Organoids generated from pluripotent stem cells are used in the development of organ replacement regenerative therapy by recapitulating the process of organogenesis. These processes are strictly regulated by morphogen signalling and transcriptional networks. However, the precise transcription factors involved in the organogenesis of exocrine glands, including salivary glands, remain unknown. Here, we identify a specific combination of two transcription factors (Sox9 and Foxc1) responsible for the differentiation of mouse embryonic stem cell-derived oral ectoderm into the salivary gland rudiment in an organoid culture system. Following orthotopic transplantation into mice whose salivary glands had been removed, the induced salivary gland rudiment not only showed a similar morphology and gene expression profile to those of the embryonic salivary gland rudiment of normal mice but also exhibited characteristics of mature salivary glands, including saliva secretion. This study suggests that exocrine glands can be induced from pluripotent stem cells for organ replacement regenerative therapy.

The protein composition of gingival crevicular fluid (GCF) may reflect the pathophysiology of periodontal diseases. A standard GCF proteomic pattern of healthy individuals would serve as a reference to identify biomarkers of periodontal diseases by proteome analyses. However, protein profiles of GCF obtained from apparently healthy individuals have not been well explored. As a step toward detection of proteomic biomarkers for periodontal diseases, we applied both gel-based and gel-free methods to analyze GCF obtained from healthy subjects as compared with supragingival saliva. To ensure optimized protein extraction from GCF, a novel protocol was developed. The proteins in GCF were extracted with high yield by urea buffer combined with ultrafiltration and the intensity of spots with supragingival saliva and GCF was compared using agarose two-dimensional electrophoresis. Eight protein spots were found to be significantly more intense in GCF. They included superoxide dismutase 1 (SOD1), apolipoprotein A-I (ApoA-I), and dermcidin (DCD). Moreover, GCF proteins from healthy subjects were broken down into small peptide fragments and then analyzed directly by LC-MS/MS analysis. A total of 327 proteins including ApoA-I, SOD1, and DCD were identified in GCF. These results may serve as reference for future proteomic studies searching for GCF biomarkers of periodontal diseases.

Data-independent acquisition (DIA)-mass spectrometry (MS)-based proteomic analysis overtop the existing data-dependent acquisition (DDA)-MS-based proteomic analysis to enable deep proteome coverage and precise relative quantitative analysis in single-shot liquid chromatography (LC)-MS/MS. However, DIA-MS-based proteomic analysis has not yet been optimized in terms of system robustness and throughput, particularly for its practical applications. We established a single-shot LC-MS/MS system with an MS measurement time of 90 min for a highly sensitive and deep proteomic analysis by optimizing the conditions of DIA and nanoLC. We identified 7020 and 4068 proteins from 200 ng and 10 ng, respectively, of tryptic floating human embryonic kidney cells 293 (HEK293F) cell digest by performing the constructed LC-MS method with a protein sequence database search. The numbers of identified proteins from 200 ng and 10 ng of tryptic HEK293F increased to 8509 and 5706, respectively, by searching the chromatogram library created by gas-phase fractionated DIA. Moreover, DIA protein quantification was highly reproducible, with median coefficients of variation of 4.3% in eight replicate analyses. We could demonstrate the power of this system by applying the proteomic analysis to detect subtle changes in protein profiles between cerebrums in germ-free and specific pathogen-free mice, which successfully showed that >40 proteins were differentially produced between the cerebrums in the presence or absence of bacteria.

Serum proteins/peptides reflect physiological or pathological states in humans and are an attractive target for the discovery of disease biomarkers. However, the existence of high-abundance proteins and the large dynamic range of serum proteins/peptides make any quantitative analysis of low-abundance proteins/peptides challenging. Furthermore, analyses of peptides, including the cleaved fragments of proteins, are difficult because of carrier protein binding. Here, we developed a differential solubilization (DS) method to extract low-molecular-weight proteins/peptides in serum with good reproducibility and yield as compared to typical peptide-extraction methods such as organic solvent precipitation and ultrafiltration. Using the DS method combined with reverse-phase HPLC fractionation followed by MALDI-TOF-MS, we performed high-quality comparative analyses of more than 1500 peptides from 1 microL of serum samples, including low-abundance peptides in the subnanomolar range and containing many peptides bound to carrier proteins such as albumin. We applied this method and successfully discovered four new biomarker candidates of colon cancer, none of which have previously been observed in serum and one of which is a fragment of the protein zyxin that possibly originated from tumor cells. Our results indicate that serum peptide analyses based on the DS method should greatly contribute to the discovery of novel low-abundance biomarkers.