Establishment of In Vitro FUS-Associated Familial Amyotrophic Lateral Sclerosis Model Using Human Induced Pluripotent Stem Cells.

Amyotrophic lateral sclerosis (ALS) is a late-onset motor neuron disorder. Although its neuropathology is well understood, the cellular and molecular mechanisms are yet to be elucidated due to limitations in the currently available human genetic data. In this study, we generated induced pluripotent stem cells (iPSC) from two familial ALS (FALS) patients with a missense mutation in the fused-in sarcoma (FUS) gene carrying the heterozygous FUS H517D mutation, and isogenic iPSCs with the homozygous FUS H517D mutation by genome editing technology. These cell-derived motor neurons mimicked several neurodegenerative phenotypes including mis-localization of FUS into cytosolic and stress granules under stress conditions, and cellular vulnerability. Moreover, exon array analysis using motor neuron precursor cells (MPCs) combined with CLIP-seq datasets revealed aberrant gene expression and/or splicing pattern in FALS MPCs. These results suggest that iPSC-derived motor neurons are a useful tool for analyzing the pathogenesis of human motor neuron disorders.

Potent lipolytic activity of lactoferrin in mature adipocytes.

Lactoferrin (LF) is a multifunctional glycoprotein found in mammalian milk. We have shown in a previous clinical study that enteric-coated bovine LF tablets decreased visceral fat accumulation. To address the underlying mechanism, we conducted in vitro studies and revealed the anti-adipogenic action of LF in pre-adipocytes. The aim of this study was to assess whether LF could increase the lipolytic activity in mature adipocytes. Pre-adipocytes were prepared from rat mesenteric fat and differentiated into mature adipocytes for assays of lipolysis. The addition of LF significantly increased the glycerol concentration in the medium in a dose-dependent manner, whereas pepsin-degraded LF did not. A DNA microarray analysis demonstrated that LF decreased the expression of perilipin and affected the cAMP pathway. These findings are supported by the results of quantitative RT-PCR of perilipin and assays of cAMP. These data collectively indicate that visceral fat reduction by LF may result from the promotion of lipolysis and the additional anti-adipogenic activity of LF.

Bovine lactoferrin reduces visceral fat and liver triglycerides in ICR mice.

Lactoferrin (LF) is a multi-functional glycoprotein found in milk. In a previous clinical trial, we showed that enteric-coated bovine LF (bLF) tablets could reduce visceral fat accumulation. We also showed that bLF had anti-adipogenic activity in vitro. However, the mechanisms responsible for these phenomena remain unclear. In this study, we established an animal model of visceral fat reduction via oral bLF administration. We used gastric intubation to ensure that LF was absorbed in the small intestine. bLF administration for 4 weeks significantly reduced mesenteric fat tissue (P < 0.05) and hepatic triglyceride levels (P < 0.01). Furthermore, these two outcomes were positively correlated (R = 0.581, P < 0.05). Overall, these findings suggest that bLF affects mesenteric adipocytes and fatty acid metabolism in the liver.

Neural stem cells directly differentiated from partially reprogrammed fibroblasts rapidly acquire gliogenic competency.

Neural stem cells (NSCs) were directly induced from mouse fibroblasts using four reprogramming factors (Oct4, Sox2, Klf4, and cMyc) without the clonal isolation of induced pluripotent stem cells (iPSCs). These NSCs gave rise to both neurons and glial cells even at early passages, while early NSCs derived from clonal embryonic stem cells (ESCs)/iPSCs differentiated mainly into neurons. Epidermal growth factor-dependent neurosphere cultivation efficiently propagated these gliogenic NSCs and eliminated residual pluripotent cells that could form teratomas in vivo. We concluded that these directly induced NSCs were derived from partially reprogrammed cells, because dissociated ESCs/iPSCs did not form neurospheres in this culture condition. These NSCs differentiated into both neurons and glial cells in vivo after being transplanted intracranially into mouse striatum. NSCs could also be directly induced from adult human fibroblasts. The direct differentiation of partially reprogrammed cells may be useful for rapidly preparing NSCs with a strongly reduced propensity for tumorigenesis.

