Generation of induced pluripotent stem cell (iPSC) from NY-ESO-I-specific cytotoxic T cells isolated from the melanoma patient with minor HLAs: The practical pilot study for the adoptive immunotherapy for melanoma using iPSC technology.

Melanoma is one of the most severe skin cancers, derived from melanocytes. Among various therapies for melanoma, adoptive immunotherapy using tumor-infiltrating lymphocytes/chimeric antigen receptor-T cells (TCs) is advanced in recent years; however, the efficacy is still limited, and major challenges remain in terms of safety and cell supply. To solve the issues of adoptive immunotherapy, we utilized induced pluripotent stem cells (iPSCs), which have an unlimited proliferative ability and various differentiation capability. First, we monoclonally isolated CD8+ TCs specifically reactive with NY-ESO-1, one of tumor antigens, from the melanoma patient's monocytes after stimulated with NY-ESO-1 peptide by manual procedure, and cultured NY-ESO-1-specific TCs until proliferated and formed colonies. iPSCs were consequently generated from colony-forming TCs by exogenous expression of reprogramming factors using Sendai virus vector. After the RAG2 gene in TC-derived iPSCs (T-iPSCs) was knocked out for preventing T-cell receptor (TCR) rearrangement, T-iPSCs were re-differentiated into rejuvenated cytotoxic TCs. We confirmed that TCR of T-iPSC-derived TC was maintained as the same of original TCs. In conclusion, T-iPSCs have a potential to be an unlimited cell source for providing cytotoxic TCs. Our study could be a "touchstone" to develop iPSC-based adoptive immunotherapy for the treatment of melanoma for the future clinical use.

Footprint-free gene mutation correction in induced pluripotent stem cell (iPSC) derived from recessive dystrophic epidermolysis bullosa (RDEB) using the CRISPR/Cas9 and piggyBac transposon system.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a monogenic skin blistering disorder caused by mutations in the type VII collagen gene. A combination of biological technologies, including induced pluripotent stem cells (iPSCs) and several gene-editing tools, allows us to develop gene and cell therapies for such inherited diseases. However, the methodologies for gene and cell therapies must be continuously innovated for safe clinical use. OBJECTIVE: In this study, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to correct the pathogenic mutation in RDEB-specific iPSCs, and the piggyBac transposon system so that no residual gene fragments remained in the genome of iPSCs after correcting the mutation. METHODS: For homologous recombination (HR)-based gene editing using CRISPR/Cas9, we designed guide RNA and template DNA including homologous sequences with drug-mediated selection cassette flanked by inverted repeat sequences of the transposon. HR reaction using CRISPR/Cas9 was induced in RDEB-specific iPSCs, and mutation-corrected iPSCs (MC-iPSCs) was obtained. Consequently, the selection cassette in the genome of MC-iPSCs was removed by transposase expression. RESULTS: After CRISPR/Cas9-induced gene editing, we confirmed that the pathogenic mutation in RDEB-specific iPSCs was properly corrected. In addition, MC-iPSCs had no genetic footprint after removing the selection cassette by transposon system, and maintained their "stemness". When differentiating MC-iPSCs into keratinocytes, the expression of type VII collagen was restored. CONCLUSIONS: Our study demonstrated one of the safer approaches to establish gene and cell therapies for skin hereditary disorders for future clinical use.

Analysis of Response Evaluation Criteria in Solid Tumors reduction ratio of primary chemotherapy in unresectable advanced or recurrent colorectal cancer.

Response Evaluation Criteria in Solid Tumors (RECIST) is used to assess the objective response of solid tumors to treatment. However, it remains unclear to what extent the response rate assessed by RECIST reflects a reduction of tumor size in multiple organs in patients with unresectable advanced or recurrent colorectal cancer (CRC) with multiple organ metastases. It is also unclear whether the management of liver metastases with systemic chemotherapy in CRC patients with multiple organ metastases improves their prognosis, although surgical resection has been shown to be the most effective treatment approach to CRC cases with liver metastases. A total of 38 CRC patients who underwent systemic chemotherapy in Kyushu Medical Center Hospital between January 2013 and April 2016 were examined. The patients had measurable lesions in multiple organs, including the liver, and did not undergo curative surgery for metastatic lesions after initiation of chemotherapy. The association between the total reduction ratio (TRR) of all lesions and liver lesion reduction ratio (LRR) was retrospectively analyzed. A total of 18 patients (47%) had H3 liver metastases, and the median liver lesion occupancy rate in the sum of the measured lesions with RECIST was 76%. TRR and LRR were strongly correlated, regardless of the volume of the liver metastases. Although a TRR of >30% was significantly associated with improved overall survival (OS), this improvement was not observed in patients with H3 liver metastases. TRR was correlated with LRR and was associated with a better OS. CRC patients with both multiple organ and H3 liver metastases exhibited poor survival, even with a high reduction ratio by chemotherapy.

