While there is great promise that iPSCs may allow us to understand typical development and the genes and cellular processes involved, another major goal of such research would be to apply that knowledge to understand and treat disease. Hence, the ability to use the iPSC technology to also examine diseases directly, with cells derived from patients and their family members, is of particular interest. For disorders of the nervous system, where the direct study of diseased tissue is limited by issues of accessibility and the inability of mature neurons to regenerate, the ability to study diseased neural cells derived from iPSCs may provide breakthroughs for diagnosis and treatment.
Disorders of the nervous system in which a genetic origin has been identified will allow immediate correlations to be made between cellular changes in development and functioning with disease. One example in which this potential of iPSCs has already been demonstrated is familial dysautonomia (FD), a severe disorder in which sensory and autonomic neurons degenerate, leading to the patient's early death. Patients with FD have a known mutation in the gene encoding I-κ-B kinase complex-associated protein (IKAP), resulting in lower levels of IKAP protein. Previous work using mouse models of related disorders and lymphocytes from patients had suggested that a cell migration defect at the early stages of the nervous system development was involved in the pathogenesis of FD (Close et al., 2006
; Iwashita et al., 2003
), but no direct link between neural pathology and cellular mechanisms in humans had been demonstrated. The use of iPSCs from three patients with FD, induced with lentiviral methods, demonstrated not only that the establishment of induced stem cells with the disease genotype was possible, but that iPSC lines differentiated to CNS lineages showed lower levels of IKAP protein, reduced neurogenesis, and impaired migration of neural crest precursors (Lee et al., 2009
). Previous work had demonstrated that lymphocytes from patients could be pharmacologically altered to increase IKAP protein levels (Slaugenhaupt et al., 2004
), but the use of patient-derived iPSCs showed for the first time that the use of candidate drugs could be used to ameliorate the pathology in neural cells, specifically by treatment with kinetin, one of three compounds theoretically able to affect IKAP protein levels and its cellular functions (Lee et al., 2009
Similarly, iPSCs have been used to investigate the degenerative processes involved in spinal muscular atrophy (SMA), a disorder of lower motor neurons known to result from mutations of the smn
genes (Ebert et al., 2009
). Electrically active human motor neurons have been generated from iPSCs (Karumbayaram et al., 2009
) and with this technique, Ebert and colleagues demonstrated that these cells became smaller and reduced in number when derived from a SMA patient compared to those derived from a healthy family member. These differences essentially reproduced in vitro
the features of in vivo
disease, including spinal motor neuron protein aggregates. In a major advancement for drug discovery, iPSCs also allowed for the testing of valproic acid and tobramycin therapeutically, which reduced pathological protein aggregating in patient-specific cells and provided a litmus test for other possible therapeutics for SMA.
Amyotrophic lateral sclerosis (ALS) has also been studied using patient-derived iPSCs (Dimos et al., 2008
). Unlike SMA and FD, ALS and many other neuropsychiatric disorders are not always the result of a mutation in a specific gene but likely have a range of different and even multiple origins in each particular patient. Dimos and colleagues derived iPSCs from an elderly patient with severe familial disease and were able to study the pathology of differentiated motor neurons in culture regardless of the advanced age or severe disease of the donor. While this patient had a known genetic mutation and the study of other patients with ALS may prove more challenging, the success of this first iPSC study on ALS holds great promise for determining even a few of the molecular mechanisms of this and other sporadic diseases.
Efforts using iPSCs to understand disorders affecting more rostral portions of the CNS are also underway, but, perhaps due to the complexity of brain cell structure and function, such investigations lag behind those examining the spinal cord and peripheral nerves. Yet, when ES cells and even iPSC-derived neuronal progenitors are cultured without growth factors and morphogens, they take on a forebrain fate and even show functional electrical activity (Hu et al.; Johnson et al., 2007
; Pankratz et al., 2007
). This appears to be a “generic” forebrain fate, conferred by the expression of OTX homeodomain genes and other transcription factors that determine brain fate early in development. The iPSC-derived cells need further patterning signals to generate specific subpopulations of forebrain neurons. How this forebrain fate occurs and what signals early neural cells and their progeny obey as they differentiate and become patterned into distinct subpopulations, are areas of active current investigation. Answering these questions will have a major impact on research into neuropsychiatric disorders which may alter the proportion of specific cell types and their fate. Published protocols suggest that mouse and even human ES cells can recapitulate the typical temporal patterns of development of cortical cells in culture, providing new models for studying this highly-coordinated sequence of events (Eiraku et al., 2008
; Gaspard et al., 2008
; Gaspard et al., 2009
Many of the first advances on disorders of the rostral CNS have been made for those with known genetic mutations. Patient specific iPSCs are in the first stages of investigation for Down syndrome, Gaucher disease type 3, Huntington disease (Park et al., 2008
