Patient-specific iPS Cells Research Articles

KIT D816V Mast Cells Derived from Induced Pluripotent Stem Cells Recapitulate Systemic Mastocytosis Transcriptional Profile.

Mast cells (MCs) represent a population of hematopoietic cells with a key role in innate and adaptive immunity and are well known for their detrimental role in allergic responses. Yet, MCs occur in low abundance, which hampers their detailed molecular analysis. Here, we capitalized on the potential of induced pluripotent stem (iPS) cells to give rise to all cells in the body and established a novel and robust protocol for human iPS cell differentiation toward MCs. Relying on a panel of systemic mastocytosis (SM) patient-specific iPS cell lines carrying the KIT D816V mutation, we generated functional MCs that recapitulate SM disease features: increased number of MCs, abnormal maturation kinetics and activated phenotype, CD25 and CD30 surface expression and a transcriptional signature characterized by upregulated expression of innate and inflammatory response genes. Therefore, human iPS cell-derived MCs are a reliable, inexhaustible, and close-to-human tool for disease modeling and pharmacological screening to explore novel MC therapeutics.

Establishment of iPS cell line (KLRMMEi002-A) by reprogramming peripheral blood mononuclear cells from a patient with USH2A-associated Usher syndrome

USH type 2 (USH2) is an autosomal recessive disorder that is characterized by inherited retinopathies and sensorineural hearing loss. USH type 2 (USH2) is frequently caused by USH2A mutations, which account for 74–90% of USH2 cases. We used peripheral blood mononuclear cells (PBMCs) from a USH2 patient with a USH2A gene mutation (c.8559-2A > G) to create an induced pluripotent stem (iPS) cell line. The patient-specific iPS cell line with the specific point mutation exhibited typical iPS cell characteristics, and it can be used as a model to investigate the pathogenic mechanisms underlying USH2A-associated retinal degeneration and sensorineural hearing loss.

A universal gene correction approach for FKRP-associated dystroglycanopathies to enable autologous cell therapy.

SUMMARYMutations in the fukutin-related protein (FKRP) gene result in a broad spectrum of muscular dystrophy (MD) phenotypes, including the severe Walker-Warburg syndrome (WWS). Here, we develop a gene-editing approach that replaces the entire mutant open reading frame with the wild-type sequence to universally correct all FKRP mutations. We apply this approach to correct FKRP mutations in induced pluripotent stem (iPS) cells derived from patients displaying broad clinical severity. Our findings show rescue of functional α-dystroglycan (α-DG) glycosylation in gene-edited WWS iPS cell-derived myotubes. Transplantation of gene-corrected myogenic progenitors in the FKRPP448L-NSG mouse model gives rise to myofiber and satellite cell engraftment and, importantly, restoration of α-DG functional glycosylation in vivo. These findings suggest the potential feasibility of using CRISPR-Cas9 technology in combination with patient-specific iPS cells for the future development of autologous cell transplantation for FKRP-associated MDs.

Keratinocytes from Gorlin Syndrome-induced pluripotent stem cells are resistant against UV radiation.

Gorlin syndrome (GS) is an autosomal dominant genetic disorder involving Patched 1 (PTCH1) mutations. The PTCH1 is a receptor as well as an inhibitor of hedgehog (Hh) to sequester downstream Hh pathway molecules called Smoothened (SMO). PTCH1 mutations causes a variety of GS conditions including falx calcification, odontogenic keratocytes and basal cell carcinomas (BCC). Because PTCH1 is a major driver gene of sporadic BCC, GS patients are characteristically prone to BCC. In order to elucidate the pathological mechanism of BCC-prone GS patients, we investigated keratinocytes derived from GS patient specific iPS cells (G-OFiPSCs) which were generated and reported previously. We found that keratinocytes derived from G-OFiPSCs (GKCs) have increased expression of Hh target molecules. GKCs were irradiated and those cells showed high resistance to UV induced apoptosis. BCL2, known as anti-apoptotic molecule as well as Hh target, significantly increased in GKCs. Several molecules involved in DNA repair, cell cycle control, senescence, and genotoxic stress such as TP53, BRCA1 and GADD45A increased only in GKCs. GKCs are indicated to be resistant to UV irradiation by upregulating molecules which control DNA repair and genotoxic even under DNA damage caused by UV. The anti-apoptotic properties of GKCs may contribute BCC.

