Human induced pluripotent stem cell-derived neuronal progenitors
are a suitable and effective drug discovery model for neurological mtDNA disorders Jonas Walter1*, Sarah Louise Nickels2*, Jens Christian Schwamborn2 1 Braingineering Technologies SARL, Esch-sur-Alzette, Luxembourg; 2University of Luxembourg, Luxembourg Centre for Systems Biomedicine, Belvaux, Luxembourg *These authors contributed equally to this work. Correspondence to: Prof. Dr. Jens Christian Schwamborn. Université du Luxembourg LCSB-Luxembourg Centre for Systems Biomedicine, 6 Avenue du Swing L-4362 Belvaux, Luxembourg. Email: jens.schwamborn@uni.lu. Provenance: This is an invited Editorial commissioned by Editor-in-Chief Zhizhuang Joe Zhao (Pathology Graduate Program, University of Oklahoma Health Sciences Center, Oklahoma City, USA). Comment on: Lorenz C, Lesimple P, Bukowiecki R, et al. Human iPSC-derived neural progenitors are an effective drug discovery model for neurological mtDNA disorders. Cell Stem Cell 2017;20:659-74.e9.
Received: 23 October 2017; Accepted: 15 November 2017; Published: 28 December 2017.
doi: 10.21037/sci.2017.11.08 View this article at: http://dx.doi.org/10.21037/sci.2017.11.08
Mitochondria are double-membraned organelles of disease relevant tissues (8), and possess incompatible endosymbiotic origin (1). Mitochondria are critically metabolic and bioenergetic states (1). Different cell types involved in cellular homeostasis and need to be tightly and subtypes strongly differ in their bioenergetic demands. controlled to maintain normal cellular function (2). Simplified, stem cells rather rely on glycolysis more than on Malfunctioning mitochondria can interfere with mitochondrial oxidative phosphorylation (OXPHOS). In cellular homeostasis in multiple ways (3). Consequently, contrast, during commitment and differentiation especially mitochondria have been associated with diverse diseases, cells from the neuronal lineage increasingly address their including neurological and neurodegenerative disorders (4). bioenergetic demands by using OXPHOS for energy Among those diseases, a group of severe disorders is generation (9). directly associated with specific mutagenesis of the Motivated by these limitations, Prigione and colleagues mitochondrial DNA (mtDNA) (5,6). Thus, despite the reached out to find a new, more advanced, and highly fact that most mitochondrial genes are encoded in the relevant human model for mtDNA-associated diseases (10). nucleus, mutagenesis of the mtDNA can have severe With the advent of induced pluripotent stem cells (iPSCs) outcomes. Hence, the status of the mtDNA is of crucial it became possible to derive patient-specific cell types, relevance for normal cellular function (7). Several studies which can be used for in vitro disease modeling (11). revealed that modeling and targeting mtDNA related iPSCs based in vitro disease modeling aims at revealing the diseases is experimentally extremely challenging. Therefore, disease underlying molecular mechanisms in order to gain the modeling of human mtDNA disorders is often a deeper understanding of the pathophysiological aspects based on so-called cybrid models (cytoplasmic hybrids), of disorders, finally resulting in the emergence of potential combining immortalized cells and the particular patient’s drug targets. Consequently, iPSC-based models bear great mitochondria. These models were able to recapitulate potential for the discovery of novel treatment strategies (12). the influence of patient specific mitochondria on cellular In particular, for neural diseases iPSC-technologies physiology, but unfortunately, cybrid models have limited revolutionized the availability and accessibility of the disease physiological relevance. Cybrid models lack the patient- relevant material. specific genetic background, are not suitable for modeling Here, Prigione and colleagues, applied for the first time
iPSC techniques for modeling a mtDNA associated disease outcome of a differentiation is bearing certain variability. and derived patient-specific iPSCs from Leigh syndrome Prigione and colleagues first characterized the derived patients (13). Since iPSC-based models were never used for iPSCs used in this study and further demonstrated that these modeling mtDNA disease before, the authors performed are able to give rise to NPCs. The commitment of NPCs to thoroughly validation steps. Importantly, inducing the neural lineage was confirmed by immunofluorescence pluripotency in somatic cells bears pitfalls for modeling staining and calcium signaling analyses. At the same mtDNA diseases, the cellular reprogramming is not only time, NPCs remained electrophysiological inactive resetting the potency