University of Groningen
Induced pluripotent stem cells: cell therapy and disease modeling Thiruvalluvan, Arun
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Chapter 6
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Recent progress in the field of stem cell research has opened up multiple opportunities in developmental biology and regenerative medicine1. Especially, iPSCs derived from many
patient cell types (fibroblasts, keratinocytes, urine cells, etc.) can be used to model different diseases and mimic an in-vivo microenvironment in dishes2, 3. In regenerative
medicine, autologous patient iPSC-derived cells can be implanted at the site of injury to replace damaged or degenerated tissue4. Another important application offered by the
iPSC technology is its use for the screening of novel therapeutic drugs in patient-derived cells5, 6. Moreover, the groundbreaking CRISPR/Cas9 gene editing technology enables us to
correct gene mutations in patient-derived cells and allows us to identify putative disease mechanisms7, 8. Thus, autologous iPS cells allow us to model complex disorders and
provide new possibilities for development of personalized therapy9-11. From the moment
the iPSC technology was discovered, it has been further improved and successfully applied in various fields of biology. In this dissertation, we have evaluated the potential use of iPSC-derived cells as a source for cell-based remyelination therapy for MS and as a tool for modeling of the SCA3 disease.
In chapter I, we describe the induced pluripotent stem cell (iPSC) technology and its application in various fields of biology. The ability to generate embryonic stem cell like cells by reprogramming somatic cells provides new opportunities for disease modeling and drug screening12, 13. Although animal models such as mice, rats, and nonhuman
primates have been used to model various human diseases, due to the complexity of some disorders, they were far from realistic.. Moreover, the physiological conditions in mice and human differ in many ways due to variations in tissue organization and cellular functions14, 15. This physiological difference can make it difficult to identify disease-initiating
mechanisms in animal models. So, we have gratefully adopted the use of patient iPSC-derived cells as a tool to identify various risk factors in diseases like multiple sclerosis (MS). In addition, patient derived iPSCs are useful to model for instance complex metabolic disorders and early- and late-onset monogenic diseases (spinocerebellar ataxia-3 and Huntington’s disease)2, 16. Additionally, the possibility of obtaining iPSCs using
non-integrative methods (employing episomal vectors) allow us to study disease phenotypes without any genome modification17. The ability to differentiate these pluripotent stem cells
into multiple cell types allows us to study pathogenic mechanisms in the specific cells that are affected in that particular disorder. Reprogramming fibroblasts from different patients with the same genetic mutation provides the possibility to analyze the effect of the same mutation in different genetic backgrounds18. In the future, the above-mentioned technical
possibilities will allow us to develop strategies for personalized stem cell therapy for various genetic- and other complex disorders. Recent advancement in reprogramming techniques allowed us to derive cell types by direct transdifferentiation of somatic cells (like fibroblasts) avoiding risky and tedious differentiation procedures19. This could provide
new possibilities in regenerative medicine. Over all, iPS technology and related (trans)differentiation techniques have provided a boost in regenerative medicine and in understanding pathogenesis by disease modelling.
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In chapter II, we described experiments concerning the potential applicability of cell replacement therapy for MS. Restoring the myelin sheath around axons damaged in MS by the implantation of exogenous remyelinating cells may protect neurons and possibly reduce the disease severity. We therefore generated iPSCs from human fibroblasts and differentiated these cells into OPCs. We tested the capacity of these human iPSC-derived OPCs to induce remyelination in-vitro and in-vivo. Previous studies have shown that human iPSC-derived oligodendrocytes are capable of complete remyelination after transplantation in a mouse model that mimics human hypomyelinating leukodystrophies (the shiverer mouse)20. However, the most common demyelinating disorder is MS and as
such the most relevant demyelinating disease for an autologous iPSC-based therapy. We implanted human iPSC-derived OPCs in a toxin-induced mouse model for demyelination and in mice in which experimental autoimmune encephalomyelitis (EAE) was induced. Our implantation experiments showed that implanted human OPCs are capable to migrate and myelinate nude axons at the demyelinating lesions in these mouse models for MS. Moreover, we observed clinical improvement in the mouse EAE model, that probably could be ascribed to an immunomodulatory effect by factors secreted by the implanted human OPCs. Previous studies have shown that rodent NSCs and OPCs produce a variety of anti-inflammatory and neurotrophic factors, responsible for a significant reduction in clinical scores in EAE animals after intraventricular or intravenous injection21, 22. The next
step in our research concerning the potential clinical application of an OPC implantation therapy for MS was to study the implantation of human iPSC-derived OPCs in a nonhuman primate model for MS, the marmoset EAE model. This EAE primate model shows clinical symptoms and pathology similar to secondary progressive stages in MS patients. We implanted human iPSC-derived OPCs intracerebrally in the marmoset EAE model and showed their capacity to myelinate denuded axons in the inflammatory environment. Although previous studies with intrathecal injection of human NSCs showed beneficial effects on the disease score in marmoset EAE model23 , our experimental set up in the
marmoset did not allow to follow the disease score for a long period of time. It is obvious that still a large number of obstacles, questions and problems need to be addressed before a similar iPSC-based cell therapy can be applied in humans. The tumorigenic capacity of the iPSCs, their potential genomic instability, inefficient differentiation and dedifferentiation of implanted cells are essential issues. Moreover, the MS pathology per se adds an additional level of complexity for an iPSC-based cell therapy in humans. In MS brain, persistent CNS inflammation, blood brain barrier leakage, infiltrating immune cells, constant secretion of proinflammatory cytokines by immune cells and other cytotoxic molecules may interfere with the survival and proper function of iPSC-derived OPCs24-26.