Establishment of induced pluripotent stem cell line CSUASOi003- A from an autosomal recessive retinitis pigmentosa patient carrying compound heterozygous mutations in CRB1 gene.

Crumbs homologue 1 (CRB1) mutations have been found in retinitis pigmentosa (RP) patients lead to severe retinal dystrophies. The human induced pluripotent stem (iPS) cell line CSUASOi003-A derived from peripheral blood mononuclear cells (PBMCs) of a patient carrying two heterozygous mutations (2249G>A p.G750D and c.2809G>A p.A937T) in CRB1 gene was generated by non-integrative reprogramming technology. Pluripotency and differentiation capacity were assessed by immunocytochemistry and quantitative polymerase chain reaction (qPCR). The RP patient-specific iPS cell line provide a powerful model for evaluating the pathological phenotypes of the disease.

Analysis of changes in the expression of dopamine-related genes in fibromyalgia patient specific-iPS cell derived-dopaminergic neurons

Analysis of changes in the expression of dopamine-related genes in fibromyalgia patient specific-iPS cell derived-dopaminergic neurons

Effects of reprogramming on genomic imprinting and the application of pluripotent stem cells.

Pluripotent stem cells are considered to be the ideal candidates for cell-based therapies in humans. In this regard, both nuclear transfer embryonic stem (ntES) cells and induced pluripotent stem (iPS) cells are particularly advantageous because patient-specific autologous ntES and iPS cells can avoid immunorejection and other side effects that may be present in the allogenic pluripotent stem cells derived from unrelated sources. However, they have been found to contain deleterious genetic and epigenetic changes that may hinder their therapeutic applications. Indeed, deregulation of genomic imprinting has been frequently observed in reprogrammed ntES and iPS cells. We will survey the recent studies on genomic imprinting in pluripotent stem cells, particularly in iPS cells. In a previous study published about six years ago, genomic imprinting was found to be variably lost in mouse iPS clones. Intriguingly, de novo DNA methylation also occurred at the previously unmethylated imprinting control regions (ICRs) in a high percentage of iPS clones. These unexpected results were confirmed by a recent independent study with a similar approach. Since dysregulation of genomic imprinting can cause many human diseases including cancer and neurological disorders, these recent findings on genomic imprinting in reprogramming may have some implications for therapeutic applications of pluripotent stem cells.

Effects of the FGF2 aptamer on growth plate cartilage development of achondroplasia patient-specific iPS cells in a xenograft model

Effects of the FGF2 aptamer on growth plate cartilage development of achondroplasia patient-specific iPS cells in a xenograft model

M89 - MODELING HUMAN CORTICAL BRAIN DEVELOPMENT BY THE GENERATION OF CEREBRAL ORGANOIDS FROM PATIENT-SPECIFIC STEM CELLS FOR THE ANALYSIS OF SCHIZOPHRENIA GENETICS

Background Characteristics of schizophrenia include the deregulation of the glutamatergic and the GABAergic neurotransmission and the degeneration of predominantly inhibitory interneurons within the cortex of the human brain. Cortical development was extensively studied in rodents, but the unique and complex architecture of the human cortex differs dramatically from brain of established animal models. Accordingly, the analysis of schizophrenia animal models is limited by the species differences, which requires the development of adequate human in vitro models mimicking the human cortical brain development. Schizophrenia is associated with DNA variations suggesting that genetic factors contribute to onset, progression and treatment of the disease. We generated induced pluripotent stem cells (iPS cells), which allow the generation of disease- and patient-specific neuronal cultures for the analysis of genetic factors. However, the functional characterization of genetic factors requires the analysis of advanced tissue-based 3D disease models. Therefore, we generated cerebral organoids for mimicking aspects of the human cortical development. Methods Human iPS cells from healthy donors and patient-specific iPS cells from schizophrenia patients were differentiated into permanent Neural Stem Cells (NSCs). Transcript and protein analysis confirmed the differentiation status of iPS cells and NSCs. NSCs were characterized in cellular 2D differentiation models. NSCs were further differentiated as free-floating neurospheres to acquire functional 3D organoids. Cells were analyzed by transcript and protein analysis. Cryosectioning of organoids was applied to visualize the distribution of different neuronal subtypes. Results Protein and transcript analysis of specific markers including OCT4 and the activity of alkaline phosphatases verified pluripotency in patient-derived iPS cells. The analysis of several NSC markers such as SOX2 and PAX6 indicated stable generation of permanent NSCs from patient-specific iPS cells. Patient-specific iPS cells were successfully differentiated into progenitor cells expressing a variety of neural lineage markers including neural makers such as TUBB3 and STX as wells as glia cell markers such as GFAP and O4. Immunostaining and whole-cell patch-clamp recordings of mature neurons showed the presence of different neuronal subtypes including inhibitory GABAergic neurons. NSCs were also successfully applied to the generation of 3D cerebral organoids. The 3D organoid differentiation model showed the RNA and protein expression of neural lineage markers similar to cells obtained from the 2D monolayer cultures. The expression pattern of cortical markers such as TBR1 observed in cryosections of organoids revealed the induction of cortical layers mimicking the developing human cortex. Discussion We successfully generated 3D cerebral organoids mimicking aspects of the early human brain development. These culture systems enable functional studies of healthy and diseased human cortical development for the analysis of neurodevelopmental diseases including schizophrenia.

