A disease-specific iPS cell resource for studying rare and intractable diseases.

BACKGROUND: Disease-specific induced pluripotent stem cells (iPSCs) are useful tools for pathological analysis and diagnosis of rare diseases. Given the limited available resources, banking such disease-derived iPSCs and promoting their widespread use would be a promising approach for untangling the mysteries of rare diseases. Herein, we comprehensively established iPSCs from patients with designated intractable diseases in Japan and evaluated their properties to enrich rare disease iPSC resources. METHODS: Patients with designated intractable diseases were recruited for the study and blood samples were collected after written informed consent was obtained from the patients or their guardians. From the obtained samples, iPSCs were established using the episomal method. The established iPSCs were deposited in a cell bank. RESULTS: We established 1,532 iPSC clones from 259 patients with 139 designated intractable diseases. The efficiency of iPSC establishment did not vary based on age and sex. Most iPSC clones originated from non-T and non-B hematopoietic cells. All iPSC clones expressed key transcription factors, OCT3/4 (range 0.27-1.51; mean 0.79) and NANOG (range 0.15-3.03; mean 1.00), relative to the reference 201B7 iPSC clone. CONCLUSIONS: These newly established iPSCs are readily available to the researchers and can prove to be a useful resource for research on rare intractable diseases.

iPS cells from Chediak-Higashi syndrome patients recapitulate the giant granules in myeloid cells.

BACKGROUND: Chediak-Higashi syndrome (CHS) is a congenital disease characterized by immunodeficiency, hemophagocytic lymphohistiocytosis, oculocutaneous albinism, and neurological symptoms. The presence of giant granules in peripheral blood leukocytes is an important hallmark of CHS. Here we prepared induced pluripotent stem cells (iPSCs) from CHS patients (CHS-iPSCs) and differentiated them into hematopoietic cells to model the disease phenotypes. METHODS: Fibroblasts were obtained from two CHS patients and then reprogrammed into iPSCs. The iPSCs were differentiated into myeloid cells; the size of the cytosolic granules was quantified by May-Grunwald Giemsa staining and myeloperoxidase staining. RESULTS: Two clones of iPSCs were established from each patient. The differentiation efficiency to CD33+ CD45+ myeloid cells was not significantly different in CHS-iPSCs compared with control iPSCs, but significantly larger granules were observed. CONCLUSIONS: We succeeded in reproducing a characteristic cellular phenotype, giant granules in myeloid cells, using CHS-iPSCs, demonstrating that iPSCs can be used to model the pathogenesis of CHS patients.

Deterioration of phagocytosis in induced pluripotent stem cell-derived retinal pigment epithelial cells established from patients with retinitis pigmentosa carrying Mer tyrosine kinase mutations.

Retinitis pigmentosa (RP) is an incurable retinal degenerative disease with an unknown mechanism of disease progression. Mer tyrosine kinase (MERTK), which encodes a receptor of the Tyro3/Axl/Mer family of tyrosine kinases, is one of the causal genes of RP. MERTK is reportedly expressed in the retinal pigment epithelium (RPE) and is essential for phagocytosis of the photoreceptor outer segment. Here, we established induced pluripotent stem cells (iPSC) from patients with RP having homozygous or compound heterozygous mutations in MERTK, and from healthy subjects; the RP patient- and healthy control-derived iPSCs were differentiated into RPE cells. Although cytoskeleton staining suggested that polarity may have been disturbed mildly, there were no apparent morphological differences between the diseased and normal RPE cells. The internalization of photoreceptor outer segments in diseased iPSC-RPE cells was significantly lower than that in normal iPSC-RPE cells. This in vitro disease model may be useful for elucidating the mechanisms of disease progression and screening treatments for the disease.

Human AK2 links intracellular bioenergetic redistribution to the fate of hematopoietic progenitors.

AK2 is an adenylate phosphotransferase that localizes at the intermembrane spaces of the mitochondria, and its mutations cause a severe combined immunodeficiency with neutrophil maturation arrest named reticular dysgenesis (RD). Although the dysfunction of hematopoietic stem cells (HSCs) has been implicated, earlier developmental events that affect the fate of HSCs and/or hematopoietic progenitors have not been reported. Here, we used RD-patient-derived induced pluripotent stem cells (iPSCs) as a model of AK2-deficient human cells. Hematopoietic differentiation from RD-iPSCs was profoundly impaired. RD-iPSC-derived hemoangiogenic progenitor cells (HAPCs) showed decreased ATP distribution in the nucleus and altered global transcriptional profiles. Thus, AK2 has a stage-specific role in maintaining the ATP supply to the nucleus during hematopoietic differentiation, which affects the transcriptional profiles necessary for controlling the fate of multipotential HAPCs. Our data suggest that maintaining the appropriate energy level of each organelle by the intracellular redistribution of ATP is important for controlling the fate of progenitor cells.

