Disease-specific Induced Pluripotent Stem Research Articles

Simplified drug efficacy evaluation system for vasopressin neurodegenerative disease using mouse disease-specific induced pluripotent stem cells

Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disorder in which vasopressin-secreting neurons degenerate over time due to the production of mutant proteins. We have demonstrated therapeutic effects of chemical chaperones in an FNDI mouse model, but the complexity and length of this evaluation were problematic. In this study, we established disease-specific mouse induced pluripotent stem cells (iPSCs) from FNDI-model mice and differentiated vasopressin neurons that produced mutant proteins. Fluorescence immunostaining showed that chemical chaperones appeared to protect vasopressin neurons generated from iPSCs derived from FNDI-model mice. Although KCL stimulation released vasopressin hormone from vasopressin neurons generated from FNDI-derived iPSCs, vasopressin hormone levels did not differ significantly between baseline and chaperone-added culture. Semi-quantification of vasopressin carrier protein and mutant protein volumes in vasopressin neurons confirmed that chaperones exerted a therapeutic effect. This research provides fundamental technology for creating in vitro disease models using human iPSCs and can be applied to therapeutic evaluation of various degenerative diseases that produce abnormal proteins.

Primary failure of tooth eruption: Etiology and management.

Primary failure of eruption (PFE) is a rare disorder defined as incomplete tooth eruption despite the presence of a clear eruption pathway. PFE is known to be caused by rare variants in the parathyroid hormone 1 receptor gene (PTH1R). Although several PTH1R variants have been reported, the etiology of PFE remains unclear. However, important studies that help elucidate the pathology of PFE have recently been published. The purpose of this review is to summarize current treatment options, clinical symptoms or phenotypes for diagnosis, genetic information including solid evidence in mouse disease models and disease-specific induced pluripotent stem cells, thus approaching the etiology of PFE from the perspective of the latest research.

A new approach to analysis of intracellular proteins and subcellular localization using cellprofiler and imageJ in combination.

Analytical pipeline, which is used for various analysis application, of CellProfiler, an open-source software for cell imaging analysis, is very important. In the present study, to examine whether intracellular proteins can be discriminated using a combination of CellProfiler and ImageJ, we analyzed neuroblastoma and monocytic cell lines, and disease-specific induced pluripotent stem cell (iPSC)-derived neurons. This revealed that scattered puncta of Rab7 and transferrin in neuroblastoma lines were clearly detectable by created analytical pipelines in CellProfiler. We then constructed pipelines for measuring the distance from the center of the nucleus to allow investigation of the intracellular localization of Rab7 or transferrin. Using CellProfiler and ImageJ in combination, we confirmed that our pipelines were applicable both quantitatively and objectively to analysis of membrane trafficking of proteins such as Rab proteins and transferrin. In addition, when applied to quantitative measurement of phagocytosis, our pipelines clearly detected monocytic cell lines that had engulfed bioparticles. Finally, we developed new pipelines for analysis of disease phenotype using iPSCs from a patient with familial Parkinson's disease (PD), harboring the I2020T LRRK2 mutation (PARK8). These were able to successfully detect Rab5 puncta and Rab7 puncta in PARK8 patient iPSC-derived neurons. Interestingly, in long-term culture, we found that the numbers of Rab7 puncta in a single PARK8 patient iPSC-derived neurons were lower than that of control iPSC-derived neurons. On the other hands, at 14days in vitro, the numbers of Rab5 puncta in PARK8 patient iPSC-derived neurons were lower than those of isogenic iPSC-derived neurons, but not Rab7 puncta. Furthermore, Rab5 puncta of PARK8 patient iPSC-derived neurons exhibited distinct localization pattern relative to isogenic iPSC-derived neurons. These present results suggest that this new analytical tool can be used as a supporting method for quantification of intracellular protein.

Pluripotent Stem Cells for Cell Therapy.

