Yoshiki Nakashima

A proteome analysis of pig pancreatic islets and exocrine tissue by liquid chromatography with tandem mass spectrometry

ABSTRACT Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is a proteome analysis method, and the shotgun analysis by LC-MS/MS comprehensively identifies proteins from tissues and cells with high resolving power. In this study, we analyzed the protein expression in pancreatic tissue by LC-MS/MS. Islets isolated from porcine pancreata (purity ≥95%) and exocrine tissue (purity ≥99%) were used in this study. LC-MS/MS showed that 13 proteins were expressed in pancreatic islets only (Group I), 43 proteins were expressed in both islets and exocrine tissue (Group I&E), and 102 proteins were expressed in exocrine tissue only (Group E). Proteins involved in islet differentiation and cell proliferation were identified in Group I (e.g. CLUS, CMGA, MIF). In addition, various functional proteins (e.g. SCG2, TBA1A) were identified in islet by using the new method of ‘principal component analysis (PCA)’. However, the function of such proteins on islets remains unclear. EPCAM was identified in Group E. Group E was found to include proteins involved in clinical inflammatory diseases such as pancreatitis (e.g. CBPA1, CGL, CYTB, ISK1 and PA21B). Many of these identified proteins were reported less frequently in previous studies, and HS71B, NEC2, PRAF3 and SCG1 were newly detected in Group I while CPNS1, DPEP1, GANAB, GDIB, GGT1, HSPB1, ICTL, VILI, MUTA, NDKB, PTGR1, UCHL3, VAPB and VINC were newly detected in Group E. These results show that comprehensive expression analysis of proteins by LC-MS/MS is useful as a method to investigate new factors constructing cellular component, biological process, and molecular function.

RCAN-11R peptide provides immunosuppression for fully mismatched islet allografts in mice

Calcineurin inhibitors have been used for transplant therapy. However, the inhibition of calcineurin outside the immune system has a number of side effects. We previously developed a cell-permeable inhibitor of NFAT (nuclear factor of activated T cells) using the polyarginine peptide delivery system. This peptide (11R-VIVIT) selectively interferes with calcineurin-NFAT interaction without affecting the activity of calcineurin phosphatase and provides immunosuppression for fully mismatched islet allografts in mice. However, our recent study showed that 11R-VIVIT affected cell viability in vitro when it was used at higher concentration because of the VIVIT sequence. The aim of this study is to develop a safer NFAT inhibitor (RCAN-11R) that does not affect cell viability, and which is less toxic than calcineurin inhibitors. The minimal sequence of the protein family of regulators of calcineurin (RCAN) that is responsible for the inhibition of calcineurin-NFAT signaling was recently characterized. The peptide could selectively interfere with the calcineurin-NFAT interaction without affecting the activity of calcineurin phosphatase, similar to 11R-VIVIT. RCAN-11R did not affect cell viability when it was used at a higher concentration than the toxic concentration of 11R-VIVIT. RCAN-11R could therefore be useful as a therapeutic agent that is less toxic than current drugs or 11R-VIVIT.

Induced Tissue-Specific Stem Cells and Epigenetic Memory in Induced Pluripotent Stem Cells

Induced pluripotent stem (iPS) cells have significant implications for overcoming most of the ethical issues associated with embryonic stem (ES) cells. The pattern of expressed genes, DNA methylation, and covalent histone modifications in iPS cells are very similar to those in ES cells. However, it has recently been shown that, following the reprogramming of mouse/human iPS cells, epigenetic memory is inherited from the parental cells. These findings suggest that the phenotype of iPS cells may be influenced by their cells of origin and that their skewed differentiation potential may prove useful in the generation of differentiated cell types that are currently difficult to produce from ES/iPS cells for the treatment of human diseases. Our recent study demonstrated the generation of induced tissue-specific stem (iTS) cells by transient overexpression of the reprogramming factors combined with tissue-specific selection. iTS cells are cells that inherit numerous components of epigenetic memory from donor tissue and acquire self-renewal potential. This review describes the “epigenetic memory” phenomenon in iPS and iTS cells and the possible clinical applications of these stem cells.