SU105 - MODELING SCHIZOPHRENIA IN VITRO BY THE GENERATION OF NEURONS FROM PATIENT-SPECIFIC STEM CELLS

Background Schizophrenia is a neurodevelopmental disease. Causes include environmental and genetic factors. Characteristics of schizophrenia include a deregulation of the glutamatergic and the GABAergic neurotransmission and the degeneration of predominantly inhibitory interneurons. Recently, 108 loci referring to Single Nucleotide Polymorphisms (SNPs) and 8 loci containing Copy Number Variations (CNVs) have been associated with schizophrenia suggesting a strong impact of genetic factors on the embryonic development of human brain. Together with other research teams, we identified CNVs in the Neurexin 1 gene (NRXN1) in patients suffering from schizophrenia, which highly recommends the detailed analysis of NRXN1-related disease mechanisms. Neurexins are presynaptic transmembrane proteins. We established patient-specific induced Pluripotent Stem (iPS) cells from schizophrenia patients and healthy donors to differentiate neurons for the investigation of disease mechanisms related to NRXN1 in schizophrenia patients. Patient-specific iPS cells carry the disease-associated genetic background of a patient, which is a CNVs affecting NRXN1. Methods Human iPS cells were generated from B-Lymphoblastoid Cell Lines (B-LCLs) using episomal vectors. B-LCLs were obtained from patients carrying CNVs in NRXN1 and healthy donors. The pluripotency of stem cells was verified by alkaline phosphatase staining, immunofluorescence analysis, transcript analysis, western blot analysis, and the induction of three germ layers. A neuronal screening protocol was applied to select iPS cell clones with a high differentiation potential towards neuronal cell lineages. Established lines were differentiated into Neural Stem Cells (NSCs) and further differentiated into functional and mature neurons. Terminal differentiated neurons were characterized by morphology, transcript analysis, western blot analysis, immunofluorescence analysis, electrophysiology, and mitochondrial respiration. Results Transcript analysis and western blot analysis of iPS cells verified permanent induction of pluripotency marker genes including OCT4. The spontaneous differentiation of iPS cells into derivatives of the three germ layers was demonstrated. The induction of neural stem cells was characterized by mRNA and protein analysis of neural stem cells markers including SOX2 and PAX6. Neural induction towards cortical neurons was monitored by a set of neurodevelopmental marker genes including NESTIN, GFAP, and NGN3. The efficient differentiation of mature neurons was shown by the expression of marker genes including glutamate and GABA receptors. Mitochondrial respiration was elevated in differentiated neurons. Patch clamp analysis verified the differentiation of functional GABAergic and glutamatergic neurons. Discussion Human iPS cells provide a powerful tool for the analysis of schizophrenia-associated and patient-specific DNA variations including CNVs. We successfully established mature and functional neuronal cultures suitable to study disease mechanisms related to NRXN1. Accordingly, this approach is an excellent opportunity for the identification of potential therapeutic targets.