In an increasingly geriatric population, in which elderly people frequently face chronic diseases and degenerative conditions, cell therapies as part of novel regenerative medicine approaches are of great interest. Even though today's cell therapies mostly rely on adult stem cells like the mesenchymal stem cells or primary somatic cells, pluripotent stem cells represent an enormously versatile cell model to explore possible new avenues in the field of regenerative medicine due to their capacity to grow indefinitely and to differentiate into the desired cell types. The discovery of reprogramming somatic cells into induced pluripotent stem cells augmented the pool of applicable cell entities so that researchers nowadays can resort to embryonic stem cells, but also to a plethora of patient- and disease-specific induced pluripotent stem cells. The ease of targeted genome engineering is an additional benefit that allows using pluripotent stem cells for disease modeling, drug discovery, and the development of cell therapies. However, the task is still demanding as the generation of subpopulations and a sufficient cell maturation for some cell entities have yet to be achieved. Likewise, even though for some applications the cells of interest can be produced in the large-scale dimensions and purity that are required for clinical purposes, proper integration, and function in the host tissue remain challenging. Nonetheless, the immense progress that has been made over the last decades warrants the prominent role of pluripotent stem cells in regenerative medicine as in vitro models to broaden our knowledge of disease onset/progression and treatment as well as in vivo as a substitution of damaged/aged tissue.

Generation of an induced pluripotent stem cell line (PUMCHi006-A) derived from a patient with pulmonary arterial hypertension carrying heterozygous c.1339 G>A mutation in PTGIS gene.

Our previous study found that mutations in the PTGIS gene contributed high susceptibility to pulmonary arterial hypertension (PAH). We have generated disease-specific induced pluripotent stem cell (iPSC) lines from a PAH patient carrying the heterozygous c.1339 G>A mutation in PTGIS gene. The generated iPSC lines can be differentiated into endothelial cells to investigate the pathogenesis of PAH associated with PTGIS gene, which could provide valuable resources for personalized medicine.

Using patient-derived iPSCs to develop humanized mouse models for chronic myelomonocytic leukemia and therapeutic drug identification, including liposomal clodronate

Chronic myelomonocytic leukemia (CMML) is an entity of myelodysplastic syndrome/myeloproliferative neoplasm. Although CMML can be cured with allogeneic stem cell transplantation, its prognosis is generally very poor due to the limited efficacy of chemotherapy and to the patient’s age, which is usually not eligible for transplantation. Comprehensive analysis of CMML pathophysiology and the development of therapeutic agents have been limited partly due to the lack of cell lines in CMML and the limited developments of mouse models. After successfully establishing patient’s derived disease-specific induced pluripotent stem cells (iPSCs) derived from a patient with CMML, we utilized these CMML-iPSCs to achieve hematopoietic re-differentiation in vitro, created a humanized CMML mouse model via teratomas, and developed a drug-testing system. The clinical characteristics of CMML were recapitulated following hematopoietic re-differentiation in vitro and a humanized CMML mouse model in vivo. The drug-testing system using CMML-iPSCs identified a MEK inhibitor, a Ras inhibitor, and liposomal clodronate as potential drugs for treating CMML. Clodronate is a drug commonly used as a bisphosphonate for osteoporosis. In this study, the liposomalization of clodronate enhanced its effectiveness in these assays, suggesting that this variation of clodronate may be adopted as a repositioned drug for CMML therapy.

Human-induced pluripotent stem cell approaches to model inborn and acquired metabolic heart diseases.

The article provides an overview of advances in the induced pluripotent stem cell field to model cardiomyopathies of inherited inborn errors of metabolism and acquired metabolic syndromes in vitro. Several inborn errors of metabolism have been studied using 'disease in a dish' models, including Pompe disease, Danon disease, Fabry disease, and Barth syndrome. Disease phenotypes of complex metabolic syndromes, such as diabetes mellitus and aldehyde dehydrogenase 2 deficiency, have also been observed. Differentiation of patient and disease-specific induced pluripotent stem cell-derived cardiomyocytes has provided the capacity to model deleterious cardiometabolic diseases to understand molecular mechanisms, perform drug screens, and identify novel drug targets.

A de novo microdeletion involving PAFAH1B (LIS1) related to lissencephaly phenotype.

Lissencephaly is a type of the congenital malformation of the brain. Due to the impairments of neuronal migration, patients show absence of brain convolution manifesting smooth brain surfaces. One of the human genes responsible for lissencephaly is the platelet-activating factor acetylhydrolase 1b gene (PAFAH1B; also known as LIS1) located on 17p13.3. Patients with heterozygous deletion of this chromosomal region exhibit lissencephaly. Recently, we encountered a male patient who showed typical lissencephaly. Using a microarray analysis, we identified a 1.3 Mb submicroscopic deletion in 17p13.3. This deletion included PAFAH1B. Both of the parents showed no deletion in this region. Therefore, this was determined to be derived from de novo origin. After obtaining the written informed consent, skin fibroblasts were provided from this patient and disease-specific induced pluripotent stem (iPS) cells were generated and used for medical research (Shimojima K, Okumura A, Hayashi M, Kondo T, Inoue H, and Yamamoto T. CHCHD2 is down-regulated in neuronal cells differentiated from iPS cells derived from patients with lissencephaly. Genomics, in press).

