Human induced pluripotent stem cells (hiPSC)
Induced pluripotent stem cells (iPSC) provide a great platform for therapeutic (regenerative medicine, patient specific personalized medicine, cell replacement therapy) and non-therapeutic applications (disease modeling, drug discovery, pharmaceutical testing and toxicology). The discovery of iPSC from mouse skin fibroblasts using retroviral mediated reactivation of four pluripotent transcription factors (OCT4, SOX2, KLF4, c-MYC) by Takahashi and Yamanaka in 2006. Late in 2007, Thomson and Yu (University of Wisconsin-Madison, USA) and Yamanaka et al. (University of Kyoto, Japan) converted human fibroblasts into hiPSC and revealed the use of two alternative factors (Nanog and Lin 28) to facilitate the programming process using a lentiviral system (1). The unique properties of iPSC, self-renewal and differentiation of three germ layers similar to embryonic stem cells (ESC) are considered as novel cell sources for studying neurodegenerative diseases (2). hiPSCs are promised to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. Neuronal model system differentiation from patient-specific hiPSC to mimic central nervous system (3) is a high-throughput screening platform for novel therapeutic targets and drugs.
An incurable neurological disorder—Rett syndrome (RTT)
RTT (OMIN # 312750), a rare and incurable postnatal neurological disorder predominantly affecting females with an incidence of 1 in 10,000 (4) is characterized by an apparently non- symptomatic phase for the first 6–18 months of age followed by apraxia, deceleration of head growth, gait abnormalities, stereotypic hand movements, and mental retardation. Classical RTT caused by MeCP2 mutations occur predominantly as C > T transitions of CpG dinucleotides mostly on the paternal X chromosome (5). There are more than 300 different mutations found in the MeCP2 gene in which 90% were classical patients. Most of these mutations are found in eight different “hot spots” such as missense or nonsense mutations within the MBD (R106W, R133C, T158M, and R168X) or TRD (R255X, R270X, R294X, and R306C) (6). Small C-terminal deletions, with one or both breakpoints located within the “deleted prone region” (DPR) of exon 4, account for 10% of the cases. Atypical RTT caused by mutations in either cyclin-dependent kinase-like 5 (CDKL5) and netrinG1 (NTNG1) or FOXG1/XBF-1 (7-9). However, at least 5% of typical forms and more other atypical forms are not linked to any of 4 genes known to be involved in the disease. Sudden unexpected death occurs in one-quarter of deaths caused by RTT (10). The course and severity of RTT are determined by the location, type, severity of mutation and X-inactivation. Therefore, two girls of the same age with the same mutation can appear quite different. We already detailed about the basic knowledge on RTT, MeCP2, and hiPSCs in our previous publication (11).
RTT & hiPSC research in India
RTT, the second most common cause of intellectual disability in girls after Down syndrome in girls and women, was first recognized in the 1960s and the first report from India was in 1994 (12). Though RTT has a prevalence of 1 per 10,000 to 22,000 (13) in India, there are very few studies and case reports of RTT in India (14). Some of the studies were detection of two deletions of 44 bp (c.1157_1200del44 or p.L386fs) and 38 bp (c.1151_1188del38 or p.P384fs) in exon 4 or C-terminal segment (CTS) region of MeCP2 in classical RTT patients of Indian origin (15); an unique family carrying non-identical MeCP2 mutations in exon 2 wherein the proband with classical RTT was carrying a de-novo early truncating frameshift mutation while her asymptomatic mother was carrying a missense mutation were both predicted as pathogenic mutations (16); c.1160C > T (P387L) in exon 4 of the MeCP2 gene homozygous mutation in Indian female patient was also reported recently (17). RTT is now recognized as one of the non-curable devastating neurodevelopmental disorders (18) that greatly affect society (19). Approximately, around 50 review articles were published about iPSC in India and a lesser number of research work done in hiPSC especially about characterization, differentiation and reprogramming factors.
There is a holistic therapy for curing the postnatal neurological disorders using the ancient medicinal systems like Siddha and Ayurveda in India. We further referred many recent in-depth reviews on hiPSC and the curative ayurvedic drugs for neurodegenerative disorders mainly for RTT.