Estimating the concentration of therapeutic range using disease-specific iPS cells: Low-dose rapamycin therapy for Pendred syndrome.

IntroductionPendred syndrome is an autosomal-recessive disease characterized by congenital hearing loss and thyroid goiter. Previously, cell stress susceptibilities were shown to increase in patient-derived cells with intracellular aggregation using an in vitro acute cochlear cell model derived from patient-specific pluripotent stem (iPS) cells. Moreover, we showed that rapamycin can relieve cell death. However, studies regarding long-term cell survival without cell stressors that mimic the natural course of disease or the rational minimum concentration of rapamycin that prevents cell death are missing.MethodsIn this report, we first investigated the rational minimum concentration of rapamycin using patient-specific iPS cells derived-cochlear cells with three different conditions of acute stress. We next confirmed the effects of rapamycin in long-term cell survival and phenotypes by using cochlear cells derived from three different patient-derived iPS cells.ResultsWe found that inner ear cells derived from Pendred syndrome patients are more vulnerable than those from healthy individuals during long-term culturing; however, this susceptibility was relieved via treatment with low-dose rapamycin. The slow progression of hearing loss in patients may be explained, in part, by the vulnerability observed in patient cells during long-term culturing. We successfully evaluated the rational minimum concentration of rapamycin for treatment of Pendred syndrome.ConclusionOur results suggest that low-dose rapamycin not only decreases acute symptoms but may prevent progression of hearing loss in Pendred syndrome patients.

Characterization of Hematopoietic Differentiation Profiles of MPN Patient-Derived Inducible Pluripotent Stem Cells Harboring Homozygous Vs Heterozygous Calreticulin Mutations

Introduction: Somatic calreticulin (CALR) mutations were discovered in patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) and have been shown to be mutually exclusive with Janus kinase 2 (JAK2) and thrombopoietin receptor (MPL) mutations. Recent studies demonstrated that the binding of CALR mutant proteins to MPL induces constitutive activation of the JAK/STAT pathway, thus causing cellular transformation and abnormal megakaryopoiesis. Additionally, it has been reported that patients carrying homozygously mutated CALR ins5 exhibit myeloperoxidase (MPO) deficiency as a result of the absence of CALR chaperone function. However, the impact of CALR mutant homozygosity vs. heterozygosity in CALR del52 mutations as well as on hematopoietic differentiation has not yet been studied. Furthermore, clonal heterogeneity of hematopoietic stem/progenitor cell (HSPC) populations in a patient, together with technical limitations isolating single clones, are major challenges, when determining the impact of CALR mutant zygosity on clonal composition and diversity in MPN. To overcome these limitations, we generated patient-specific iPS cells carrying homozygous or heterozygous CALR mutations or their wild-type counterparts to study their roles in hematopoietic differentiation.Methods: iPS cells were generated by reprogramming peripheral blood-derived mononuclear cells from three patients carrying CALR del52, ins5, or del31 mutations using a CytoTune iPS 2.0 Sendai Reprogramming Kit. Individual colonies were picked and screened for CALR genotypes by PCR. Pluripotency of iPS cells was confirmed by immunofluorescences, and the clones were screened for additional mutations using panel-based next generation sequencing (NGS). Subsequently, CALR iPS cells were subjected to embryonic body formation, mesoderm commitment, and hematopoietic differentiation using our standard in vitro differentiation protocol. CD34+ HSPCs were MACS-sorted and characterized by flow cytometry, cytospins and RNA expression analysis on days 10, 15, and 20 during differentiation. Hematopoietic progenitors, erythrocytes, granulocytes, and megakaryocytes were identified by defined lineage markers. MPO expression was assessed by flow cytometry and cytochemical staining.Results: We established patient-specific iPS cells carrying CALR del52, ins5 or del31 mutation after written informed consent (Table 1). Pluripotency markers OCT4, Tra-1-60 and Tra-1-81 expression were confirmed in all iPS cell clones. In accordance with findings in peripheral blood cells, we detected MPO deficiency in homozygous iPS cell-derived CD15+ cells from CALRins5- and, in addition, also from CALRdel52-mutated patients (pMFI=0.0106 and pMFI=0.0187, resp.). Intriguingly, in vitro hematopoietic differentiation assays revealed additional abnormalities, such as decreased CD66b+ granulocytes derived from homozygous CALR del52 or ins5 iPS cells vs. heterozygous iPS cells on day 10 (pdel52=0.0303 and pins5=0.0253, resp.) and a trend towards increased KIThigh+CD45+ cells. Megakaryopoiesis, defined by CD41+CD42b+ cells, was increased in CALRins5 homozygous vs. heterozygous clones (p=0.0031). However, this bias was not observed in all clones, indicating clone-specific megakaryocytic differentiation potential. No phenotypic differences during hematopoietic differentiation were observed in iPS cell-derived progenitors carrying heterozygous CALRdel31 mutation and its isogenic unmutated CALR controls. Furthermore, our NGS data revealed patient-specific sets of co-occurring mutations in iPS cell clones, which may have contributed to the observed patient-specific phenotypes. As an example, the IDH2 R140Q mutation, reported to block cell differentiation, was found in approximately half of the CALRdel52 iPS clones, and these clones failed to differentiate into the hematopoietic lineage in vitro.Conclusions: We successfully generated patient-specific CALR mutant iPS cells. Upon in vitro differentiation, we detected MPO deficiency and aberrant granulocytic differentiation in CALR homozygous but not heterozygous or wild-type clones. Thus, it is now possible at the single stem cell level to further analyze the molecular mechanisms of CALR-mutant induced MPO deficiency and altered hematopoietic differentiation, in order to better understand disease biology in ET and PMF. [Display omitted] DisclosuresBrümmendorf:Merck: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Janssen: Consultancy; Takeda: Consultancy.