This hostile environment in the CNS may inhibit the myelin production, mediated by both exogenous (transplanted) and endogenous OPCs. In spite of all these difficulties it may be expected that recent advances in iPSC technology, gene editing, and in highly efficient cell differentiation and purification will eventually lead to an effective cell therapy for MS.
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In chapter III, we reported our attempts to generate iPSCs from skin fibroblasts taken from MS patient and to differentiate them into OPCs. The rationale to generate iPSCs (and OPCs differentiated from them) from different types of MS patients was provided by genome-wide association studies (GWAS) on MS patients: Several of these GWA studies have identified a number of MS risk factors, associated with various disease initiation mechanisms for MS27, 28. GWAS studies have uncovered risk loci related to functions other
than immune-related genes, such as cell metabolism and oligodendrocyte differentiation29. These findings suggest that intrinsic, genetic, differences in MS
oligodendrocytes and neurons may provide a primary cause of the disease and so MS patient derived iPSCs may provide ideal tools to study these intrinsic mechanisms. Moreover, the ability to generate unrestricted amounts of functional OPCs and neurons may serve as a tool for high throughput screening for therapeutic drug development and the development of a personalized pharmaceutical therapy30. In addition, development of
MS iPSC-derived neurons will furthermore allow investigation of neuronal homeostasis in-vitro by testing other possible disease initiation mechanisms such as glutamate-induced excitotoxicity and mitochondrial dysfunction31, 32. Our first attempts demonstrated that it
was indeed possible to generate iPSCs from skin fibroblasts of MS patients and that these iPSCs can be differentiated efficiently into oligodendrocytes and neurons, corroborating similar finding by others11. In-vitro and in-vivo studies with the iPSC-derived OPCs from MS
patients will be necessary to establish their proper myelination function.
In chapter IV, we have attempted to convert reactive astrocytes in-vitro into functional oligodendrocytes by inducing the overexpression of a specific set of transcription factors via gene transfection. Deriving functional cells from pluripotent stem cells has been a major goal in biomedical research for the last decade35, 36. Currently, various strategies have
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been developed to generate specific functional cell types for clinical application and disease modeling19. Since the advent of iPS cells, researchers have developed novel
methods to derive specific cell phenotypes by the direct transdifferentiation of somatic cells via the overexpression of cell-type-specific transcription factors37-40. This strategy has
now been used for generating several cell types such as neural cells (astrocytes and neurons), cardiomyocytes, and hepatocytes, etc. Moreover, reprogramming by transcription factors was quickly adapted from in-vitro to in-vivo in various disease models41, 42. Rapid progress and development of novel strategies show that in-vivo
reprogramming could become the future cell-based therapy for many neurodegenerative diseases. In this respect, we attempted to convert astrocytes into functional oligodendrocytes in-vitro by the conditional (tetracycline inducible) expression of a set of four transcription factors (Sox10, Olig2, Ezh2, and Zfp536)43, 44. We showed that these
induced oligodendrocyte precursors (iOPC) express markers similar to normal (NSC-derived) OPCs and that they mature into myelin producing cells in-vitro. The next logical step will be to convert resident reactive astrocytes in the MS lesion to OPCs with our transcription factors to promote local myelin repair, which would comprise an novel cell-based remyelination therapy approach. Several studies have reported that cuprizone-induced mouse model for demyelination and the mouse EAE model (experimental autoimmune encephalomyelitis) have abundant reactive astrocytes in the demyelinating lesions45, 46. Testing transfection in-vivo with the aforementioned transcription factors in
these MS models will allow us to evaluate whether lineage reprogramming of astrocytes is possible in MS lesions. Even though lineage reprogramming has rapidly progressed in recent years, a number of issues remain to be resolved including: constitutive activation of the reprogramming cassette, potential tumor formation, functional maturation of the converted cells, inefficient conversion, and cell type specificity of the transfection47.