Pluripotent stem cell models of Blau syndrome reveal an IFN-γ-dependent inflammatory response in macrophages.

BACKGROUND: Blau syndrome, or early-onset sarcoidosis, is a juvenile-onset systemic granulomatosis associated with a mutation in nucleotide-binding oligomerization domain 2 (NOD2). The underlying mechanisms of Blau syndrome leading to autoinflammation are still unclear, and there is currently no effective specific treatment for Blau syndrome. OBJECTIVES: To elucidate the mechanisms of autoinflammation in patients with Blau syndrome, we sought to clarify the relation between disease-associated mutant NOD2 and the inflammatory response in human samples. METHODS: Blau syndrome-specific induced pluripotent stem cell (iPSC) lines were established. The disease-associated NOD2 mutation of iPSCs was corrected by using a CRISPR-Cas9 system to precisely evaluate the in vitro phenotype of iPSC-derived cells. We also introduced the same NOD2 mutation into a control iPSC line. These isogenic iPSCs were then differentiated into monocytic cell lineages, and the statuses of nuclear factor κB pathway and proinflammatory cytokine secretion were investigated. RESULTS: IFN-γ acted as a priming signal through upregulation of NOD2. In iPSC-derived macrophages with mutant NOD2, IFN-γ treatment induced ligand-independent nuclear factor κB activation and proinflammatory cytokine production. RNA sequencing analysis revealed distinct transcriptional profiles of mutant macrophages both before and after IFN-γ treatment. Patient-derived macrophages demonstrated a similar IFN-γ-dependent inflammatory response. CONCLUSIONS: Our data support the significance of ligand-independent autoinflammation in the pathophysiology of Blau syndrome. Our comprehensive isogenic disease-specific iPSC panel provides a useful platform for probing therapeutic and diagnostic clues for the treatment of patients with Blau syndrome.

Insulin-producing cells derived from 'induced pluripotent stem cells' of patients with fulminant type 1 diabetes: Vulnerability to cytokine insults and increased expression of apoptosis-related genes.

AIMS/INTRODUCTION: The present study was carried out to generate induced pluripotent stem cells (iPSCs) from patients with fulminant type 1 diabetes, and evaluate the cytokine-induced apoptotic reactions of ß-like insulin-producing cells differentiated from the iPSCs. MATERIALS AND METHODS: iPSCs were generated from fibroblasts of patients with fulminant type 1 diabetes by inducing six reprogramming factors. Insulin-producing cells were differentiated from the iPSCs in vitro. The proportion of cleaved caspase-3-positive or terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling-positive cells among insulin (INS)-positive cells derived from fulminant type 1 diabetes iPSC and control human iPSC lines was evaluated under treatment with tumor necrosis factor-α, interleukin-1ß and interferon-γ. Ribonucleic acid sequencing was carried out to compare gene expressions in INS-positive cells derived from fulminant type 1 diabetes iPSC and control human iPSC lines. RESULTS: Two iPSC clones were established from each of three patients with fulminant type 1 diabetes. The differentiation of insulin-producing cells from fulminant type 1 diabetes iPSC was confirmed by immunofluorescence analysis and KCl-induced C-peptide secretion. After treatment with pro-inflammatory cytokines, these INS-positive cells showed higher expression of cleaved caspase-3 than those derived from control human iPSCs. Altered expression levels of several apoptosis-related genes were observed in INS-positive cells derived from the fulminant type 1 diabetes iPSCs by ribonucleic acid sequencing. CONCLUSIONS: We generated iPSCs from patients with fulminant type 1 diabetes and differentiated them into insulin-producing cells. This in vitro disease model can be used to elucidate the disease mechanisms of fulminant type 1 diabetes.

Identification of a High-Frequency Somatic NLRC4 Mutation as a Cause of Autoinflammation by Pluripotent Cell-Based Phenotype Dissection.

