) differenziazione aggregata, diversi gruppi hanno sviluppato sofisticati protocolli di differenziazione 3D, spesso definiti “cultura organoide” per la loro capacità di formare strutture organizzate che ricordano gli organi in via di sviluppo. In particolare, per la coltura di organoidi del sistema nervoso centrale, Lancaster et al. ha dimostrato che le iPSC derivate da un paziente microcefalico formavano effettivamente un organoide cerebrale più piccolo rispetto alle iPSC di un controllo sano 25. Allo stesso modo, diverse tecniche di coltura di organoidi per iPSC si sono evolute per generare altri tipi di tessuti e organi (coppa ottica, ghiandola pituitaria) 26, 27. Indubbiamente, these breakthrough discoveries will provide necessary complexity to more accurately model disorders and allow for greater opportunity for preclinical testing of treatment options for human cells in vitro.

iPSCs to Define Further Phenotypic Variations in Monogenic Disorders
In human monogenic disorders, a single gene mutation is predominantly responsible for the phenotype of the disease. In many cases, we can predict how a specific mutation in a single gene affects protein function (per esempio., residual enzyme activity), which correlates with severity and presentation of a disease. È, Tuttavia, still challenging to accurately predict clinical symptoms, severity and onset of the disease from the type of mutation. An example of this challenge is Gaucher disease (GD), una malattia autosomica recessiva causata da mutazioni nel gene GBA che codifica per la glucocerebrosidasi (GCase) 28. GCase è un enzima lisosomiale che catalizza l'idrolisi del glicolipide glucocerebroside a ceramide e glucosio. I pazienti con GD mostrano un ampio spettro di sintomi clinici inclusa l'epatosplenomegalia, deformità ossea, anomalia ematologica, e sintomi neurologici. La mutazione N370S nel GBA si trova spesso nel tipo 1 GD, che si presenta con sintomi non neuronali. D'altra parte, la mutazione L444P si trova spesso nel tipo 2 O 3 GD, che si presenta con sintomi neurologici. Anche gli eventi di ricombinazione del locus GBA con uno pseudogene vicino sono stati collegati ad alcune presentazioni cliniche insolite 29. Variabilità fenotipiche, Tuttavia, sono stati osservati tra pazienti con mutazioni GBA identiche, come ad esempio tra coppie di fratelli affetti, and even between identical twins. In a monozygotic twins case, one was affected with GD but the other had no clinical symptoms even with low GCase activity 30. In GD, deficiency of GCase leads to accumulation of the intermediate metabolite glucosphingolipids glucosylceramide, which is further metabolized into sphingosine by an extra lysosomal GCase, GBA2. interestingly, deletion of GBA2 in a GD mouse model rescued visceral and bone symptoms, suggesting that GBA2 could potentially be targeted to ameliorate certain debilitating manifestations of GD 31.

In another study, Awad et al. uncovered involvement of lysosomal dysfunctions and an autophagy block during the neurodegenerative process of GD by using neuronal cells derived from the iPSCs of patients with type 2 GD (neuropathic form). Upon rapamycin treatment, neuronal death was preferentially induced in neurons from type 2 GD‐iPSCs, but not type 1 GD‐iPSCs. Although expression of the transcription factor EB (TFEB), the master regulator of lysosomal genes was downregulated, overexpression of TFEB only partially restored the neurodegenerative process in neurons from type 2 GD‐iPSCs 32. These findings represent a promising avenue to identify genetic and nongenetic (epigenetic and/or environmental) modulators that influence disease‐causing mutations. Since iPSCs can be generated from individuals with various genetic backgrounds, and genomic loci can be targeted in iPSCs, disease‐relevant cell types obtained from such iPSCs will be an indispensable tool to validate newly proposed disease mechanisms and to screen environmental factors/small compounds to modulate disease phenotypes.

