Expression levels and DNA methylation profiles of the growth gene SHOX in cartilage tissues and chondrocytes.

All attempts to identify male-specific growth genes in humans have failed. This study aimed to clarify why men are taller than women. Microarray-based transcriptome analysis of the cartilage tissues of four adults and chondrocytes of 12 children showed that the median expression levels of SHOX, a growth gene in the pseudoautosomal region (PAR), were higher in male samples than in female samples. Male-dominant SHOX expression was confirmed by quantitative RT-PCR for 36 cartilage samples. Reduced representation bisulfite sequencing of four cartilage samples revealed sex-biased DNA methylation in the SHOX-flanking regions, and pyrosequencing of 22 cartilage samples confirmed male-dominant DNA methylation at the CpG sites in the SHOX upstream region and exon 6a. DNA methylation indexes of these regions were positively correlated with SHOX expression levels. These results, together with prior findings that PAR genes often exhibit male-dominant expression, imply that the relatively low SHOX expression in female cartilage tissues reflects the partial spread of X chromosome inactivation into PAR. Altogether, this study provides the first indication that sex differences in height are ascribed, at least in part, to the sex-dependent epigenetic regulation of SHOX. Our findings deserve further validation.

Gorlin syndrome-induced pluripotent stem cells form medulloblastoma with loss of heterozygosity in PTCH1.

Gorlin syndrome is a rare autosomal dominant hereditary disease with a high incidence of tumors such as basal cell carcinoma and medulloblastoma. Disease-specific induced pluripotent stem cells (iPSCs) and an animal model have been used to analyze disease pathogenesis. In this study, we generated iPSCs derived from fibroblasts of four patients with Gorlin syndrome (Gln-iPSCs) with heterozygous mutations of the PTCH1 gene. Gln-iPSCs from the four patients developed into medulloblastoma, a manifestation of Gorlin syndrome, in 100% (four out of four), of teratomas after implantation into immunodeficient mice, but none (0/584) of the other iPSC-teratomas did so. One of the medulloblastomas showed loss of heterozygosity in the PTCH1 gene while the benign teratoma, i.e. the non-medulloblastoma portion, did not, indicating a close clinical correlation between tumorigenesis in Gorlin syndrome patients and Gln-iPSCs.

Effect of cell culture biomaterials for completely xeno-free generation of human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) were generated on several biomaterials from human amniotic fluid in completely xeno-free and feeder-free conditions via the transfection of pluripotent genes using a nonintegrating RNA Sendai virus vector. The effect of xeno-free culture medium on the efficiency of the establishment of human amniotic fluid stem cells from amniotic fluid was evaluated. Subsequently, the effect of cell culture biomaterials on the reprogramming efficiency was investigated during the reprogramming of human amniotic fluid stem cells into hiPSCs. Cells cultured in laminin-511, laminin-521, and Synthemax II-coated dishes and hydrogels having optimal elasticity that were engrafted with specific oligopeptides derived from vitronectin could be reprogrammed into hiPSCs with high efficiency. The reprogrammed cells expressed pluripotency proteins and had the capability to differentiate into cells derived from all three germ layers in vitro and in vivo. Human iPSCs could be generated successfully and at high efficiency (0.15-0.25%) in completely xeno-free conditions from the selection of optimal cell culture biomaterials.

Xeno-free and feeder-free culture and differentiation of human embryonic stem cells on recombinant vitronectin-grafted hydrogels.

Recombinant vitronectin-grafted hydrogels were developed by adjusting surface charge of the hydrogels with grafting of poly-l-lysine for optimal culture of human embryonic stem cells (hESCs) under xeno- and feeder-free culture conditions, with elasticity regulated by crosslinking time (10-30 kPa), in contrast to conventional recombinant vitronectin coating dishes, which have a fixed stiff surface (3 GPa). hESCs proliferated on the hydrogels for over 10 passages and differentiated into the cells derived from three germ layers indicating the maintenance of pluripotency. hESCs on the hydrogels differentiated into cardiomyocytes under xeno-free culture conditions with much higher efficiency (80% of cTnT+ cells) than those on conventional recombinant vitronectin or Matrigel-coating dishes just only after 12 days of induction. It is important to have an optimal design of cell culture biomaterials where biological cues (recombinant vitronectin) and physical cues (optimal elasticity) are combined for high differentiation of hESCs into specific cell lineages, such as cardiomyocytes, under xeno-free and feeder-free culture conditions.