of the cells but also extensively and consequently non-neuronal. Further, the authors remodels the cells’ metabolism and mitochondria (14,15). performed transcriptional analyses and demonstrate the In addition, the conventional reprogramming process high similarity of the generated NPCs to other published starts from somatic cells and includes clonal expansion. NPC variants. In this context the authors confirmed that Clonal expansion is bearing the likelihood of an enrichment the NPC transcriptome, indeed, overlaps the most with of highly mutagenic mtDNAs during mitochondrial the transcriptional state reported for the early embryonic inheritance due to the presence of heteroplasmy of neurodevelopment (20) emphasizing the physiological mtDNA. This bottleneck could potentially result in the validity of the NPC model. expansion of a pool of cells carrying a variety of additional, After successful and thorough characterization of the not disease-relevant mtDNA mutations, aggravating iPSCs and the derived NPC model, Prigione and colleagues the identification of pathophysiological mechanisms (6). continued and assessed the metabolic status in the NPC Another question that arises during an iPSC-based disease model. In particular, the authors validated the mitochondrial modeling is the selection of the most relevant cell type microstructural remodeling taking place during the available for modeling the particular disease. Prigione derivation of NPCs and confirmed a high similarity to and colleagues decided to utilize neural progenitor cells neurons and dissimilarity to immature mitochondria in (NPCs) for modeling the impact of Leigh syndrome on iPSCs. In addition to structural remodeling, the authors normal neurodevelopment (16). The use of iPSC derived also confirmed a bioenergetic shift of NPCs towards neural stem cells for modeling neurological diseases has neurons rather than iPSCs; this is specifically prominent been extensively reviewed before (17,18). In this study, the in the downregulation of glycolytic activity. Next, and authors demonstrate that NPCs represent a physiologically most importantly, Prigione and colleagues were able to relevant cellular model for neurological mtDNA disorders. validate that NPCs, despite potential pitfalls during the Accordingly, Prigione and colleagues dedicate the reprogramming and clonal expansion processes, retain the first part of their manuscript to the characterization and disease-specific mtDNA mutation pattern. In particular, validation of the applicability of iPSC-based NPCs for the authors describe the preservation of the spectrum of the mtDNA disease modeling. The authors describe the D310 tract hyperplasmy in the hypervariable region (57–372) derivation of the pool of iPSCs used throughout the of the D-loop. Due to the continuous advancement of study. The authors further characterized and validated reprogramming techniques, the authors used iPSCs that the neural identity of their neural precursor cell model. were generated by different techniques. However, in the NPCs were derived from iPSCs using a small molecules context of the mtDNA analyses the authors were able to based derivation protocol, which represents a robust, cost- demonstrate that the preservation of mtDNA patterns is efficient and highly defined patterning strategy (16,19). The independent of the iPSC derivation technique. NPCs are reminiscent of the early neural tube development After these model validation steps Prigione and colleagues and retain the differentiation potential into all cell types of continued with the derivation and characterization of a pool the central nervous system (16). Apart from their disease of Leigh syndrome specific NPCs. In a first step, the authors specific relevance NPCs in comparison to iPSCs are easier focus on ATP-related plasma membrane resting potential, to handle, cheaper in their maintenance, and already proliferation, and mitochondria specific reactive oxygen committed to the neural lineage. Specifically, the neural phenotypes. They demonstrate that NPCs retain Leigh commitment is important for studying the recapitulation syndrome specific pathogenicity in comparison to specific of the disease relevant neural alterations. The advantage cybrids and parental fibroblasts. Further, the authors based of neural progenitor cultures over neuronal differentiation their mitochondria related phenotyping on the assessment cultures is clearly their high definition and purity, while the of the mitochondrial membrane potential (MMP). Using
MMP analysis, the authors verify a hyperpolarization of Acknowledgements