Therapeutic target for treating RTT
The MeCP2 gene is important for healthy brain activity. Mutations in MeCP2 gene alter the level of MeCP2 and some downstream factors, which can cause neuronal defects in RTT.
There are two types of neurons play a key role in RTT such as inhibitory and excitatory neurons. RTT affects every part of the brain. Generally, excitatory neurons are thought to carry information flow while inhibitory neurons are required for tuning the circuits. Restoring MeCP2 gene expression only in glutamatergic neurons leads to complementary phenotypes of tremor, anxiety, and acoustic startle response (20).
The inhibitory GABAergic neurons results in social problems and repetitive behaviors. Genetically restoring MeCP2 expression only in GABAergic neurons of male MeCP2 null mice enhanced inhibitory signaling, extended lifespan, rescued ataxia, apraxia, and social abnormalities but did not rescue tremor or anxiety. Restoring MeCP2 in neither excitatory nor inhibitory neurons alone is sufficient to fully rescue premature lethality, RTT-like symptoms, and RTT. Hence, modulating the excitatory/inhibitory balance through GABAergic neurons could prove a viable therapeutic option in RTT (21).
MeCP2 in cholinergic neurons
MeCP2 in cholinergic neurons is necessary and sufficient for autonomic cardiac control, thermoregulation, and survival. Targeting the overactive parasympathetic system may be a useful therapeutic strategy to prevent sudden unexpected death in RTT (10).
Downstream gene target
Loss of MeCP2 function in RTT elevates PTP1B levels and thereby impairs insulin signaling and glucose metabolism. PTPN1 encodes PTP1B which is a major metabolic regulator that inhibits insulin signaling by directly dephosphorylating the insulin receptor and IRS1. Therefore, inhibition of PTP1B is a direct target of MeCP2. Animal study by Krishnan et al. 2015 (22) revealed that the small- molecule inhibitors of PTP1B, the difluoromethyl phosphonic acid CPT157633 and the ursolic acid derivative UA0713 act as a potential downstream gene target for RTT (23).
RTT shows a significant deficit in neuron-specific K+-Cl- cotransporter 2 (KCC2) expression, resulting in a delayed GABA functional switch from excitation to inhibition. Restoring KCC2 level rescues GABA functional deficits and therefore acts as a downstream gene target to treat RTT (18).
Loss of MeCP2 function leads to upregulation of Irak1 gene, which is downstream of MeCP2 resulting abnormal activation of NF-κB signaling. Macklis et al. in 2016 found the reduction of NF-κB signaling by genes can increase the dendritic complexity of cortical callosal projection neurons (CPN) and extends lifespan in RTT, indicating potential therapeutic strategies of RTT pathogenesis (24).
Clinical trials and drug discovery in RTT
Current therapeutic approaches aim to increase the expression of brain-derived neurotrophic factor (BDNF), a neurotrophin involved in brain development and plasticity to counteract the deficiency in MeCP2. BDNF does not cross the blood brain barrier (BBB), and therefore it has no immediate therapeutic application for RTT patients; however an alternative growth factor, insulin-like growth factor 1 (IGF1), crosses the BBB and shares similar properties and functions with BDNF. Animal research reveals that systemic administration of the active peptide of IGF1 improves physiological behavior and survival in MeCP2 mutant mice suggesting that IGF1 can be a treatment in RTT patients (25). IGF1 can also rescue the impaired KCC2 level as a downstream gene target in RTT. Phase I clinical trials of recombinant IGF-1 treatment in patients with RTT have produced encouraging outcomes leads to ongoing Phase II trials. There are about 40 studies on RTT present in the clinicaltrial.gov, the drug based studies were listed in the below Table 1. There is no drug marketed for treating RTT till now.