Tumorigenicity-associated characteristics of human iPS cell lines.

Human induced pluripotent stem cells (hiPSCs) represent promising raw materials of human cell-based therapeutic products (hCTPs). As undifferentiated hiPSCs exhibit intrinsic tumorigenicity properties that enable them to form teratomas, hCTPs containing residual undifferentiated hiPSCs may cause tumor formation following transplantation. We first established quantitative and sensitive tumorigenicity testing of hiPSCs dissociated into single cells using NOD/Shi-scid IL2Rγnull (NOG) mice by inhibiting apoptosis of hiPSCs with a Rho kinase inhibitor. To examine different features in tumorigenicity of various hiPSCs, 10 commonly available hiPSC lines were subjected to in vivo tumorigenicity testing. Transplanted hiPSC lines showed remarkable variation in tumor incidence, formation latency, and volumes. Most of the tumors formed were classified as immature teratomas. However, no signs of malignancies, such as carcinoma and sarcoma, were recognized in the tumors. Characteristics associated tumorigenicity of hiPSCs were investigated with microarray analysis, karyotype analysis, and whole exome sequencing. Gene expression profiling and pathway analysis supported different features of hiPSC lines in tumorigenicity. hiPSC lines showed chromosomal abnormalities in some lines and 61-77 variants of cancer-related genes carrying effective nonsynonymous mutations, which were confirmed in the COSMIC databases. In this study, the chromosomal abnormalities and cancer-related gene mutations observed in hiPSC lines did not lead to the malignancy of tumors derived from hiPSCs. Our results suggest that the potential tumorigenicity risk of hCTPs containing residual undifferentiated hiPSCs is dependent on not only amounts of undifferentiated hiPSCs but also features of the cell lines used as raw materials, a finding that should be considered from the perspective of quality of hCTPs used.

Efficiency of Human Epiphyseal Chondrocytes with Differential Replication Numbers for Cellular Therapy Products.

The cell-based therapy for cartilage or bone requires a large number of cells; serial passages of chondrocytes are, therefore, needed. However, fates of expanded chondrocytes from extra fingers remain unclarified. The chondrocytes from human epiphyses morphologically changed from small polygonal cells to bipolar elongated spindle cells and to large polygonal cells with degeneration at early passages. Gene of type II collagen was expressed in the cells only at a primary culture (Passage 0) and Passage 1 (P1) cells. The nodules by implantation of P0 to P8 cells were composed of cartilage and perichondrium. The cartilage consisted of chondrocytes with round nuclei and type II collagen-positive matrix, and the perichondrium consisted of spindle cells with type I collage-positive matrix. The cartilage and perichondrium developed to bone with marrow cavity through enchondral ossification. Chondrogenesis and osteogenesis by epiphyseal chondrocytes depended on replication number in culture. It is noteworthy to take population doubling level in correlation with pharmaceutical efficacy into consideration when we use chondrocytes for cell-based therapies.

In vivo maturation of human embryonic stem cell-derived teratoma over time.