Integration-free T cell-derived human induced pluripotent stem cells (iPSCs) from a healthy individual: WT-iPSC2.

Expanded human T cells from a Japanese healthy male were used to generate integration-free induced pluripotent stem cells (iPSCs) by exogenous expression of four reprogramming factors, OCT3/4, SOX2, cMYC, KLF4, using Sendai virus vector (SeVdp). The authenticity of established iPSC line, WT-iPSC2, was confirmed by the expressions of stem cell markers and the differentiation capability into three germ layer. WT-iPSC2 may be a useful cell resource as a normal control for the comparative study using disease-specific iPSCs.

Integration-free T cell-derived human induced pluripotent stem cells (iPSCs) from a healthy individual: WT-iPSC4.

Expanded human T cells from a Japanese healthy male were used to generate integration-free induced pluripotent stem cells (iPSCs) by exogenous expression of four reprogramming factors, OCT3/4, SOX2, cMYC, KLF4, using Sendai virus vector (SeVdp). The authenticity of established iPSC line, WT-iPSC4, was confirmed by the expressions of stem cell markers and the differentiation capability into three germ layer. WT-iPSC4 may be a useful cell resource as a normal control for the comparative study using disease-specific iPSCs.

Integration-free T cell-derived human induced pluripotent stem cells (iPSCs) from a healthy individual: WT-iPSC1.

Expanded human T cells from a Japanese healthy male were used to generate integration-free induced pluripotent stem cells (iPSCs) by exogenous expression of four reprogramming factors, OCT3/4, SOX2, cMYC, KLF4, using Sendai virus vector (SeVdp). The authenticity of established iPSC line, WT-iPSC1, was confirmed by the expressions of stem cell markers and the differentiation capability into three germ layers. WT-iPSC1 may be a useful cell resource as a normal control for the comparative study using disease-specific iPSCs.

Integration-free T cell-derived human induced pluripotent stem cells (iPSCs) from a patient with recessive dystrophic epidermolysis bullosa (RDEB) carrying two compound heterozygous mutations in the COL7A1 gene.

Expanded human T cells from a Japanese female with recessive dystrophic epidermolysis bullosa (RDBE) were used to generate integration-free induced pluripotent stem cells (iPSCs) by exogenous expression of four reprogramming factors, OCT3/4, SOX2, cMYC, KLF4, using Sendai virus vector (SeVdp). The authenticity of established iPSC line, RDEB-iPSC26, was confirmed by the expressions of stem cell markers and the differentiation capability into three germ layer. RDEB-iPSC26 may be a useful cell resource for the establishment of in vitro RDEB modeling and the study for developing gene and cell therapy.

Integration-free T cell-derived human induced pluripotent stem cells (iPSCs) from a patient with lymphedema-distichiasis syndrome (LDS) carrying an insertion-deletion complex mutation in the FOXC2 gene.

Expanded human T cells from a Japanese male with lymphedema-distichiasis syndrome (LDS) were used to generate integration-free induced pluripotent stem cells (iPSCs) by exogenous expression of four reprogramming factors, OCT3/4, SOX2, cMYC, KLF4, using Sendai virus vector (SeVdp). The authenticity of established iPSC line, LDS-iPSC8, was confirmed by the expression of stem cell markers and the differentiation capability into three germ layers. LDS-iPSC8 may be a useful cell resource for the establishment of in vitro LDS modeling and the study for vascular and lymph vessel development.

The generation of induced pluripotent stem cells (iPSCs) from patients with infantile and late-onset types of Pompe disease and the effects of treatment with acid-α-glucosidase in Pompe's iPSCs.