Furthermore, a keen awareness is required with respect to linage reprogramming of neural cells such as preservation of genetic and epigenetic identity and recapitulating various aspects of neurodevelopment.
In chapter V, we generated iPSCs from SCA3 and HD patients by non-integrative methods17 and differentiated them into neurons as a tool to examine cell type and
patient-related sensitivity to protein aggregation of the respective endogenously expressed polyQ proteins. IPSC generation and neuronal differentiation were found to be unaffected by the expression of the polyQ in both SCA3 and HD cells. Moreover, iPSC-derived SCA3 and HD neurons were both morphologically and functionally indistinguishable from those derived from controls. Based on the previous study by Koch et al48, we exposed SCA3
patient-derived NSCs and neurons to the excitatory neurotransmitter L-glutamate. We observed glutamate-induced polyQ aggregation only in the SCA3 iPSC-derived neuronal population. Glutamate treatment did not result in aggregation of ataxin-3 polyQ in the iPSCs or in NSCs. Interestingly, analysis of chaperone proteins expression revealed a drastic reorganization of the chaperone network during differentiation of iPSC to neurons, including an almost complete loss of expression of the anti-amyloidogenic chaperone
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DNAJB6. Previous studies have identified DNAJB6 as a highly potent anti-amyloidogenic protein. Upon neuronal overexpression, DNAJB6 also was shown to suppress polyQ aggregation in animal models49. To test the anti-amyloidogenic property of DNAJB6, we
knocked down DNAJB6 in SCA3-derived NSCs and HD-iPSCs. Reduced expression of DNAJB6 resulted in the spontaneous formation of SCA3 and HTT aggregation. This not only highlights the importance of endogenously expressed DNAJB6 for polyQ aggregate prevention but also adds to a more general emerging concept that stem cells are equipped with a highly efficient protein quality control (PQC) system, that includes crucial chaperones like DNAJB6, to ensure their resistance to situations that may cause imbalances in protein homeostasis. PQC mechanisms being important to stem cell function and more specifically to neural stem cell (NSC) self-renewal, proliferation and neurogenesis in vivo50. Especially the finding that stem cells also have elevated
proteasomal activity51 is interesting in the context of our findings that DNAJB6 is part of
the PQC elements that are elevated in stem cells. The Kampinga & Bergink lab has evidence that DNAJB6 activity is directly linked to proteasomal activity, providing a direct link between the chaperone system and the proteasomal system as responsible for maintaining stem cell fitness to ensure they can maintain tissue homeostasis throughout the entire human lifespan. Our study also suggest that the lowered expression of DNAJB6 in neurons is a key factor in the neuronal hypersensitivity to polyQ-mediated protein aggregation and likely also the aggregation of other disease-associated proteins since it was found to also act on prions52, amyloid beta53 and α-synuclein aggregation54. The
question as to why PQC and chaperones are re-wired upon differentiation as we show in Chapter V remains an enigma. All in all, our data in Chapter V not only illustrate the mechanistic power of iPSC research, but also re-emphasize that (re)activation of DNAJB6 in neurons in SCA3 and HD patients and maybe patients with other neurodegenerative diseases could be a possible therapeutic option.
In conclusion, in this thesis we examined the potential use of human iPSCs derived cells as a tool for stem cell-based therapy and disease modeling. We provided evidence that iPSC-derived oligodendrocytes can potentially turn into a valid cell therapy for MS. In addition, we demonstrated the feasibility for in-situ direct conversion of reactive astrocytes into OPCs that may lead to a new therapy promoting local remyelination. Moreover, we established MS patient derived iPSC cell lines that may serve as disease modeling tools to study intrinsic mechanisms underlying MS pathogenesis.
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Chapter 6 : Summary and discussion