Transformation of human embryonic stem cells (hESC) is of interest to scientists who use them as a raw material for cell-processed therapeutic products. However, the WHO and ICH guidelines provide only study design advice and general principles for tumorigenicity tests. In this study, we performed in vivo tumorigenicity tests (teratoma formation) and genome-wide sequencing analysis of undifferentiated hESCs i.e. SEES-1, -2 and -3 cells. We followed up with teratoma formation histopathologically after subcutaneous injection of SEES cells into immunodeficient mice in a qualitative manner and investigated the transforming potential of the teratomas. Maturity of SEES-teratomas perceptibly increased after long-term implantation, while areas of each tissue component remained unchanged. We found neither atypical cells/structures nor cancer in the teratomas even after long-term implantation. The teratomas generated by SEES cells matured histologically over time and did not increase in size. We also analyzed genomic structures and sequences of SEES cells during cultivation by SNP bead arrays and next-generation sequencing, respectively. The nucleotide substitution rate was 3.1 × 10-9, 4.0 × 10-9, and 4.6 × 10-9 per each division in SEES-1, SEES-2, and SEES-3 cells, respectively. Heterozygous single-nucleotide variations were detected, but no significant homologous mutations were found. Taken together, these results imply that SEES-1, -2, and -3 cells do not exhibit in vivo transformation and in vitro genomic instability.

Endochondral ossification model system: designed cell fate of human epiphyseal chondrocytes during long-term implantation.

The aim of this study is to establish a recapitulation system of human endochondral ossification as a paradigm of developmental engineering. Chondrocytes were isolated from the epiphyseal cartilage of the supernumerary digits of infants with polydactyly. In vivo studies showed that implanted chondrocytes exhibited cartilaginous regeneration over a short period of time and subsequent endochondral ossification with a marrow cavity. Tracing studies revealed that cells of donor origin at the periphery of the cartilage migrated into the center of the cartilage and transformed into osteoblasts, adipocytes, and endothelial cells. Bone marrow was formed through anastomosis with the recipient endothelial system at 13 weeks, and from the migration of recipient hematopoietic cells at 50 weeks. This study provides a human endochondral ossification model system with transdifferentiation of the donor cells at the periphery of the cartilage. J. Cell. Physiol. 230: 1376-1388, 2015. © 2015 Wiley Periodicals, Inc., A Wiley Company.

Phosphorus-insufficient maternal milk is associated with ectopic expression of collagen I and female-specific bony changes in infant mouse cartilages.

Excessive phosphorus intake causes diseases such as hyperphosphatemia and hypocalcaemia, but the effect of dietary insufficiency of phosphorus is unclear. Here, we explored the effect of phosphorus dietary insufficiency on tissue growth and maintenance by using C57BL/6J mice fed a low phosphorus diet, which contained 18.5% of the phosphorus of a normal diet. We demonstrated that the phosphorus content in the maternal milk of mother mice was significantly reduced due to the consumption of a low phosphorus diet, which further resulted in bone deformation in infant mice in a female-specific manner. Polarizing microscopic analysis of low-phosphorus milk (LPM)-induced bone deformation resulted in unusually formed crystals inside cartilage. Furthermore, immunohistochemical analysis revealed ectopic expression of collagen I in the region where crystals were ectopically formed. Electron microscopic analysis showed morphological features similar to bone tissues. Immunochemical analysis demonstrated that the amount of interleukin-6 (IL-6), a cytokine known to trigger osteoclast formation, was significantly reduced in the maternal milk of mice fed the low-phosphorus diet. Our results suggest that phosphorus intake from maternal milk is involved in infant cartilage formation.

Ataxia telangiectasia derived iPS cells show preserved x-ray sensitivity and decreased chromosomal instability.

Ataxia telangiectasia is a neurodegenerative inherited disease with chromosomal instability and hypersensitivity to ionizing radiation. iPS cells lacking ATM (AT-iPS cells) exhibited hypersensitivity to X-ray irradiation, one of the characteristics of the disease. While parental ataxia telangiectasia cells exhibited significant chromosomal abnormalities, AT-iPS cells did not show any chromosomal instability in vitro for at least 80 passages (560 days). Whole exome analysis also showed a comparable nucleotide substitution rate in AT-iPS cells. Taken together, these data show that ATM is involved in protection from irradiation-induced cell death.

Marked hepatomegaly due to AA type amyloidosis in a case with Castleman's disease.