iPSCs to Dissect the Roles of SNPs in Polygenic Disorders and Differential Drug Responses
Many common human diseases and traits are influenced by several genetic and environmental factors. Polygenic diseases result from the additive inheritance of multiple subtle polymorphisms, culminating in an affected phenotype. In 2016, nearly 5,000 disease phenotypes have been cataloged and linked with causal genetic loci in OMIM (http://omim.org/statistics/entry). GWAS have successfully identified hundreds of genetic variants associated with various conditions and have provided valuable insights into diagnostics, prognosis, and therapeutic optimization for complex human diseases 33. An example of a common, complex and polyfactorial disease is hypertension (HTN). HTN is a major health burden in the U.S. that affects approximately 80 million people 34 e il trattamento diretto del paziente ammonta quasi a 40 miliardi di dollari all'anno 35. Inoltre, L'HTN aumenta il rischio di malattie cardiovascolari avanzate come ictus e insufficienza cardiaca 34, 36 Numerosi agenti antipertensivi come i diuretici, ACE inibitori, bloccanti dei recettori dell’angiotensina, Attualmente sono disponibili beta-bloccanti e inibitori dei canali del calcio, ma la loro efficacia sulla pressione sanguigna varia da individuo a individuo. GWAS e casi di studio sui geni candidati hanno identificato diverse varianti genetiche che possono regolare la pressione sanguigna o contribuire al percorso farmacologico di un farmaco 37. I modelli animali sono stati utilizzati intensamente per studiare malattie sistemiche come l'HTN, tuttavia potrebbero non essere sempre adatti a comprendere l'impatto biologico delle varianti genetiche umane. È anche difficile ottenere un gran numero di tessuti appropriati rilevanti per il fenotipo di interesse (per esempio., muscolatura liscia vascolare o endotelio) da una persona con un genotipo specifico per testare le conseguenze biologiche o funzionali di queste variazioni genetiche. Per combattere tali sfide, Biel et al. costruito un repository iPSC da 17 Pazienti HTN, di cui erano disponibili le variazioni SNP dell’intero genoma e le risposte cliniche ai farmaci antipertensivi 38. Le iPSC sono state generate da un prelievo di sangue di cellule mononucleate del sangue periferico raccolte dai partecipanti alla valutazione farmacogenomica della risposta antipertensiva (PERA) studio 39 (https://clinictrials.gov/NCT00246519). Biel et al. quindi differenziato queste iPSC in cellule muscolari lisce vascolari e quantificata la loro contrazione in risposta a vari stimoli fisiologici 38. Furthermore, the study also demonstrated the ability of iPSCs to recapitulate a SNP‐associated modification of PRKCA expression. The SNP rs16960228 has been well‐documented in multiple GWAS cohorts to associate with a hypertensive drug response as well as differential expression levels of PRKCA. These data support the applicability and translational value of iPSCs in modeling GWAS findings.

Another example of cardiovascular disease modeling using iPSCs is presented by Ebert et al. 40. Ebert et al. studied a SNP in the gene coding for aldehydronease 2 enzyme, which confers a loss of cardioprotective effects and increases risk for coronary artery and ischemic heart disease. Cardiomyocytes (CM) differentiated from iPSCs derived from an east Asian population genotyped for a common ALDH2* SNP (MAF = 0.08), demonstrated that CMs carrying the ALDH2* genotype had increased levels of oxidative stress and aldehyde byproduct 4HNE buildup. Accumulation of these two byproducts resulted in dysregulated cell cycle and apoptosis signaling, which exacerbated damage and reduced cellular recovery to ischemic challenge in the CMs of ALDH2* carriers, thereby establishing the cellular mechanisms for increased disease susceptibility for a single SNP.

Finalmente, it is well established that differences in susceptibility and drug response to HTN and multiple polygenic diseases varies by ethnic group (cioè., African American vs. Western European American), it will be important to understand the utility of such iPSCs libraries based on ethnic background. To address the challenge of diversity in disease genetics using iPSCs, Chang et al. ha segnalato la costruzione di una banca iPSC composta da popolazioni etnicamente diverse 41. Presi insieme, questi studi dimostrano che una libreria iPSC con SNP definiti e dati fenotipici sarà una risorsa utile per convalidare gli effetti degli SNP identificati da GWAS e per facilitare la comprensione meccanicistica delle condizioni fisiologiche e patologiche umane.

È sempre più importante capire come specifiche varianti di rischio contribuiscono funzionalmente alla patogenesi sottostante. Rispetto alla mutazione di un singolo gene riscontrata nelle malattie monogeniche, gli effetti delle varianti SNP possono spesso essere minori o sottili. È importante utilizzare cellule isogeniche per decodificare il significato di tali varianti genetiche. Recenti progressi nella tecnologia di modifica del genoma (per esempio., Sistemi CRISPR/Cas9) have simplified the ability to target specific genetic loci for functional studies. Gene‐editing methods in iPSC’s has been reviewed in detail elsewhere 42, 43. Soldner et al. demonstrated functional connect of GWAS‐identified risk variants of Parkinson’s disease in neurons derived from human iPSCs 44. They focused on Parkinson’s disease associated risk SNPs, which were located in an α‐synuclein (SNCA) regulatory region based on genome‐wide epigenetic information. By establishing TaqMan SNP genotyping assays for quantitative reverse transcription polymerase chain reaction, they were able to monitor subtle changes in allele‐specific transcription of SNCA between two SNPs located in the SCNA enhancer region. As a follow up approach, they knocked‐out the single allele of the SNPs using the CRISPR/Cas9 system to see how the SNPs affect SNCA expression. They found that allele‐specific expression roughly translated to an increase of total SNCA expression of 1.06 times in neurons and 1.18 times in neural precursors. Furthermore, sequence‐dependent binding of the brain‐specific transcription factors EMX2 and NKX6‐1 on this locus was revealed.