Pompe disease (PD), which is also called glycogen storage disease type II (GSDII), is one of the lysosomal storage diseases (LSDs) caused by a deficiency in acid-α-glucosidase (GAA) in the lysosome and is characterized by the accumulation of glycogen in various cells. PD has been treated by enzyme replacement therapy (ERT). We generated induced pluripotent stem cells (iPSCs) from the cells of patients with infantile-type and late-onset-type PD using a retrovirus vector to deliver transgenes encoding four reprogramming factors, namely, OCT4, SOX2, c-MYC, and KLF4. We confirmed that the two types of PD-iPSCs exhibited an undifferentiated state, alkaline phosphatase staining, and the presence of SSEA-4, TRA-1-60, and TRA-1-81. The PD-iPSCs exhibited strong positive staining with Periodic acid-Schiff (PAS). Moreover, ultrastructural features of these iPSCs exhibited massive glycogen granules in the cytoplasm, particularly in the infantile-type but to a lesser degree in the late-onset type. Glycogen granules of the infantile-type iPSCs treated with rhGAA were markedly decreased in a dose-dependent manner. Human induced pluripotent stem cell provides an opportunity to build up glycogen storage of Pompe disease in vitro. It represents a promising resource to study disease mechanisms, screen new drug compounds and develop new therapies for Pompe disease.

Morphological features of iPS cells generated from Fabry disease skin fibroblasts using Sendai virus vector (SeVdp).

We generated iPS cells from human dermal fibroblasts (HDFs) of Fabry disease using a Sendai virus (SeVdp) vector; this method has been established by Nakanishi et al. for pathogenic evaluation. We received SeVdp vector from Nakanishi and loaded it simultaneously with four reprogramming factors (Klf4, Oct4, Sox2, and c-Myc) to HDFs of Fabry disease; subsequently, we observed the presence of human iPS-like cells. The Sendai virus nucleocapsid protein was not detected in the fibroblasts by RT-PCR analysis. Additionally, we confirmed an undifferentiated state, alkaline phosphatase staining, and the presence of SSEA-4, TRA-1-60, and TRA-1-81. Moreover, ultrastructural features of these iPS cells included massive membranous cytoplasmic bodies typical of HDFs of Fabry disease. Thus, we successfully generated human iPS cells from HDFs of Fabry disease that retained the genetic conditions of Fabry disease; also, these abnormal iPS cells could not be easily differentiated into mature cell types such as neuronal cells, cardiomyocytes, etc. because of a massive accumulation of membranous cytoplasmic bodies in lysosomes, possibly the persistent damages of intracellular architecture.

Enzyme replacement therapy (ERT) procedure for mucopolysaccharidosis type II (MPS II) by intraventricular administration (IVA) in murine MPS II.

Mucopolysaccharidosis type II (MPS II), or Hunter syndrome, is a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS) and is characterized by the accumulation of glycosaminoglycans (GAGs). MPS II has been treated by hematopoietic stem cell therapy (HSCT)/enzyme replacement therapy (ERT), but its effectiveness in the central nervous system (CNS) is limited because of poor enzyme uptake across the blood-brain barrier (BBB). To increase the efficacy of ERT in the brain, we tested an intraventricular ERT procedure consisting of repeated administrations of IDS (20 µg/mouse/3 weeks) in IDS-knockout, MPS II model mice. The IDS enzyme activity and the accumulation of total GAGs were measured in mouse brains. The IDS activity was significantly increased, and the accumulation of total GAGs was decreased in the MPS II mouse brains treated with multiple administrations of IDS via intraventricular ERT. Additionally, a high level of IDS enzyme activity was appreciated in other MPS II mouse tissues, such as the liver, spleen, testis and others. A Y-maze was used to test learning and memory after repeated intraventricular ERT with IDS. The IDS-treated mouse groups recovered the capacity for short-term memory and activity. Although large and small vacuoles were found at the margin of the cerebellar Purkinje cells in the disease-control mice, these vacuoles disappeared upon treated with IDS. Loss of vacuoles was also observed in other tissues (liver, kidney and testis). These results demonstrate the possible efficacy of an ERT procedure with intraventricular administration of IDS for the treatment of MPS II.

Endoplasmic reticulum stress induces autophagy through activation of p38 MAPK in fibroblasts from Pompe disease patients carrying c.546G>T mutation.