OBJECTIVE: To elucidate the genetic background of a patient with neonatal-onset multisystem inflammatory disease (NOMID) with no NLRP3 mutation. METHODS: A Japanese male child diagnosed as having NOMID was studied. The patient did not have any NLRP3 mutation, even as low-frequency mosaicism. We performed whole-exome sequencing on the patient and his parents. Induced pluripotent stem cells (iPSCs) were established from the patient's fibroblasts. The iPSCs were then differentiated into monocyte lineage to evaluate the cytokine profile. RESULTS: We established multiple iPSC clones from a patient with NOMID and incidentally found that the phenotypes of monocytes from iPSC clones were heterogeneous and could be grouped into disease and normal phenotypes. Because each iPSC clone was derived from a single somatic cell, we hypothesized that the patient had somatic mosaicism of an interleukin-1ß-related gene. Whole-exome sequencing of both representative iPSC clones and the patient's blood revealed a novel heterozygous NLRC4 mutation, p.T177A (c.529A>G), as a specific mutation in diseased iPSC clones. Knockout of the NLRC4 gene using the clustered regularly interspaced short palindromic repeat/Cas9 system in a mutant iPSC clone abrogated the pathogenic phenotype. CONCLUSION: Our findings indicate that the patient has somatic mosaicism of a novel NLRC4 mutation. To our knowledge, this is the first case showing that somatic mutation of NLRC4 causes autoinflammatory symptoms compatible with NOMID. The present study demonstrates the significance of prospective genetic screening combined with iPSC-based phenotype dissection for individualized diagnoses.

BMP-SMAD-ID promotes reprogramming to pluripotency by inhibiting p16/INK4A-dependent senescence.

Fibrodysplasia ossificans progressiva (FOP) patients carry a missense mutation in ACVR1 [617G > A (R206H)] that leads to hyperactivation of BMP-SMAD signaling. Contrary to a previous study, here we show that FOP fibroblasts showed an increased efficiency of induced pluripotent stem cell (iPSC) generation. This positive effect was attenuated by inhibitors of BMP-SMAD signaling (Dorsomorphin or LDN1931890) or transducing inhibitory SMADs (SMAD6 or SMAD7). In normal fibroblasts, the efficiency of iPSC generation was enhanced by transducing mutant ACVR1 (617G > A) or SMAD1 or adding BMP4 protein at early times during the reprogramming. In contrast, adding BMP4 at later times decreased iPSC generation. ID genes, transcriptional targets of BMP-SMAD signaling, were critical for iPSC generation. The BMP-SMAD-ID signaling axis suppressed p16/INK4A-mediated cell senescence, a major barrier to reprogramming. These results using patient cells carrying the ACVR1 R206H mutation reveal how cellular signaling and gene expression change during the reprogramming processes.

Impaired adipogenic capacity in induced pluripotent stem cells from lipodystrophic patients with BSCL2 mutations.

OBJECTIVE: Congenital generalized lipodystrophy (CGL) is an autosomal recessive disorder characterized by marked scarcity of adipose tissue, extreme insulin resistance, hypertriglyceridemia, hepatic steatosis and early-onset diabetes. Mutation of the BSCL2/SEIPIN gene causes the most severe form of CGL. The aim of this study was to generate induced pluripotent stem (iPS) cells from patients with CGL harboring BSCL2/SEIPIN mutations. METHODS: Skin biopsies were obtained from two Japanese patients with CGL harboring different nonsense mutations (E189X and R275X) in BSCL2/SEIPIN. The fibroblasts thus obtained were infected with retroviruses encoding OCT4, SOX2, c-MYC, and KLF4. The generated iPS cells were evaluated for pluripotency by examining the expression of pluripotency markers (alkaline phosphatase, SSEA-4, TRA-1-60, and NANOG) and their ability to differentiate to three germ layers in vitro by forming embryoid bodies, and to form teratomas in vivo. Adipogenic capacity of differentiated BSCL2-iPS cells was determined by oil red O and adipose differentiation-related protein (ADRP) staining. Rescue experiments were also performed using stable expression of wild-type BSCL2. A coimmunoprecipitation assay was conducted to investigate the interaction of SEIPIN with ADRP. RESULTS: iPS cells were generated from fibroblasts of the two patients with CGL. Each of the patient-derived iPS (BSCL2-iPS) clones showed all of the hallmarks of pluripotency and could differentiate into derivatives of all three germ layers in vitro by forming embryoid bodies, and form teratomas after injection into mouse testes. BSCL2-iPS cells maintained the mutations in BSCL2 and lacked intact BSCL2. Upon adipogenic differentiation, BSCL2-iPS cells exhibited marked reduction of lipid droplet formation concomitant with diffuse cytoplasmic distribution of ADRP, compared with iPS cells from healthy individuals. Forced expression of BSCL2 not only rescued the lipid accumulation defects, but also restored cytoplasmic punctate localization of ADRP in BSCL2-iPS cells. Coimmunoprecipitation indicated SEIPIN interacted with ADRP. CONCLUSION: BSCL2-iPS cells that recapitulate the lipodystrophic phenotypes in vitro could provide valuable models with which to study the physiology of lipid accumulation and the pathology of human lipodystrophy. We found that BSCL2 defines the localization of ADRP, which has a role in lipid accumulation and adipogenic differentiation.

The moyamoya disease susceptibility variant RNF213 R4810K (rs112735431) induces genomic instability by mitotic abnormality.

