Extracellular acidity in tumor tissue upregulates programmed cell death protein 1 expression on tumor cells via proton-sensing G protein-coupled receptors.

Acidity in the tumor microenvironment has been reported to promote cancer growth and metastasis. In our study, we examined a potential relation between extracellular acidity and expression level of the immune checkpoint molecule programmed cell death protein 1 (PD-L1) in murine squamous cell carcinoma (SCC) and melanoma cell lines. PD-L1 expression in the tumor cells was upregulated by culturing in a low pH culture medium. Tumor-bearing mice were allowed to ingest sodium bicarbonate, resulting in neutralization of acidity in the tumor tissue, a decrease in PD-L1 expression in tumor cells and suppression of tumor growth in vivo. Proton-sensing G protein-coupled receptors, T-cell death-associated gene 8 (TDAG8) and ovarian cancer G-protein-coupled receptor 1 (OGR1), were upregulated by low pH, and essentially involved in the acidity-induced elevation of PD-L1 expression in the tumor cells. Human head and neck SCC RNAseq data from the Cancer Genome Atlas also suggested a statistically significant correlation between expression levels of the proton sensors and PD-L1 mRNA expression. These findings strongly suggest that neutralization of acidity in tumor tissue may result in reduction of PD-L1 expression, potentially leading to inhibition of an immune checkpoint and augmentation of antitumor immunity.

Interferon-ß signal may up-regulate PD-L1 expression through IRF9-dependent and independent pathways in lung cancer cells.

The programmed death ligand-1 (PD-L1) (also called B7-H1 and CD274) belonging to the CD28 family of co-stimulatory molecules is ectopically expressed on the surface of various cancer cells. PD-L1 interacts with programmed death-1 (PD-1) on T cells to trigger an inhibitory signal that suppresses anti-tumor T cell responses as an important mechanism of tumor escape from anti-tumor immune response. Recent development of PD-1/PD-L1 blockades has provided novel immunotherapy strategies for cancers including non-small cell lung cancer (NSCLC). Although the therapy is quite effective for some patients with NSCLC, others are resistant to the treatment, so that regulatory mechanisms of PD-L1 in lung cancer cells need to be understood in detail. Here we analyzed effect of interferon-ß (IFN-ß) that can be produced in cancer microenvironment on PD-L1 expression in lung tumor cells. An addition of IFN-ß elevated PD-L1 expression in mouse and human lung cancer cell lines in culture. This phenomenon was totally dependent on JAK signaling molecules, while IRF9 deficiency in murine lung cancer cells partially attenuated the IFN-ß-induced increase in PD-L1. mTOR may not be significantly involved in the regulation of PD-L1, whereas PI3-K pathway played differential roles on PD-L1 mRNA and cell-surface PD-L1 expression, in the cells treated with IFN-ß. These results strongly suggest that the type I IFN receptor signal elicits an increase in PD-L1 expression in lung cancer cells through IRF9-dependent and independent pathways.

Efficient direct conversion of human fibroblasts into myogenic lineage induced by co-transduction with MYCL and MYOD1.

The skeletal muscle consists of contractile myofibers and plays essential roles for maintenance of body posture, movement, and metabolic regulation. During the development and regeneration of the skeletal muscle tissue, the myoblasts fuse into multinucleated myotubes that subsequently form myofibers. Transplantation of myoblasts may make possible a novel regenerative therapy against defects or dysfunction of the skeletal muscle. It is reported that rodent fibroblasts are converted into myoblast-like cells and fuse to form syncytium after forced expression of exogenous myogenic differentiation 1 (MYOD1) that is a key transcription factor for myoblast differentiation. But human fibroblasts are less efficiently converted into myoblasts and rarely fused by MYOD1 alone. Here we found that transduction of v-myc avian myelocytomatosis viral oncogene lung carcinoma derived homolog (MYCL) gene in combination with MYOD1 gene induced myoblast-like phenotypes in human fibroblasts more strongly than MYOD1 gene alone. The rate of conversion was approximately 90%. The directly converted myoblasts (dMBs) underwent fusion in an ERK5 pathway-dependent manner. The dMBs also formed myofiber-like structure in vivo after an inoculation into mice at the subcutaneous tissue. The present results strongly suggest that the combination of MYCL plus MYOD1 may promote direct conversion of human fibroblasts into functional myoblasts that could potentially be used for regenerative therapy for muscle diseases and congenital muscle defects.