Limited hair cell induction from human induced pluripotent stem cells using a simple stepwise method

Disease-specific induced pluripotent stem cells (iPS) cells are expected to contribute to exploring useful tools for studying the pathophysiology of inner ear diseases and to drug discovery for treating inner ear diseases. For this purpose, stable induction methods for the differentiation of human iPS cells into inner ear hair cells are required. In the present study, we examined the efficacy of a simple induction method for inducing the differentiation of human iPS cells into hair cells. The induction of inner ear hair cell-like cells was performed using a stepwise method mimicking inner ear development. Human iPS cells were sequentially transformed into the preplacodal ectoderm, otic placode, and hair cell-like cells. As a first step, preplacodal ectoderm induction, human iPS cells were seeded on a Matrigel-coated plate and cultured in a serum free N2/B27 medium for 8 days according to a previous study that demonstrated spontaneous differentiation of human ES cells into the preplacodal ectoderm. As the second step, the cells after preplacodal ectoderm induction were treated with basic fibroblast growth factor (bFGF) for induction of differentiation into otic-placode-like cells for 15 days. As the final step, cultured cells were incubated in a serum free medium containing Matrigel for 48 days. After preplacodal ectoderm induction, over 90% of cultured cells expressed the genes that express in preplacodal ectoderm. By culture with bFGF, otic placode marker-positive cells were obtained, although their number was limited. Further 48-day culture in serum free media resulted in the induction of hair cell-like cells, which expressed a hair cell marker and had stereocilia bundle-like constructions on their apical surface. Our results indicate that hair cell-like cells are induced from human iPS cells using a simple stepwise method with only bFGF, without the use of xenogeneic cells.

Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy.

Disease-specific induced pluripotent stem (iPS) cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3)-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening.

Disease modeling and lentiviral gene transfer in patient-specific induced pluripotent stem cells from late-onset Pompe disease patient

Pompe disease is an autosomal recessive inherited metabolic disease caused by deficiency of acid α-glucosidase (GAA). Glycogen accumulation is seen in the affected organ such as skeletal muscle, heart, and liver. Hypertrophic cardiomyopathy is frequently seen in the infantile onset Pompe disease. On the other hand, cardiovascular complication of the late-onset Pompe disease is considered as less frequent and severe than that of infantile onset. There are few investigations which show cardiovascular complication of late onset Pompe disease due to the shortage of appropriate disease model. We have generated late-onset Pompe disease-specific induced pluripotent stem cell (iPSC) and differentiated them into cardiomyocytes. Differentiated cardiomyocyte shows glycogen accumulation and lysosomal enlargement. Lentiviral GAA rescue improves GAA enzyme activity and glycogen accumulation in iPSC. The efficacy of gene therapy is maintained following the cardiomyocyte differentiation. Lentiviral GAA transfer ameliorates the disease-specific change in cardiomyocyote. It is suggested that Pompe disease iPSC-derived cardiomyocyte is replicating disease-specific changes in the context of disease modeling, drug screening, and cell therapy.

Your Heart on a Chip: iPSC-Based Modeling of Barth-Syndrome-Associated Cardiomyopathy

Disease-specific induced pluripotent stem cells (iPSCs) are invaluable tools for studying genetic disorders in a dish. A recent paper by Wang etal. (2014) powerfully combines analysis of human iPSCs with genome editing and tissue engineering, in conjunction with biochemical and physiological assays, to provide insights into Barth-syndrome-associated cardiomyopathy.