Hepatic amyloidosis complicated with Castleman's disease is quite rare. A 48-year-old woman was referred to our hospital with general fatigue, low-grade fever, anemia, thrombocythemia, and liver dysfunction. Physical examination revealed anemia and hepatomegaly and abdominal computed tomography showed marked hepatomegaly and right upper abdominal masses. Technetium-99m pyrophosphate (99mTc-PYP) scintigraphy revealed the diffuse abnormal uptake of the enlarged liver, suggesting amyloid deposition. Liver biopsy showed destruction of the liver structure and the massive deposition of AA type amyloid protein. Surgical resection was performed on the abdominal masses. Histological examination of the masses showed Castleman's disease (plasma cell type). After resection, her fever resolved and the liver size gradually decreased to within the normal range. This case shows that surgical resection of the main lesion is effective for hepatomegaly due to AA type amyloidosis associated with Castleman's disease.

Different transformation of mature teratoma in a patient with mixed germ cell tumor of the testis.

A 44-year-old male was referred with a left supraclavicular lymphadenopathy. A biopsy of the lymph node showed metastatic embryonal carcinoma. Tumor markers were present at high levels: alpha-fetoprotein 253.9 ng/mL, beta-human chorionic gonadotrophin 62 ng/mL. Computed tomography (CT) showed retroperitoneal adenopathy. High orchiectomy was done. The patient was treated with three cycles of etoposide plus cisplatin, achieved normalization of the serum tumor markers and underwent retroperitoneal lymph node dissection. Pathological findings of multiple lymph nodes showed teratomatous glands without viable cells. At follow-ups performed every 3 months, tumor markers remained within normal limits and no evidence of recurrence was observed. Eight years after first admission a CT scan revealed a cystic tumor 1 cm in diameter in the para-aortic region. The cystic tumor continued to slowly grow, expanding by 1 cm in diameter per year without elevation of tumor markers. The para-aortic tumor had grown to 4 cm in diameter and a left supraclavicular lymphadennopathy recurred. A resection of the supraclavicular cystic tumor showed mucinous cystadenocarcinoma, but a cystic tumor in the para-aortic region revealed mature teratoma. Here we report a case of mature teratoma with metastases at supraclavicular and para-aortic lymph nodes which had different transformations in spite of both regions consisting of cystic tumors.

[A 95-year-old female with autopsy-proven cerebral necrosis due to candidiasis who developed stroke-like manifestations].

A 95-year-old woman complained of sudden onset of disturbance of consciousness and right hemiparesis on April 20, 2003 and was admitted on the next day. She was drowsy and showed moderate right motor and sensory hemiparesis. The blood laboratory tests showed slight inflammatory reaction. A low density area was found in the left basal ganglia by brain CT, which was also coincided with the high signal region in T2, FLAIR and diffusion-MR images. The MRA of the intracerebral arteries presented no remarkable abnormality. The hemiparesis and impaired consciousness improved partially in the following week. However, she did not fully recover, since aspiration pneumonia and mild generalized inflammation continued. Percutaneous gastrostomy and intravenous hyperalimentation were started to improve her nutrition. The moderate inflammatory state persisted for several weeks. Her blood pressure suddenly fell and she died on June 12. Autopsy showed a mildly brownish and necrotic lesion from the left caudate to the putamen through the internal capsule. There was no liquefaction. On the microscopic examination, the necrosis surrounded by small vessels was consisted of numerous neutrophils and macrophages with pseudohypha and blastospore of candida. Small fragments of fungus were phagocytosed by macrophages. Small abscesses and necrotic foci due to candidiasis were observed in the bladder, kidneys, lungs, myocardium and thyroid gland. In this case, cerebral candidiasis probably occurred via hematogenous dissemination from a primary focus in the urinary tract. The intracerebral arteries revealed rather mild atherosclerotic changes and there was no occlusion by thromboembolism. Intracerebral lesion was diagnosed as candidiasis and there was no cerebral infarction by thromboembolism. If the infection occurred after cerebral infarction, there should not be any inflammatory reaction in the center of necrotic area. There have been few reports of cerebral candidal infection in patients without diabetes mellitus or immunosuppressive conditions. None of them had been diagnosed before death. Caution should be exercised for the presence of systemic candidiasis in elderly patients who are bedridden and with continuous low grade inflammatory reactions.