TGF-ß inhibitor treatment of H2O2-induced cystitis models provides biochemical mechanism for elucidating interstitial cystitis/painful bladder syndrome patients.

Interstitial cystitis/painful bladder syndrome (IC/PBS) is a chronic disease for which no effective treatment is available. Transforming growth factor-ß (TGF-ß) is thought to be involved in the pathogenesis of IC/PBS, and previous studies have suggested that administrations of a TGF-ß inhibitor significantly ameliorated IC/PBS in a mouse model. However, the molecular mechanisms underlying the therapeutic effect of a TGF-b inhibitor on IC/PBS has not been comprehensively analyzed. TGF-ß has a variety of actions, such as regulation of immune cells and fibrosis. In our study, we induced IC/PBS-like disease in mice by an intravesical administration of hydrogen peroxide (H2O2) and examined the effects of three TGF-ß inhibitors, Repsox, SB431542, and SB505124, on the urinary functions as well as histological and gene expression profiles in the bladder. TGF-ß inhibitor treatment improved urinary function and histological changes in the IC/PBS mouse model, and SB431542 was most effective among the TGF-ß inhibitors. In our present study, TGF-ß inhibitor treatment improved abnormal enhancement of nociceptive mechanisms, immunity and inflammation, fibrosis, and dysfunction of bladder urothelium. These results show that multiple mechanisms are involved in the improvement of urinary function by TGF-ß inhibitor.

ALK5 i II Accelerates Induction of Adipose-Derived Stem Cells toward Schwann Cells through a Non-Smad Signaling Pathway.

Schwann cells (SCs) are likely to be a vital component of cell-based therapies for nerve regeneration. There are various methods for inducing SC-like cells (SCLCs) from adipose-derived stem cells (ADSCs), but their phenotypic and functional characteristics remain unsatisfactory. Here, we report a novel efficient procedure to induce SCLCs by culturing ADSCs with ALK5 inhibitor (ALK5 i) II, a specific inhibitor of activin-like kinase 5 (ALK5) (transforming growth factor-ß receptor 1 (TGFßR1)) that is also known as Repsox. The resultant cells that we named "modified SCLCs (mSCLCs)" expressed SC-specific genes more strongly than conventional SCLCs (cSCLCs) and displayed a neurosupportive capacity in vitro, similarly to genuine SCs. Regarding the mechanism of the mSCLC induction by ALK5 i II, knockdown of Smad2 and Smad3, key proteins in the TGFß/Smad signaling pathway, did not induce SC markers. Meanwhile, expression of multipotent stem cell markers such as Sex-determining region Y- (SRY-) box 2 (Sox2) was upregulated during induction. These findings imply that ALK5 i II exerts its effect via the non-Smad pathway and following upregulation of undifferentiated cell-related genes such as Sox2. The procedure described here results in highly efficient induction of ADSCs into transgene-free and highly functional SCLCs. This approach might be applicable to regeneration therapy for peripheral nerve injury.

Osteogenic Response to Polysaccharide Nanogel Sheets of Human Fibroblasts After Conversion Into Functional Osteoblasts by Direct Phenotypic Cell Reprogramming.

Human dermal fibroblasts (HDFs) were converted into osteoblasts using a ALK inhibitor II (inhibitor of transforming growth factor-ß signal) on freeze-dried nanogel-cross-linked porous (FD-NanoClip) polysaccharide sheets or fibers. Then, the ability of these directly converted osteoblasts (dOBs) to produce calcified substrates and the expression of osteoblast genes were analyzed in comparison with osteoblasts converted by exactly the same procedure but seeded onto a conventional atelocollagen scaffold. dOBs exposed to FD-NanoClip in both sheet and fiber morphologies produced a significantly higher concentration of calcium deposits as compared to a control cell sample (i.e., unconverted fibroblasts), while there was no statistically significant difference in calcification level between dOBs exposed to atelocollagen sheets and the control group. The observed differences in osteogenic behaviors were interpreted according to Raman spectroscopic analyses comparing different polysaccharide scaffolds and Fourier transform infrared spectroscopy analyses of dOB cultures. This study substantiates a possible new path to repair large bone defects through a simplified transplantation procedure using FD-NanoClip sheets with better osteogenic outputs as compared to the existing atelocollagen scaffolding material.