Modeling hallmark pathology using motor neurons derived from the family and sporadic amyotrophic lateral sclerosis patient-specific iPS cells

BackgroundAmyotrophic lateral sclerosis (ALS) represents a devastating, progressive, heterogeneous, and the most common motor neuron (MN) disease. To date, no cure has been available for the condition. Studies with transgenic mice have yielded significant results that help us understand the underlying mechanisms of ALS. Nonetheless, none of more than 30 large clinical trials over the past 20 years proved successful, which led some researchers to challenge the validity of the preclinical models.MethodsHuman-induced pluripotent cells (iPSCs) were established by introducing Sendai virus into fibroblast cells. We established TDP-43 HES by inserting CAG-TDP43 (G298S) cassette or the CAG-EGFP cassette into PPP1R12C-locus of human embryonic stem cells (ESC, H9) by TALEN-mediated homologous recombination. iPSCs or HESC were differentiated to motor neurons and non-motor neuron as control. Relevant biomarkers were detected in different differentiated stages. TDP-43 aggregates, neurofilament, and mitochondria analyses were performed.ResultsIn this study, using iPSCs-derived human MN from an ALS patient with a TDP43 G298S mutation and two sporadic ALS patients, we showed that both sporadic and familial ALS were characterized by TDP-43 aggregates in the surviving MN. Significantly higher neurofilament (NF) inclusion was also found in ALS MN compared with wild-type (WT) GM15 controls (P < 0.05). The neurite mitochondria density was significantly lower in ALS MN than that in the control MNs. Transgenesis of TDP-43 G298S into AAVS locus in human embryonic stem cells reproduced phenotype of patient-derived G289S MN. By challenging MNs with a proteasome inhibitor, we found that MNs were more vulnerable to MG132, with some accompanying phenotype changes, such as TDP43 translocation, NF inclusion, mitochondria distribution impairment, and activation of caspase3.ConclusionsOur results suggested that changes in TDP43 protein, NF inclusion, and distribution impairment of mitochondria are common early pathology both in familial and sporadic ALS. These findings will help us gain insight into the pathogenesis of the condition and screen relevant drugs for the disease.

Polarized Human Retinal Pigment Epithelium Exhibits Distinct Surface Proteome on Apical and Basal Plasma Membranes.

