Osteoblasts produce calcified bone matrix and contribute to bone formation and remodeling. In this study, we established a procedure to directly convert human fibroblasts into osteoblasts by transducing some defined factors and culturing in osteogenic medium. Osteoblast-specific transcription factors, Runt-related transcription factor 2 (Runx2), and Osterix, in combination with Octamer-binding transcription factor 3/4 (Oct4) and L-Myc (RXOL) transduction, converted ∼80% of the fibroblasts into osteocalcin-producing cells. The directly converted osteoblasts (dOBs) induced by RXOL displayed a similar gene expression profile as normal human osteoblasts and contributed to bone repair after transplantation into immunodeficient mice at artificial bone defect lesions. The dOBs expressed endogenous Runx2 and Osterix, and did not require continuous expression of the exogenous genes to maintain their phenotype. Another combination, Oct4 plus L-Myc (OL), also induced fibroblasts to produce bone matrix, but the OL-transduced cells did not express Osterix and exhibited a more distant gene expression profile to osteoblasts compared with RXOL-transduced cells. These findings strongly suggest successful direct reprogramming of fibroblasts into functional osteoblasts by RXOL, a technology that may provide bone regeneration therapy against bone disorders.O steoblasts play a central role in bone formation and remodeling by producing type I collagen, osteopontin, osteocalcin, and bone sialoprotein (BSP), and calcifying these bone matrixes (1). They are also involved in hematopoiesis, phosphate metabolism, and glucose metabolism (2). Osteoblasts are derived from mesenchymal progenitor cells that are common precursors shared by chondrocytes, adipocytes, and myoblasts (3). The differentiation of osteoblasts is regulated by various transcription factors, including Runt-related transcription factor 2 [Runx2, also known as core-binding factor subunit α-1 (Cbfα-1)] (4, 5), Osterix (6, 7), Distal-less homeobox 5 (Dlx5) (8), and activation transcription factor 4 (ATF-4) (8). A functional decline in osteoblasts relative to osteoclasts results in imbalance between bone formation and resorption and may cause osteolytic pathological conditions, such as osteoporosis (9), alveolar bone resorption associated with periodontitis (10), and bone lysing associated with bone tumors, including multiple myeloma (11).It has been demonstrated that forced expression of combinations of some transcription factors, such as Octamer-binding transcription factor 3/4 (Oct4), Sox2, Klf-4, and c-Myc (reprogramming factors), induces immortality and pluripotency in mammalian somatic cells (12, 13). The generation of induced pluripotent stem (iPS) cells clearly indicates that genome-wide epigenetic programming can be drastically changed in somatic cells by a small number of transcription factors that may have key regulatory roles in cell fate decisions (14,15).Recent studies have reported that direct conversion, or direct reprogramming, of somatic cells into another dif...

Objective. Synovial fluid pH is decreased in patients with rheumatoid arthritis (RA); however, the underlying mechanisms are unclear. We undertook this study to examine the mechanism by which synovial fluid pH is regulated and to explore the possibility of a therapeutic strategy by manipulating this mechanism.Methods. We determined the pH and lactate concentration in synovial fluid from 16 RA patients. Cultured synovial fibroblasts (SFs) from the inflamed joints of 9 RA patients (RASFs) were examined for the expression of ion transporters that regulate intracellular and extracellular pH. The ion transporter up-regulated in RASF lines was then suppressed in RASFs by small interfering RNA (siRNA), and the effect of transfection on viability and proliferation was investigated. Finally, we examined the therapeutic effect of electrotransfer of monocarboxylate transporter 4 (MCT4)-specific siRNA into the articular synovium of mice with collagen-induced arthritis (CIA).Results. Synovial fluid pH correlated inversely with both the Disease Activity Score in 28 joints using the C-reactive protein level and the synovial fluid lactate levels. RASFs exhibited up-regulated transcription of MCT4 messenger RNA. MCT4 exported intracellular lactate into the extracellular space. RASFs had significantly higher MCT4 protein levels than did SFs from patients with osteoarthritis. Knockdown of MCT4 induced intrinsic apoptosis of RASFs, thereby inhibiting their proliferation. Moreover, electrotransfer of MCT4-specific siRNA into the articular synovium of mice with CIA significantly reduced the severity of arthritis.Conclusion. RA activity correlated with decreased synovial fluid pH. This may be due to increased MCT4 expression in RASFs. Silencing MCT4 induced apoptosis in RASFs and reduced the severity of CIA, suggesting that MCT4 is a potential therapeutic target for inflammatory arthritis.

Bioactive peptides in the body after ingestion of plant protein hydrolysates have been speculated but not identified. We aimed to establish an approach to identify small amounts of food-derived peptides in humans after ingestion of non-extracellular matrix protein hydrolysates. Corn and wheat gluten hydrolysates were digested using pancreatin and leucine aminopeptidase; the resultant peptides were identified via size-exclusion chromatography and reverse-phase HPLC-tandem mass spectrometry (MS/MS). Structures of indigestible peptides were confirmed via LC-MS/MS in multi-reaction monitoring mode. All indigestible peptides in the exopeptidase digest were diprolyl and di- and tripyroglutamyl peptides. Blood collected from healthy volunteers (n = 4) before and after ingestion of 9 g of the hydrolysates was assessed for the indigestible peptides via LC-MS/MS. Six peptides (Pro-Ala, Pro-Gly, Pro-Gln, pyroGlu-Pro, pyroGlu-Leu-Pro, and pyroGlu-Gln-Pro) significantly increased in human plasma up to 10–100 nM compared to the baseline. This may hence be a powerful tool for identifying foodderived peptides in blood.

SummaryBrown adipocytes (BAs) play important roles in body temperature regulation, energy balance, and carbohydrate and lipid metabolism. Activities of BAs are remarkably diminished in obese and diabetic patients, providing possibilities of transplanting functional BAs resulting in therapeutic benefit. Here, we show generation of functional BAs by cellular reprogramming procedures. Transduction of the PRDM16 gene into iPSC-derived embryoid bodies induced BA phenotypes (iBAs). Moreover, normal human fibroblasts were directly converted into BAs (dBAs) by C/EBP-β and C-MYC gene transduction. Approximately 90% of the fibroblasts were successfully converted within 12 days. The dBAs were highly active in mitochondrial biogenesis and oxidative metabolism. Mouse dBAs were induced by Prdm16, C/ebp-β, and L-myc genes, and after transplantation, they significantly reduced diet-induced obesity and insulin resistance in an UCP1-dependent manner. Thus, highly functional BAs can be generated by cellular reprogramming, suggesting a promising tailor-made cell therapy against metabolic disorders including type 2 diabetes mellitus.

The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases.