Analysis of splice variants of the human protein disulfide isomerase (P4HB) gene.

BACKGROUND: Protein Disulfide Isomerases are thiol oxidoreductase chaperones from thioredoxin superfamily with crucial roles in endoplasmic reticulum proteostasis, implicated in many diseases. The family prototype PDIA1 is also involved in vascular redox cell signaling. PDIA1 is coded by the P4HB gene. While forced changes in P4HB gene expression promote physiological effects, little is known about endogenous P4HB gene regulation and, in particular, gene modulation by alternative splicing. This study addressed the P4HB splice variant landscape. RESULTS: Ten protein coding sequences (Ensembl) of the P4HB gene originating from alternative splicing were characterized. Structural features suggest that except for P4HB-021, other splice variants are unlikely to exert thiol isomerase activity at the endoplasmic reticulum. Extensive analyses using FANTOM5, ENCODE Consortium and GTEx project databases as RNA-seq data sources were performed. These indicated widespread expression but significant variability in the degree of isoform expression among distinct tissues and even among distinct locations of the same cell, e.g., vascular smooth muscle cells from different origins. P4HB-02, P4HB-027 and P4HB-021 were relatively more expressed across each database, the latter particularly in vascular smooth muscle. Expression of such variants was validated by qRT-PCR in some cell types. The most consistently expressed splice variant was P4HB-021 in human mammary artery vascular smooth muscle which, together with canonical P4HB gene, had its expression enhanced by serum starvation. CONCLUSIONS: Our study details the splice variant landscape of the P4HB gene, indicating their potential role to diversify the functional reach of this crucial gene. P4HB-021 splice variant deserves further investigation in vascular smooth muscle cells.

Optimization of culture conditions for the efficient differentiation of mouse-induced pluripotent stem cells into dental epithelial-like cells.

The establishment of a method to derive dental epithelial cells seems to be an important challenge toward realizing the whole tooth regeneration. In order to obtain a source of dental epithelial-like cells, a new methodology has been previously developed by our research group. In the method, induced pluripotent stem cells are cultured in suspension in the presence of neurotrophin-4 to form embryoid bodies followed by further adherent culture of the embryoid bodies in DMEM basal nutrient medium. The present study was directed to improve the efficiency of dental epithelial-like cell production, by focusing on the optimization of initial cell number for the formation of embryoid bodies and the addition of epidermal growth factor as well as its timing. Our results demonstrated that an initial cell number of 1000 cells/drop gives the highest efficiency of dental epithelial-like cell production. It appears that, under this condition, medium deterioration is moderated, and that cell-cell interactions are optimized within embryoid bodies. On the other hand, epidermal growth factor serves to increase the abundance of dental epithelial-like cells when added to the medium together with neurotrophin-4 during embryoid body formation. The promotive effect of epidermal growth factor may involve the transactivation of TrkB, mediated by the effectors of epidermal growth factor receptor signaling.

Differentiation of mouse-induced pluripotent stem cells into dental epithelial-like cells in the absence of added serum.

Recent studies have successfully generated tooth-like structure by mimicking the reciprocal interaction between dental epithelial and mesenchymal cells in tooth organogenesis. However, clinical applications of these methods are limited primarily due to the lack of appropriate sources for dental epithelial cells. Induced pluripotent stem cells (iPSCs) are attractive as a source for dental epithelial cells due to their unique characteristics. In this study, we examined the effect of neurotrophin-4 (NT-4) on the differentiation of mouse iPSCs (miPSCs) into dental epithelial cells. Our results showed that the addition of NT-4 during the formation of embryoid body significantly triggered the upregulation of epithelial markers such as p63 and CK14, suggesting that NT-4 provides an inductive condition for the differentiation of miPSCs into epithelial cells. Expansion of the NT-4-treated cells under serum-free culture conditions improves the formation of cells with cobblestone-like morphology and significantly downregulated the expression of pluripotent and ectodermal markers. Phenotypic analysis revealed that a dental epithelial surface marker, CD49f, was highly expressed on these cells. Formation of miPSCs-derived dental epithelial-like cells was further confirmed by the expression of ameloblast-specific markers. These results suggest that the addition of NT-4 during the formation of embryoid body together with the serum-free culture condition promoted the differentiation of miPSCs into dental epithelial-like cells.

Human platelet lysate permits scale-up of dental pulp stromal cells for clinical applications.

BACKGROUND AIMS. Dental pulp stromal cells (DPSC) are considered to be a promising source of stem cells in the field of regenerative therapy. However, the usage of DPSC in transplantation requires large-scale expansion to cater for the need for clinical quantity without compromising current good manufacturing practice (cGMP). Existing protocols for cell culturing make use of fetal bovine serum (FBS) as a nutritional supplement. Unfortunately, FBS is an undesirable additive to cells because it carries the risk of transmitting viral and prion diseases. Therefore, the present study was undertaken to examine the efficacy of human platelet lysate (HPL) as a substitute for FBS in a large-scale set-up. METHODS. We expanded the DPSC in Dulbecco's modified Eagle's medium-knock-out (DMEM-KO) with either 10% FBS or 10% HPL, and studied the characteristics of DPSC at pre- (T25 culture flask) and post- (5-STACK chamber) large-scale expansion in terms of their identity, quality, functionality, molecular signatures and cytogenetic stability. RESULTS. In both pre- and post-large-scale expansion, DPSC expanded in HPL showed extensive proliferation of cells (c. 2-fold) compared with FBS; the purity, immune phenotype, colony-forming unit potential and differentiation were comparable. Furthermore, to understand the gene expression profiling, the transcriptomes and cytogenetics of DPSC expanded under HPL and FBS were compared, revealing similar expression profiles. CONCLUSIONS. We present a highly economized expansion of DPSC in HPL, yielding double the amount of cells while retaining their basic characteristics during a shorter time period under cGMP conditions, making it suitable for therapeutic applications.

Inherent differential propensity of dental pulp stem cells derived from human deciduous and permanent teeth.

INTRODUCTION: Lately, several new stem cell sources and their effective isolation have been reported that claim to have potential for therapeutic applications. However, it is not yet clear which type of stem cell sources are most potent and best for targeted therapy. Lack of understanding of nature of these cells and their lineage-specific propensity might hinder their full potential. Therefore, understanding the gene expression profile that indicates their lineage-specific proclivity is fundamental to the development of successful cell-based therapies. METHODS: We compared proliferation rate, gene expression profile, and lineage-specific propensity of stem cells derived from human deciduous (SCD) and permanent teeth (DPSCs) over 5 passages. RESULTS: The proliferation rate of SCD was higher (cell number, 25 x 10(6) cells/mL; percent colony-forming units [CFUs], 151.67 +/- 10.5; percent cells in S/G2 phase, 12.4 +/- 1.48) than that of DPSCs (cell number, 21 x 10(6) cells/mL; percent CFUs, 133 +/- 17.62; percent cells in S/G2 phase, 10.4 +/- 1.18). It was observed that fold expression of several pluripotent markers such as OCT4, SOX2, NANOG, and REX1 were higher (>2) in SCD as compared with DPSCs. However, DPSCs showed higher expression of neuroectodermal markers PAX6, GBX2, and nestin (fold expression >100). Similarly, higher neurosphere formation and neuronal marker expression (NF, GFAP) were found in the differentiated DPSCs into neuron-like cells as compared with SCD. CONCLUSIONS: This study thus demonstrates that both SCD and DPSCs exhibit specific gene expression profile, with clear-cut inclination of DPSCs toward neuronal lineage.