Surface proteins localized on the apical and basal plasma membranes are required for a cell to sense its environment and relay changes in ionic, cytokine, chemokine, and hormone levels to the inside of the cell. In a polarized cell, surface proteins are differentially localized on the apical or the basolateral sides of the cell. The retinal pigment epithelium (RPE) is an example of a polarized cell that performs a variety of functions that are dependent on its polarized state including trafficking of ions, fluid, and metabolites across the RPE monolayer. These functions are absolutely crucial for maintaining the health and integrity of adjacent photoreceptors, the photosensitive cells of the retina. Here we present a series of approaches to identify and validate the polarization state of cultured primary human RPE cells using immunostaining for RPE apical/basolateral markers, polarized cytokine secretion, electrophysiology, fluid transport, phagocytosis, and identification of plasma membrane proteins through cell surface capturing technology. These approaches are currently being used to validate the polarized state and the epithelial phenotype of human induced pluripotent stem (iPS) cell derived RPE cells. This work provides the basis for developing an autologous cell therapy for age-related macular degeneration using patient specific iPS cell derived RPE.

A Skeletal Muscle Model of Infantile-onset Pompe Disease with Patient-specific iPS Cells

Pompe disease is caused by an inborn defect of lysosomal acid α-glucosidase (GAA) and is characterized by lysosomal glycogen accumulation primarily in the skeletal muscle and heart. Patients with the severe type of the disease, infantile-onset Pompe disease (IOPD), show generalized muscle weakness and heart failure in early infancy. They cannot survive over two years. Enzyme replacement therapy with recombinant human GAA (rhGAA) improves the survival rate, but its effect on skeletal muscle is insufficient compared to other organs. Moreover, the patho-mechanism of skeletal muscle damage in IOPD is still unclear. Here we generated induced pluripotent stem cells (iPSCs) from patients with IOPD and differentiated them into myocytes. Differentiated myocytes showed lysosomal glycogen accumulation, which was dose-dependently rescued by rhGAA. We further demonstrated that mammalian/mechanistic target of rapamycin complex 1 (mTORC1) activity was impaired in IOPD iPSC-derived myocytes. Comprehensive metabolomic and transcriptomic analyses suggested the disturbance of mTORC1-related signaling, including deteriorated energy status and suppressed mitochondrial oxidative function. In summary, we successfully established an in vitro skeletal muscle model of IOPD using patient-specific iPSCs. Disturbed mTORC1 signaling may contribute to the pathogenesis of skeletal muscle damage in IOPD, and may be a potential therapeutic target for Pompe disease.

Generation of induced pluripotent stem cells derived from an autosomal dominant polycystic kidney disease patient with a p.Ser1457fs mutation in PKD1

Autosomal dominant polycystic kidney disease is one of the most prevalent forms of inherited cystic kidney disease, and can be characterized by kidney cyst formation and enlargement. Here we report the generation of a Type 1 ADPKD disease iPS cell line, IBMS-iPSC-012-12, which retains the conserved deletion of PKD1, normal karyotype and exhibits the properties of pluripotent stem cells such as ES-like morphology, expression of pluripotent markers and capacity to differentiate into all three germ layers. Our results show that we have successfully generated a patient-specific iPS cell line with a mutation in PKD1 for study of renal disease pathophysiology.

Modeling Human Neurological and Neurodegenerative Diseases: From Induced Pluripotent Stem Cells to Neuronal Differentiation and Its Applications in Neurotrauma.

With the help of several inducing factors, somatic cells can be reprogrammed to become induced pluripotent stem cell (iPSCs) lines. The success is in obtaining iPSCs almost identical to embryonic stem cells (ESCs), therefore various approaches have been tested and ultimately several ones have succeeded. The importance of these cells is in how they serve as models to unveil the molecular pathways and mechanisms underlying several human diseases, and also in its potential roles in the development of regenerative medicine. They further aid in the development of regenerative medicine, autologous cell therapy and drug or toxicity screening. Here, we provide a comprehensive overview of the recent development in the field of iPSCs research, specifically for modeling human neurological and neurodegenerative diseases, and its applications in neurotrauma. These are mainly characterized by progressive functional or structural neuronal loss rendering them extremely challenging to manage. Many of these diseases, including Parkinson's disease (PD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD) have been explored in vitro. The main purpose is to generate patient-specific iPS cell lines from the somatic cells that carry mutations or genetic instabilities for the aim of studying their differentiation potential and behavior. This new technology will pave the way for future development in the field of stem cell research anticipating its use in clinical settings and in regenerative medicine in order to treat various human diseases, including neurological and neurodegenerative diseases.