Current trends in hiPSC technology
Recent advances in stem cell research, especially in the development of iPSC technology, provide a new paradigm for drug screening by permitting the use of human cells with the same genetic makeup as the patients without the typical quantity constraints associated with patient primary cells (26). The delivery of iPS cells treated with mitomycin C (MMC) loading with gold nanorods (AuNRs) for the targeted photothermal treatment of gastric cancer resulted in killing the tumor cells by the heat generated from the gold nanorods. This suggested that pre-treated iPS cells with MMC can be used as a novel and safe approach for targeted tumor therapy (27). Nanoplatform by the combination of Au nanorods, SiO2, CXCR4 nanoparticles with hiPSC for the photothermal treatment paves a great promise for clinical translation in the near future (28). hiPSCs can be used as an autologous source for cell therapy. Transplantation of human iPSC-derived cells is a safe and efficient approach to promoting recovery after stroke and can be used to supply the injured brain with new neurons for replacement (29). Furthermore, the ability to simulate organ systems with hiPSCs may allow for studying the effects of a drug’s metabolites on target and non-target cell types. This is an important part of a drug’s side effect profile that can currently be observed only in animal models or clinical trials. hiPSCs plays a larger role in studying the effects of polypharmacy, both in the general population and specific patient. Recently, “tissue chip” devices have become prominent because these cellular models are designed to recapitulate the structure and function of human organs, such as the lung, liver and heart; therefore, they are often called “organs-on-a-chip” or “human-on-a-chip” (30). Three-dimensional model systems are expected to be able to mimic human physiology more accurately than traditional two-dimensional cultures, among which “organ-on-a-chip” and “body-on-a-chip” are advanced formats of “tissue-on-a-chip” devices (31). Once “tissue-on-a-chip” devices are developed and accepted as a standard part of the preclinical drug development process, they can be used to predict whether a drug candidate is safe or toxic to humans in the safety of the laboratory setting (32). This is an increasingly important aspect of drug development, taking into account the commonality of multiple medication regimens and a patient population that will likely increase the number of medications as they age (33). A total of 1,500 compounds were tested on wild-type hiPSC-derived neural progenitor cells for their effect on the Wnt/β-catenin signaling pathway for identifying a potential novel neuropsychiatric drug (34).
Limitations associated with drug screening for RTT
Drug screening for neurological disorders is a tedious process and it is insignificant to confirm the efficacy of the respective drug dosage. The main issue is the difference between the mice and human neurons activity against the drug. Drug treatment using hiPSC derived neuron model system is more successful for neurodevelopmental disorders than late-onset neurodegenerative disorders, likely because of the foetal-like properties of the cells (35). It is difficult to correlate the drug pharmacodynamics in the neurodegenerative disorder animal models. There are also some limitations associated with drug screening even in hiPSC such as genetic aberration and teratoma formation by retroviral or lentiviral systems of gene incorporation in the generation of iPSCs. Five oncogenes were found to overexpress in iPSCs whereas the oncogene RAB25 was found to express in cells derived from iPSCs (36). Apart from these limitations, generation of patient specific or mutation specific hiPSC model system is a great challenge and cost effective but ultimately it can be the immortal modal system as compared with animal models. It is unlikely that hiPSC technology will successfully model for all disorders (37).
Indian traditional ayurvedic system: curative drugs for neurological disorders
Ayurveda is a science of life and science of longevity to health and personalized medicine. India is known for its traditional medicinal systems—Ayurveda, Siddha, and Unani. Ayurveda has an extensive pharmacopeia, predominantly herbs and minerals. The Ayurvedic concept appeared and developed between 2,500 and 500 BC in India, which is the largest producer of medicinal plants. There are currently about 20,000 medicinal plants have been recorded. Clinical trials are ongoing in more than 100 natural product derived drugs and at least 100 molecules/compounds are in the preclinical development stage (38). In the Indian system of medicine, the following medicinal plants have shown promising activity in neuropsychopharmacology: Allium sativum, Bacopamonnierae, Centellaasiatica, Celastruspaniculatus, Nicotianatabaccum, Withaniasomnifera, Ricinuscommunis, Salvia officinalis, Ginkgo biloba, Huperizaserrata, Angelica sinensis, Uncariatomentosa, Hypericumperforatum, Physostigmavenosum, Acorus Calamus, Curcuma longa, Terminaliachebula, Crocus sativus, Enhydrafluctuans, Valerianawallichii, Glycyrrhizaglabra etc. (39). The Medhya Rasayanas are group of medicinal plants described in Ayurveda, known to improve mental health, intellect ability, immunity and hence longevity. Medha means intellect/retention and Rasayana means therapeutic procedure. Medhya Rasayana is a group of 4 medicinal plants that can be used singly or in combinations.