Pompe disease (glycogen storage disease type II) is an autosomal recessive myopathic disorder arising from the deficiency of lysosomal acid α-glucosidase (GAA). Activation of autophagy is a key pathophysiological feature in skeletal muscle fibers and fibroblasts from patients with Pompe disease. The accumulation of autophagic vacuoles has been shown to interfere with the efficacy of enzyme replacement therapy with recombinant human GAA. However, the induction mechanism of autophagy in Pompe disease is still unclear. In this study, we show that misfolded GAA-induced endoplasmic reticulum (ER) stress triggers autophagy in a manner regulated by p38 MAPK signaling pathways in fibroblasts from late-onset patients with Pompe disease. By studying normal fibroblasts and patient fibroblasts carrying a c.546G>T mutation, we uncovered that mutant GAA was rapidly degraded by proteasome. In addition, we found both activation of ER stress response and autophagy in these patient fibroblasts. Treatment with N-butyl-deoxynojirimycin (NB-DNJ), which acts as a pharmacological chaperone for certain mutant forms of GAA, led to attenuation of not only ER stress, but also autophagy in patient fibroblasts. Levels of phosphorylated p38 MAPK observed in patient fibroblasts were decreased after treatment with NB-DNJ. The autophagic response in patient fibroblasts was also negatively regulated by treatment with the p38 MAPK inhibitor SB203580. These findings define a critical role for ER stress in the activation of autophagy due to GAA mutation, and provide evidence that chaperone therapy may be a useful treatment for alleviation of autophagy in Pompe disease patients carrying a chaperon-responsive mutation.

Generation of induced pluripotent stem (iPS) cells derived from a murine model of Pompe disease and differentiation of Pompe-iPS cells into skeletal muscle cells.

Our study is the first to demonstrate the ability to generate iPS cells from a mouse model of Pompe disease. Initially, mouse tail tip fibroblasts were harvested from male, 8-week-old (GAA) knockout mice, and three reprogramming factors (Oct3/4, Sox2 and Klf4) were transfected into the isolated donor cells using a retroviral vector. These iPS cells also showed decreased levels of GAA enzymatic activity and strong positive staining with periodic acid-Schiff (indicating the accumulation of glycogen) and acid phosphatase (lysosomal activation marker). Pompe-iPS cells were differentiated into skeletal muscle cells in Matrigel®-coated plates. Spindle-shaped skeletal muscle cells were successfully generated from Pompe-iPS cells and showed spontaneous contraction and positive staining with the myosin heavy chain antibody. Electron microscopic analysis of the skeletal muscle cells showed typical morphological features, including Z-bands, I-bands, A-bands and H-bands, which were visible in wild-type and Pompe cells. Furthermore, Pompe skeletal muscle cells accumulated massive glycogen in lysosomes. This study indicates that the iPS and skeletal muscle cells generated in this study could also be a useful disease model for studies investigating the pathogenesis and treatment of skeletal muscle in Pompe disease.

Induced pluripotent stem cells derived from mouse models of lysosomal storage disorders.

Most lysosomal storage diseases (LSDs) are life-threatening genetic diseases. The pathogenesis of these diseases is poorly understood. Induced pluripotent stem (iPS) cell technology offers new opportunities for both mechanistic studies and development of stem cell- based therapies. Here we report the generation of disease-specific iPS cells from mouse models of Fabry disease, globoid cell leukodystrophy (GLD), and mucopolysaccharidosis VII (MPSVII). These mouse model-derived iPS cells showed defects in disease-specific enzyme activities and significant accumulation of substrates for these enzymes. In the lineage-directed differentiation studies, Fabry-iPS and GLD-iPS cells were efficiently differentiated into disease-relevant cell types, such as cardiomyocytes and neural stem cells, which might be useful in mechanistic and therapeutic studies. Notably, MPSVII-iPS cells demonstrated a markedly impaired ability to form embryoid bodies (EBs) in vitro. MPSVII-EBs exibited elevated levels of hyaluronan and its receptor CD44, and markedly reduced expression levels of E-cadherin and cell-proliferating marker. Partial correction of enzyme deficiency in MSPVII-iPS cells led to improved EB formation and reversal of aberrant protein expression. These data indicate a potential mechanism for the partial lethality of MPSVII mice in utero, and suggest a possible abnormality of embryonic development in MPSVII patients. Thus, our study demonstrates the unique promise of iPS cells for studying the pathogenesis and treatment of LSDs.