As part of the Next Generation Genetic Association Studies (Next Gen) Programma, various fields of researchers are now depositing iPSC resources, generated from individuals representing various conditions as well as healthy controls, with the goal of following up findings from functional genomics with mechanistic investigations. The program is aimed at generating iPSC lines from more than 1,500 individuals some of which are available through a public iPSC bank (http://www.wicell.org/home/stem-cell-lines/collections/collections.cmsx). Each iPSC line is linked with clinical data (per esempio., lipid condition, QT interval and ECG cardiac trait, pulmonary HTN) as well as age, gender and ethnic background. SNP genotyping, espressione genica, and ‐omics analysis data will be available for these lines in the future.

It is critically important that high quality iPSC lines are also paired with high quality genetic and clinical data. This can be facilitated through large collaborations that generate harmonized phenotypes through established criteria for diagnosis and accurate phenotype definition, with an ultimate goal of reducing phenotype variability. The more accurately a phenotype is defined, the higher the likelihood of identifying the culprit gene and genetic variants 45. With such standardized phenotypes, advancement of genetic discoveries and their replication can be made, which can be carried forward to iPSC studies using the tissues of relevance. A study by Akawi et al. shows that the value of deep sequencing information is decreased if it is not coupled with high quality phenotype data from patients 46. An analogy can be made here as we think of the diminished value of iPSC if we do not have an accurately defined clinical phenotype that will be ultimately translated into a cellular phenotype in a dish. Perciò, it is increasingly important for the collaborative genetic consortia to establish procedures for phenotype ascertainment to reap the maximum benefit of iPSC modeling.

iPSCs to Understand Genetic and Phenotypic Variations Beyond GWAS
It has been recently shown that especially rare genetic variants, such as homozygous variant defects resulting in rare pathologies, can associate for increased risk of more common maladies as well. Per esempio, having a pathogenic GBA mutation for Gaucher’s Disease (GD) 28 (per esempio., N370S, L444P) in one allele (carrier) will not usually manifest the full symptoms of GD, but does increase risk for Parkinson’s disease 47, 48. The odds ratio for the GBA mutation in PD was greater than 5, which is unusually high compared to risk loci found from GWAS 49. D'altra parte, there is an example where a rare genetic variant has a protective effect on a complex disease. SLC30A8 encodes an islet zinc transporter (ZnT8) and ZnT8 has been known as a key regulator of insulin secretion in pancreatic beta cells. 50. Furthermore, large scale GWAS identified a common variant (p.Trp325Arg) on SLC30A8 that results in an increased risk for type 2 diabete (T2D) 51-53. Animal studies with this variant, Tuttavia, showed conflicting results for pathogenesis of T2D. Breakthrough have been made through international collaborative studies, which aimed to find loss‐of‐function variants protective against T2D. Sequencing data from more than 150,000 people identified heterozygous individuals for a nonsense variant (P. Arg 138*) in a Finnish cohort exhibited a 60% reduced risk of type 2 diabete 54. Recentemente, Chen et al. proposed the reverse approach to find healthy individuals resilient to highly penetrant forms of genetic childhood disorders. They sequenced 874 genes in 589,306 genomes and found 13 adults carried mutations for 8 severe Mendelian conditions with no reported clinical manifestation of the indicated disease 55. This could be a first step toward uncovering protective genetic variants, and further mechanistic studies are anticipated. As discussed above, iPSCs will serve as a powerful tool here as well to dissect molecular mechanisms of the genetic associations, hopefully leading to novel therapeutic discoveries.

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Conclusione
Gathering our knowledge of human disease genetics, we start to realize that each person has a unique set of variants that contribute to susceptibility and protection for a variety to disorders. Phenotypes vary even within rare monogenic diseases based on their mutation types, genetic background and environmental factors. To further advance precision medicine, it will become increasingly important to dissect molecular mechanisms underlying these genotype‐phenotype associations. Human iPSCs provide a unique opportunity to fill these knowledge gaps, and their anticipated increase in utilization by researchers via cellular repositories position them as a crucial reagent for the next generation of disease genomics studies (Fig. 1).

Figure 1
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Stem cell tactic to advance precision medicine. Each person has a unique set of gene variations that affect susceptibility to and protection from both common & rare disorders. Human iPSCs provide a unique opportunity to dissect the roles of genetic variants for pathogenesis. Abbreviation: iPSC, induced pluripotent stem cells.

Acknowledgments
This work was supported in part by Japan Agency for Medical Research and Development, AMED, Practical Research Project for Rare/Intractable Diseases, National Institutes of Health (GM119977 and DK104194), American Heart Association (16GRNT30980002), and the University of Florida Clinical and Translational Science Institute (UL1TR001427). NCF is a recipient of postdoctoral fellowship T32 DK074367.


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