) and epilepsy, with investigations of the latter particularly focusing on patients with known sodium channel mutations such as SCNA1, the most commonly mutated gene in seizure disorders (Meisler et al., 2010
). For Huntington disease, monkey iPSCs have already shown that differentiated neurons have disease-specific accumulation of huntingtin aggregates (Chan et al., 2010
Work on iPSCs of other brain disorders with known genetic mutations has also demonstrated the need to be systematic in the evaluation of these reprogrammed cells, which may not consistently reproduce the development of typical stem cells. Skin and lung fibroblasts from three patients with Fragile X, a disorder characterized by intellectual disability and expanded triplet repeats in the fragile X mental retardation (FMR1
) gene, were used to create iPSCs (Urbach et al., 2010
) . While Fragile X iPSCs met all basic criteria of stem cells, they differed from both wild type and Fragile X embryonic stem cells, in which the fmr1
gene is active regardless of triplet repeats; the reprogramming process using lentiviruses was not able to reverse the methylation of fmr1
that typically inactivates the gene as cells differentiate. However, iPSCs taken from Rett syndrome patients with MeCP2 gene mutations show that the reprogramming process can induce the normal active status of both X chromosomes in pluripotent cells (Marchetto et al., 2010
). When cell lines were established from cells that had these “normal” reactivated pairs of X chromosomes, X chromosome silencing occurred with differentiation. Interestingly, Rett syndrome neurons showed abnormal synapse structure and function, a measurable phenotype for drug discovery trials, even in cells displaying normal expression of the MeCP2 gene from the active X chromosome, suggesting a non-cell autonomous mechanism of disease (Marchetto et al., 2010
). These results showing varied gene silencing during reprogramming suggest that different techniques and/or different genotypes will have to be more thoroughly understood to fully utilize iPSCs, even in the case of disorders with known genetic mutations.
The most progress with iPSCs has been made on Parkinson disease (PD), an example of a disorder in which the cellular pathology—loss of dopaminergic neurons in the substansia nigra—is well understood, making it amenable to treatment with transplantation. iPSCs represent a potential source of transplantable cells that, if derived from the patient themselves, would be immunologically compatible. Dopamine neurons and cells expressing tyrosine hydroxylase have been derived from human and rodent iPSCs, albeit with low and variable levels of efficiency (Chambers et al., 2009
). More recently, iPSCs have been generated from patients with PD (Soldner et al., 2009
). Both undifferentiated and differentiated cells from these and non-patient derived iPSCs have been successfully transplanted into rodent models of PD showing the potential to correct behavioral and anatomical pathology (Cai et al., 2009
; Hargus et al., 2010
; Wernig et al., 2008
). While iPSCs have not been demonstrated to be significantly better than embryonic stem cells in terms of this normalization, the early potential shown in these studies suggest that there may be equal promise for iPSCs in late-appearing neurodegenerative and early-appearing neurodevelopmental disorders.
Interestingly, the first disorder for which transplanted cells became an approved treatment option falls into a separate category entirely—spinal cord injury. Starting in 2009, the FDA granted approval to use human ES cells as an experimental treatment for spinal cord injury and, more recently, ALS. ES cells have a longer track record of success, with resolution of many of the risks yet to be resolved before iPSCs can be used in human disease treatment--namely the risk for tumor formation, graft rejection, DNA recombination in culture and appropriate telomerase levels to ensure cell survival (Naegele et al., 2010
). Thus, several goals must be met before we may be able to apply the iPSC technology to tissue treatments for CNS disorders. However, as discussed above, iPSCs promise significant advances in cell-based regenerative medicine and other methods for improving treatments. Indeed, even the early reports discussed here demonstrate how iPSCs have pinpointed disease mechanisms and tested successful candidate drugs for multiple disorders.
The most ambitious future application of iPSC technologies is to derive isogenic, relatively differentiated, transplantable cells to assist in the cure of neurodegenerative disorders. The hope is to provide cell replacement for neurological conditions such as Parkinson, Huntington and Alzheimer disorders. If the neurodegenerative conditions are driven by an intrinsic genetic defect, this will be likely reproduced in the transplanted isogenic cells, and therefore this strategy depends upon the ability to fix genetic disorders by in vitro gene replacement. However, there are concerns that will need to be addressed before iPSC and ES can be used for human neural cell replacement therapy, particularly the potential of these cells to accumulate point mutations and CNV/SVs during culture. Hence, their long-term genomic stability will need to be examined. Other concerns are potential tumor formation, arising from the intrinsic pluripotency of these cells or from the oncogenes that are used for their derivation. It is also significant that the differentiation of iPSCs in vitro will necessarily be different from that which occurs in vivo. Even proposals to use more naturalistic culture conditions may be limited by the complexity of the interactions that occur in the developing brain that involve neural, vascular, and immune cells and which are not fully understood. The neural cells derived from iPSCs may therefore differ from their in vivo counterparts in their stability and potential to continue or change development in response to extracellular cues. This may affect their potential as transplantable tissue.