- Mandukaparni (Centella asiatica). Useful in treating mental retardation (improvement in performance IQ), Social Quotient, immediate memory span.
- Yashtimadhu (Glycyrrhiza glabra). Spatial learning, preliminary free radical scavenging, cerebral ischemia and antioxidant capacity towards LDL oxidation. It increases the circulation into the CNS system, improves learning and memory on scopolamine-induced dementia.
- Guduchi (Tinospora cordifolia). Strong free radical scavenging properties against reactive oxygen and nitrogen species. It is useful for treatment of improving behavior disorders, mental deficit and IQ levels.
- Shankhpushpi (Convolvulus pluricaulis). Anxiolytic and memory enhancing, mood elevating, retard brain aging (40).
Phytochemical based antioxidants may have neuroprotective (preventing apoptosis) and neuroregenerative roles, by reducing or reversing cellular damage and by slowing progression of neuronal cell loss. There is ample scientific and empirical evidence supporting the use of antioxidants for the control of neurological disorders. As the focus of medicine shifts from the treatment of manifest disease to prevention, herbal medicine is coming into consideration, being a renaissance of age-old human tradition (41).
Projected curative drugs for RTT: Ayurveda with hiPSC technology
Medhya Rasayana will cure the symptoms of RTT. The treatment is cost effective and devoid of side effects. As so far the drugs tested for RTT using hiPSC neuronal modal system were having action on synapse number, spine number and soma size. The role of ‘Medhya drugs’ in neuronal stem cells differentiation is also described earlier (42). Earlier reports indicate that ‘Rasayan drugs’ could be used in stem cell therapy based on the regeneration and cell renewal properties. A tablet prepared from four Medhya Rasayana herbs aid in yielding concentrated medicament with the same efficacy as per the classically proposed drug dosage at lower dose (43). The active compounds in Medhya drugs will reverse neurological disorders and psychomotor activities (40). Hence, these active compounds pave a way to cure the RTT symptoms like repetitive hand movements and mental impairment. Figure 1 depicts the drugs tested and projected Madhya drugs need to test in the hiPSC-RTT neuronal model system.
Way forward and conclusions
Comprehensive research on the discovery of novel neuroprotective drug candidates has proven that natural products, such as plant extracts and their bioactive compounds, can have tremendous potential as lead neuroprotective candidates. A source of herb-based drugs severely compromises proper understanding of Ayurveda, which insists on restoring balance to doshas and dhatus, and preventing a repetition of their vitiation, as the first priority. Identification and characterization of new medicinal plants to cure neurological diseases and brain injuries are the major and increasing scientific interest in recent years. There are more than 120 traditional medicines that are being used for the therapy of central nervous system (CNS) disorders in Asian countries (49). Solely using herbal drugs is against the ethos of Ayurveda. Sadly, even in India, current research and practice of Ayurveda are moving on the lines of allopathy where drugs take center stage. Unfortunately, modern medicine based psychoactive drugs have met with limited success in the treatment of various neurological and psychiatric disorders due to multi-factorial nature of these diseases. The world is trying to move towards holistic and integrative approaches, which represent the core of Ayurveda. Using hiPSCs in phase I clinical trials may provide a more sensitive assay for a candidate drug’s toxicity and safety compared with conventional clinical trial phases. Hence, in future the integrative approach of novel drug discovery from Ayurveda using hiPSC will pave the wave for drugging the undruggable diseases.
The author would like to thank the Human Genetics Laboratory, Bharathiar University, India for providing necessary infrastructure facilities to conduct this article.
Conflicts of Interest: The authors have no conflicts of interest to declare.
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Cite this article as: Gomathi M, Balachandar V. Novel therapeutic approaches: Rett syndrome and human induced pluripotent stem cell technology. Stem Cell Investig 2017;4:20.