Identification of PITX1 as a TERT suppressor gene located on human chromosome 5.

Telomerase, a ribonucleoprotein enzyme that maintains telomere length, is crucial for cellular immortalization and cancer progression. Telomerase activity is attributed primarily to the expression of telomerase reverse transcriptase (TERT). Using microcell-mediated chromosome transfer (MMCT) into the mouse melanoma cell line B16F10, we previously found that human chromosome 5 carries a gene, or genes, that can negatively regulate TERT expression (H. Kugoh, K. Shigenami, K. Funaki, J. Barrett, and M. Oshimura, Genes Chromosome Cancer 36:37-47, 2003). To identify the gene responsible for the regulation of TERT transcription, we performed cDNA microarray analysis using parental B16F10 cells, telomerase-negative B16F10 microcell hybrids with a human chromosome 5 (B16F10MH5), and its revertant clones (MH5R) with reactivated telomerase. Here, we report the identification of PITX1, whose expression leads to the downregulation of mouse tert (mtert) transcription, as a TERT suppressor gene. Additionally, both human TERT (hTERT) and mouse TERT (mtert) promoter activity can be suppressed by PITX1. We show that three and one binding site within the hTERT and mtert promoters, respectively, that express a unique conserved region are responsible for the transcriptional activation of TERT. Furthermore, we showed that PITX1 binds to the TERT promoter both in vitro and in vivo. Thus, PITX1 suppresses TERT transcription through direct binding to the TERT promoter, which ultimately regulates telomerase activity.

Effects of pepsin and trypsin on the anti-adipogenic action of lactoferrin against pre-adipocytes derived from rat mesenteric fat.

Lactoferrin (LF) is a multifunctional glycoprotein in mammalian milk. In a previous report, we showed that enteric-coated bovine LF tablets can decrease visceral fat accumulation, hypothesising that the enteric coating is critical to the functional peptides reaching the visceral fat tissue and exerting their anti-adipogenic activity. The aim of the present study was to assess whether ingested LF can retain its anti-adipogenic activity. We therefore investigated the effects of LF and LF treated with digestive enzymes (the stomach enzyme pepsin and the small intestine enzyme trypsin) on lipid accumulation in pre-adipocytes derived from the mesenteric fat tissue of male Sprague-Dawley rats. Lipid accumulation in pre-adipocytes was significantly reduced by LF in a dose-dependent manner and was associated with reduction in gene expression of CCAAT/enhancer binding protein delta, CCAAT/enhancer binding protein alpha and PPARγ as revealed by DNA microarray analysis. Trypsin-treated LF continued to show anti-adipogenic action, whereas pepsin-treated LF abrogated the activity. When an LF solution (1000 mg bovine LF) was administered by gastric intubation to Sprague-Dawley rats, immunoreactive LF determined by ELISA could be detected in mesenteric fat tissue at a concentration of 14·4 µg/g fat after 15 min. The overall results point to the importance of enteric coating for action of LF as a visceral fat-reducing agent when administered in oral form.

Localization of an hTERT repressor region on human chromosome 3p21.3 using chromosome engineering.

Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA. The reactivation of telomerase activity by aberrant upregulation/expression of its catalytic subunit hTERT is a major pathway in human tumorigenesis. However, regulatory mechanisms that control hTERT expression are largely unknown. Previously, we and others have demonstrated that the introduction of human chromosome 3, via microcell-mediated chromosome transfer (MMCT), repressed transcription of the hTERT gene. These results suggested that human chromosome 3 contains a regulatory factor(s) involved in the repression of hTERT. To further localize this putative hTERT repressor(s), we have developed a unique experimental approach by introducing various truncated chromosome 3 regions produced by a novel chromosomal engineering technology into the renal cell carcinoma cell line (RCC23 cells). These cells autonomously express ectopic hTERT (exohTERT) promoted by a retroviral LTR promoter in order to permit cellular division after repression of endogenous hTERT. We found a telomerase repressor region located within a 7-Mb interval on chromosome 3p21.3. These results provide important information regarding hTERT regulation and a unique method to identify hTERT repressor elements.