Modeling Human Brain Development And Disease In Stem Cell Derived 3D Organoid Culture

The human brain is highly unique in size and complexity. While many of its characteristics have been successfully studied in model organisms, recent experiments have emphasized unique features that cannot easily be modeled in animals. We have therefore developed a 3D organoid culture system derived from human pluripotent stem cells that recapitulates many aspects of human brain development. These cerebral organoids are capable of generating several brain regions including a well-organized cerebral cortex. Furthermore, human cerebral organoids display stem cell properties and progenitor zone organization that show characteristics specific to humans. Finally, we use both RNAi and patient specific iPS cells to model microcephaly, a human neurodevelopmental disorder that has been difficult to recapitulate in mice. This approach reveals premature neuronal differentiation with loss of the microcephaly protein CDK5RAP2, a defect that could explain the disease phenotype. Our data demonstrate an in vitro approach that recapitulates development of even this most complex organ, which can be used to gain insights into disease mechanisms.

Towards a Safer, More Randomized Lentiviral Vector Integration Profile Exploring Artificial LEDGF Chimeras.

The capacity to integrate transgenes into the host cell genome makes retroviral vectors an interesting tool for gene therapy. Although stable insertion resulted in successful correction of several monogenic disorders, it also accounts for insertional mutagenesis, a major setback in otherwise successful clinical gene therapy trials due to leukemia development in a subset of treated patients. Despite improvements in vector design, their use is still not risk-free. Lentiviral vector (LV) integration is directed into active transcription units by LEDGF/p75, a host-cell protein co-opted by the viral integrase. We engineered LEDGF/p75-based hybrid tethers in an effort to elicit a more random integration pattern to increase biosafety, and potentially reduce proto-oncogene activation. We therefore truncated LEDGF/p75 by deleting the N-terminal chromatin-reading PWWP-domain, and replaced this domain with alternative pan-chromatin binding peptides. Expression of these LEDGF-hybrids in LEDGF-depleted cells efficiently rescued LV transduction and resulted in LV integrations that distributed more randomly throughout the host-cell genome. In addition, when considering safe harbor criteria, LV integration sites for these LEDGF-hybrids distributed more safely compared to LEDGF/p75-mediated integration in wild-type cells. This approach should be broadly applicable to introduce therapeutic or suicide genes for cell therapy, such as patient-specific iPS cells.

MPS 01-09 THE APPLICATION OF VASCULAR CELL DIFFERENTIATION SYSTEM FROM HUMAN ES/iPS CELLS FOR VASCULAR BIOLOGY

Objective: Previously, we have succeeded in inducing and isolating vascular endothelial cells (ECs) and mural cells (MCs) from human iPS cells. Recently, we modified our vascular differentiation system and we have induced vascular cells more effectively and stably from various ES/iPS cell clones. Our vascular differentiation system could be applied for the clarification of vascular biology or pathology, because we can observe the change of gene expressions in the process of human vascular cell differentiation, not mouse or other species. Design and Method: We have researched about the expressions of cardiovascular hormones and their receptors through our vascular differentiation kinetics, aiming to research the meanings of these hormones into vascular cells. At first, we examined mRNA expressions in undifferentiated ES/iPS cells, ES/iPS-derived immature vascular cells, ES/iPS-derived ECs, ES/iPS-derived MCs. Also we used cell line aortic ECs and smooth muscle cells(SMCs) as control. Results: hES/iPS derived ECs expressed adrenomedullin and endothelin, which mean that we succeed to induce mature ECs endocrinologically. Also I examined the expressions of natriuretic peptides family. Interestingly, the mRNA expression of brain natriuretic peptide (BNP) was highly detected only in sorted Flk1-positive cells, which are considered as immature vascular cells. After differentiation, these BNP expressions were decreased. Also we could measure BNP at high concentration in the culture medium of Flk1 positive cells. By doing fraction assay, the majority of measured BNP turned out to be not BNP-32 but pro-BNP. As for NP receptors, GC-A were expressed only in ES or iPS – derived EC. Conclusions: These results suggest the possibility that BNP (which would be secreted from immature vascular cells) has some paracrine functions to vascular endothelial cells and now we examine the function of BNP for vascular differentiation/development. Also I would introduce other application of our vascular differentiation system, using patient specific-iPS cells.