Moyamoya disease (MMD) is a cerebrovascular disease characterized by occlusive lesions in the Circle of Willis. The RNF213 R4810K polymorphism increases susceptibility to MMD. In the present study, we characterized phenotypes caused by overexpression of RNF213 wild type and R4810K variant in the cell cycle to investigate the mechanism of proliferation inhibition. Overexpression of RNF213 R4810K in HeLa cells inhibited cell proliferation and extended the time of mitosis 4-fold. Ablation of spindle checkpoint by depletion of mitotic arrest deficiency 2 (MAD2) did not shorten the time of mitosis. Mitotic morphology in HeLa cells revealed that MAD2 colocalized with RNF213 R4810K. Immunoprecipitation revealed an RNF213/MAD2 complex: R4810K formed a complex with MAD2 more readily than RNF213 wild-type. Desynchronized localization of MAD2 was observed more frequently during mitosis in fibroblasts from patients (n=3, 61.0 ± 8.2%) compared with wild-type subjects (n=6, 13.1 ± 7.7%; p<0.01). Aneuploidy was observed more frequently in fibroblasts (p<0.01) and induced pluripotent stem cells (iPSCs) (p<0.03) from patients than from wild-type subjects. Vascular endothelial cells differentiated from iPSCs (iPSECs) of patients and an unaffected carrier had a longer time from prometaphase to metaphase than those from controls (p<0.05). iPSECs from the patients and unaffected carrier had significantly increased mitotic failure rates compared with controls (p<0.05). Thus, RNF213 R4810K induced mitotic abnormalities and increased risk of genomic instability.

Downregulation of Securin by the variant RNF213 R4810K (rs112735431, G>A) reduces angiogenic activity of induced pluripotent stem cell-derived vascular endothelial cells from moyamoya patients.

Moyamoya disease (MMD) is a cerebrovascular disease characterized by occlusive lesions in the circle of Willis. The RNF213 R4810K polymorphism increases susceptibility to MMD. Induced pluripotent stem cells (iPSCs) were established from unaffected fibroblast donors with wild-type RNF213 alleles, and from carriers/patients with one or two RNF213 R4810K alleles. Angiogenic activities of iPSC-derived vascular endothelial cells (iPSECs) from patients and carriers were lower (49.0 ± 19.4%) than from wild-type subjects (p<0.01). Gene expression profiles in iPSECs showed that Securin was down-regulated (p<0.01) in carriers and patients. Overexpression of RNF213 R4810K downregulated Securin, inhibited angiogenic activity (36.0 ± 16.9%) and proliferation of humanumbilical vein endothelial cells (HUVECs) while overexpression of RNF213 wild type did not. Securin expression was downregulated using RNA interference techniques, which reduced the level of tube formation in iPSECs and HUVECs without inhibition of proliferation. RNF213 R4810K reduced angiogenic activities of iPSECs from patients with MMD, suggesting that it is a promising in vitro model for MMD.

Robust and highly-efficient differentiation of functional monocytic cells from human pluripotent stem cells under serum- and feeder cell-free conditions.

Monocytic lineage cells (monocytes, macrophages and dendritic cells) play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established, these methods depend on the use of xenogeneic materials and, therefore, have a relatively poor-reproducibility. Here, we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3 × 10(6) ± 0.3 × 10(6) floating monocytes from approximately 30 clusters of ESCs/iPSCs 5-6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine, disease-specific iPSC studies and drug discovery.

Induced pluripotent stem cells from CINCA syndrome patients as a model for dissecting somatic mosaicism and drug discovery.

Chronic infantile neurologic cutaneous and articular (CINCA) syndrome is an IL-1-driven autoinflammatory disorder caused mainly by NLRP3 mutations. The pathogenesis of CINCA syndrome patients who carry NLRP3 mutations as somatic mosaicism has not been precisely described because of the difficulty in separating individual cells based on the presence or absence of the mutation. Here we report the generation of NLRP3-mutant and nonmutant-induced pluripotent stem cell (iPSC) lines from 2 CINCA syndrome patients with somatic mosaicism, and describe their differentiation into macrophages (iPS-MPs). We found that mutant cells are predominantly responsible for the pathogenesis in these mosaic patients because only mutant iPS-MPs showed the disease relevant phenotype of abnormal IL-1ß secretion. We also confirmed that the existing anti-inflammatory compounds inhibited the abnormal IL-1ß secretion, indicating that mutant iPS-MPs are applicable for drug screening for CINCA syndrome and other NLRP3-related inflammatory conditions. Our results illustrate that patient-derived iPSCs are useful for dissecting somatic mosaicism and that NLRP3-mutant iPSCs can provide a valuable platform for drug discovery for multiple NLRP3-related disorders.