Establishment of hepatic and neural differentiation platforms of Wilson’s disease specific induced pluripotent stem cells

The combination of disease-specific human induced pluripotent stem cells (iPSC) and directed cell differentiation offers an ideal platform for modeling and studying many inherited human diseases. Wilson's disease (WD) is a monogenic disorder of toxic copper accumulation caused by pathologic mutations of the ATP7B gene. WD affects multiple organs with primary manifestations in the liver and central nervous system (CNS). In order to better investigate the cellular pathogenesis of WD and to develop novel therapies against various WD syndromes, we sought to establish a comprehensive platform to differentiate WD patient iPSC into both hepatic and neural lineages. Here we report the generation of patient iPSC bearing a Caucasian population hotspot mutation of ATP7B. Combining with directed cell differentiation strategies, we successfully differentiated WD iPSC into hepatocyte-like cells, neural stem cells and neurons. Gene expression analysis and cDNA sequencing confirmed the expression of the mutant ATP7B gene in all differentiated cells. Hence we established a platform for studying both hepatic and neural abnormalities of WD, which may provide a new tool for tissue-specific disease modeling and drug screening in the future.

High efficiency of neural induction in Parkinson's disease-specific induced pluripotent stem cells

1. Chung, S., et al., Stem Cells, 2006. 24(6): p. 1583-93. 2. Swistowski, A., et al., PLoS One, 2009. 4(7): p. e6233. 3. Kim, D.S., et al., Stem Cell Rev, 2010. 6(2): p. 270-81. 4. Morizane, A., et al., J Neurosci Res, 2011. 89(2): p. 117-26. ACKNOWLEDGMENTS Special thanks to all patients, family members, spouses, and healthy volunteers participating in this study and “putting their skin in the game”. Sponsors: California Institute of Regenerative Medicine, Parkinson Alliance, Blume foundation, Brin Wojcicki Foundation High Efficiency of Neural Induction in Parkinson’s Disease-Specific Induced Pluripotent Stem Cells

Induced pluripotent stem cells from hair follicles as a cellular model for neurodevelopmental disorders

Disease-specific induced pluripotent stem cells (iPSC) allow unprecedented experimental platforms for basic research as well as high-throughput screening. This may be particularly relevant for neuropsychiatric disorders, in which the affected neuronal cells are not accessible. Keratinocytes isolated from hair follicles are an ideal source of patients' cells for reprogramming, due to their non-invasive accessibility and their common neuroectodermal origin with neurons, which can be important for potential epigenetic memory. From a small number of plucked human hair follicles obtained from two healthy donors we reprogrammed keratinocytes to pluripotent iPSC. We further differentiated these hair follicle-derived iPSC to neural progenitors, forebrain neurons and functional dopaminergic neurons. This study shows that human hair follicle-derived iPSC can be differentiated into various neural lineages, suggesting this experimental system as a promising in vitro model to study normal and pathological neural developments, avoiding the invasiveness of commonly used skin biopsies.

Abstract 4225: Expression of NANOG in a prostate cancer cell line increases CD24 expression and enhances cell invasion

Abstract The transcription factor NANOG is critical for the maintenance of embryonic stem cell properties including self-renewal and pluripotency. NANOG performs these functions by regulating the expression of genes required for the preservation of the stem cell phenotype. Introduction of NANOG and other embryonic stem cell genes has successfully established disease-specific induced pluripotent stem cells in several types of human cancers. Although the expression of NANOG has been documented in cancer cells, the function of NANOG in cancer has not been extensively investigated. We hypothesized that NANOG plays a role in prostate cancer cell biology based on our observation of NANOG protein expression in prostate cancer cell lines. To test this hypothesis, we transduced the PC3 prostate cancer cell line with a lentivirus capable of directing the expression of NANOG. A stable cell line containing the NANOG expression cassette was established by selection with puromycin, and expression of NANOG protein was verified by Western blot analysis. Analysis of stem cell surface markers by flow cytometry in the PC3-NANOG cells and PC3 cells transduced with empty virus revealed an increase in the percentage of CD24 positive cells in the PC3-NANOG sample as compared with the control. In addition, the density of CD24 per cell appeared to increase. This increase in CD24 expression was confirmed by Western blot analysis. In multiple cancers expression of CD24, a mucin-like adhesion protein, correlates with invasion and metastatic potential. Therefore, we conducted an invasion assay to assess the ability of PC3-NANOG cells to invade through a matrix assaying invasion by migration of cells from one chamber of a bicameral chamber through a porous membrane. The results showed that PC3-NANOG cells exhibited a 30-40% increase in invasion as compared to the control cells. (p<0.001). These results suggest that expression of NANOG in a prostate cancer cell line contributes to increased invasive capacity, possibly through enhanced expression of CD24. Therefore, NANOG could be a potential mediator of prostate cancer cell invasion and maneuvers to decrease NANOG expression could be a new therapeutic target for treatment of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4225.