mitochondria in association with the Leigh syndrome. None. A subsequent transcriptional analysis revealed calcium signaling pathway related genes as differently regulated, a phenotype that is conserved between fibroblasts, iPSCs, Footnote and NPCs. Among the differently regulated calcium Conflicts of Interest: JW is employee of the biotech company signaling genes specifically LETM1, VDAC3, ATP50 were Braingineering Technologies SARL. JCS is co-founder and strongly altered between control and Leigh syndrome CSO of Braingineering Technologies SARL. The other NPCs. Consequently, the authors applied calcium-imaging author has no conflicts of interest to declare. analysis to assess the pathophysiological impact of the disease associated transcriptional changes. Thereby they verified the negative impact of the transcriptional changes References on the time-resolved calcium signaling activity. Next, 1. Carelli V, Chan DC. Mitochondrial DNA: impacting specific compound modulation of the calcium signalling central and peripheral nervous systems. Neuron revealed the mitochondrial specificity of the phenotype. 2014;84:1126-42. In a final step, the authors transferred the image based 2. Suen DF, Norris KL, Youle RJ. Mitochondrial dynamics MMP phenotyping to a high-throughput analysis pipeline and screened a compound library of 130 selected FDA and apoptosis. Genes Dev 2008;22:1577-90. approved compounds. After 24 h of treatment, the effect 3. Wang X. The expanding role of mitochondria in apoptosis. of the compounds on the MMP was analyzed. This screen Genes Dev 2001;15:2922-33. revealed several potential rescue compounds. Among these 4. Winklhofer KF, Haass C. Mitochondrial dysfunction in hits Avanafil, a PDE 5 inhibitor, which is approved for Parkinson’s disease. Biochim Biophys Acta 2010;1802:29-44. treating pulmonary arterial hypertension was of particular 5. Taylor RW, Turnbull DM. Mitochondrial DNA mutations interest. Using Avanafil the authors, in addition to the in human disease. Nat Rev Genet 2005;6:389-402. MMP rescue, also verified a rescue of ATP production and 6. Tuppen HA, Blakely EL, Turnbull DM, et al. calcium signaling. Mitochondrial DNA mutations and human disease. In summary, Prigione and colleagues reached several Biochim Biophys Acta 2010;1797:113-28. milestones advancing the field of mtDNA diseases. Most 7. Gilkerson R, Bravo L, Garcia I, et al. The mitochondrial importantly, despite the potential pitfalls, the authors nucleoid: integrating mitochondrial DNA into verified iPSC-based modeling approaches for studying cellular homeostasis. Cold Spring Harb Perspect Biol mtDNA diseases. The authors demonstrate that iPSC-based 2013;5:a011080. models perfectly retain the characteristics of the mtDNA. 8. D’Aurelio M, Vives-Bauza C, Davidson MM, et A finding of outstanding importance for all iPSC-based al. Mitochondrial DNA background modifies the studies involving mitochondria. The authors further verified bioenergetics of NARP/MILS ATP6 mutant cells. Hum that patient-specific iPSCs derived NPCs recapitulate Mol Genet 2010;19:374-86. the most important Leigh syndrome related alterations 9. Xu X, Duan S, Yi F, et al. Mitochondrial regulation in in mitochondria. They also demonstrate that NPCs pluripotent stem cells. Cell Metab 2013;18:325-32. represent a valuable tool for high throughput screening 10. Lorenz C, Lesimple P, Bukowiecki R, et al. Human iPSC- approaches and drug discovery strategies. Compared to Derived neural progenitors are an effective drug discovery neuron based screening this shortcut proves NPC models model for neurological mtDNA disorders. Cell Stem Cell an ideal tool for the implementation of new treatment and 2017;20:659-74.e9. prevention strategies in precision medicine. Conclusively, 11. Takahashi K, Yamanaka S. Induction of pluripotent stem the study holds great promises not only for mtDNA related cells from mouse embryonic and adult fibroblast cultures disease but also for other diseases involving mitochondrial by defined factors. Cell 2006;126:663-76. dysfunctions such as seen in Parkinson’s disease (21). Only 12. Reinhardt P, Schmid B, Burbulla LF, et al. Genetic further studies will be able to reveal the outcome of such correction of a LRRK2 mutation in human iPSCs links alterations e.g., on neurodevelopment or the definition of parkinsonian neurodegeneration to ERK-dependent an appropriate treatment window. changes in gene expression. Cell Stem Cell 2013;12:354-67.
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doi: 10.21037/sci.2017.11.08 Cite this article as: Walter J, Nickels SL, Schwamborn JC. Human induced pluripotent stem cell-derived neuronal progenitors are a suitable and effective drug discovery model for neurological mtDNA disorders. Stem Cell Investig 2017;4:101.