Direct conversion of fibroblasts into urothelial cells that may be recruited to regenerating mucosa of injured urinary bladder.

Urothelial cells play essential roles in protection of urine exudation and bacterial invasion at the urothelial mucosa, so that defect or damage of urothelial cells associated with urinary tract diseases may cause serious problems. If a sufficient number of functional urothelial cells are prepared in culture and transplanted into the damaged urothelial lesions, such technology may provide beneficial effects to patients with diseases of the urinary tract. Here we found that human adult dermal fibroblasts were converted into urothelial cells by transducing genes for four transcription factors, FOXA1, TP63, MYCL and KLF4 (FTLK). The directly converted urothelial cells (dUCs) formed cobblestone-like colonies and expressed urothelium-specific markers. dUCs were successfully expanded and enriched after serial passages using a specific medium that we optimized for the cells. The passaged dUCs showed similar genome-wide gene expression profiles to normal urothelial cells and had a barrier function. The FTLK-transduced fibroblasts were also converted into urothelial cells in vivo and recruited to the regenerating urothelial tissue after they were transplanted into the bladder of mice with interstitial cystitis. Our technology may provide a promising solution for a number of patients with urinary tract disorders.

Direct phenotypic conversion of human fibroblasts into functional osteoblasts triggered by a blockade of the transforming growth factor-ß signal.

A procedure to generate functional osteoblasts from human somatic cells may pave the way to a novel and effective transplantation therapy in bone disorders. Here, we report that human fibroblasts were induced to show osteoblast phenotypes by culturing with ALK5 i II, which is a specific inhibitor for activin-like kinase 5 (ALK5) (tumor growth factor-ß receptor 1 (TGF-ß R1)). Cells cultured with ALK5 i II expressed osteoblast-specific genes and massively produced calcified bone matrix, similar to the osteoblasts induced from mesenchymal stem cells (MSC-OBs). Treatment with vitamin D3 in addition to ALK5 i II induced more osteoblast-like characters, and the efficiency of the conversion reached approximately 90%. The chemical compound-mediated directly converted osteoblasts (cOBs) were similar to human primary osteoblasts in terms of expression profiles of osteoblast-related genes. The cOBs abundantly produced bone matrix in vivo and facilitated bone healing after they were transplanted into immunodeficient mice at an artificially induced defect lesion in femoral bone. The present procedure realizes a highly efficient direct conversion of human fibroblasts into transgene-free and highly functional osteoblasts, which might be applied in a novel strategy of bone regeneration therapy in bone diseases.

Nanogel tectonic porous 3D scaffold for direct reprogramming fibroblasts into osteoblasts and bone regeneration.

Transplantation of engineered three-dimensional (3D) bone tissue may provide therapeutic benefits to patients with various bone diseases. To achieve this goal, appropriate 3D scaffolds and cells are required. In the present study, we devised a novel nanogel tectonic material for artificial 3D scaffold, namely the nanogel-cross-linked porous (NanoCliP)-freeze-dried (FD) gel, and estimated its potential as a 3D scaffold for bone tissue engineering. As the osteoblasts, directly converted osteoblasts (dOBs) were used, because a large number of highly functional osteoblasts could be induced from fibroblasts that can be collected from patients with a minimally invasive procedure. The NanoCliP-FD gel was highly porous, and fibronectin coating of the gel allowed efficient adhesion of the dOBs, so that the cells occupied the almost entire surface of the walls of the pores after culturing for 7 days. The dOBs massively produced calcified bone matrix, and the culture could be continued for at least 28 days. The NanoCliP-FD gel with dOBs remarkably promoted bone regeneration in vivo after having been grafted to bone defect lesions that were artificially created in mice. The present findings suggest that the combination of the NanoCliP-FD gel and dOBs may provide a feasible therapeutic modality for bone diseases.