A Comparison of the Preservation of Mouse Adipose Tissue-Derived Mesenchymal Stem Cells Using the University of Wisconsin Solution and Hank’s Balanced Salt Solution

Preservation of adipose tissue before the isolation of cells is one of the most important steps in maintaining the cell viability of adipose tissue-derived mesenchymal stem cells (ADSCs) for clinical use. Hanks balanced salt solution (HBSS) is one of the main ADSC preservation solutions used clinically. However, this step is known to lead to decreased cell viability. The University of Wisconsin (UW) solution is recognized by transplant physicians as an excellent organ preservation solution. We aimed to investigate the effectiveness of UW solution in preservation of the viability of ADSCs. We collected adipose tissue from the inguinal fat pad of mice and compared preservation in UW solution and HBSS overnight by measuring cell viability after isolation. We found that the number of viable cells harvested per gram of adipose tissue mass was higher in UW solution- than HBSS-preserved tissue.

Modified cell-permeable JNK inhibitors efficiently prevents islet apoptosis and improves the outcome of islet transplantation

We previously reported that treatment with a JNK inhibitory peptide (11R-JNKI) prevents islet apoptosis and enhances the islet function in vivo. In the present study, we explored more efficient JNK inhibitors. The inhibition of the JNK activity by five types of deletion peptides in 11R-JNKI was investigated. One of the peptides, 8R-sJNKI(-9), significantly prevented JNK activation. At a concentration of 1 µM, 8R-sJNKI(-9) inhibited JNK activity similarly to 10 µM 11R-JNKI and the inhibition of the JNK activity by 10 µM 8R-sJNKI(-9) was significantly greater than that by 10 µM 11R-JNK. To evaluate the effects of 8R-sJNKI(-9), porcine islets were cultured with 1 µM of 8R-sJNKI(-9) or 8R-mutant sJNKI(-9) (8R-mJNKI(-9)). After 1 day of culture, the numbers of islets in the 8R-sJNKI(-9)-treated group was significantly higher than that in the 8R-mJNKI(-9)-treated group. After islet transplantation, the blood glucose levels reached the normoglycemic range in 58.3% of streptozotocin-induced diabetic mice in the 8R-sJNKI(-9) group and 0% of the mice in the 8R-mJNKI(-9)-treated group. These data suggest that 8R-sJNKI(-9) inhibits islet apoptosis and improves islet function.

Characterization of induced tissue-specific stem cells from pancreas by a synthetic self-replicative RNA

Induced pluripotent stem (iPS) cells have significant implications for overcoming most of the ethical issues associated with embryonic stem (ES) cells. Furthermore, our recent study demonstrated the generation of induced tissue-specific stem (iTS) cells by transient overexpression of the reprogramming factors using a plasmid combined with tissue-specific selection. In this study, we were able to generate RNA-based iTS cells that utilize a single, synthetic, self-replicating VEE-RF RNA replicon expressing four reprogramming factors (OCT4, KLF4, SOX2, and GLIS1). A single VEE-RF RNA transfection into mouse pancreatic tissue resulted in efficient generation of iTS cells from pancreas (iTS-P cells) with genetic markers of endoderm and pancreatic progenitors and differentiation into insulin-producing cells more efficiently than ES cells. Subcutaneous transplantation of iTS-P cells into immunodeficient mice resulted in no teratoma formation. Bisulfite genomic sequencing demonstrated that the promoters of Oct4 and Nanog remained partially methylated in iTS-P cells. We compared the global gene-expression profiles of ES cells, iTS-P cells, and pancreatic islets. Microarray analyses confirmed that the iTS-P cells were similar but not identical to ES cells compared with islets. These data suggest that iTS-P cells are cells that inherit numerous components of epigenetic memory from pancreas cells and acquire self-renewal potential. The generation of iTS cells may have important implications for the clinical application of stem cells.

Adhesion characteristics of porcine pancreatic islets and exocrine tissue to coating materials

ABSTRACT Since the report of the Edmonton protocol in 2000, islet transplantation has been implemented worldwide, and xenotransplantation using porcine islets has also been reported. In addition, many basic experiments using pancreatic islets and exocrine tissue after isolation have been reported. Recently, exocrine cells have been found to be essential for inducing the differentiation of pancreatic islets. Therefore, the importance of the culture conditions for pancreatic tissue when conducting experiments using pancreatic tissue is also increasing. In this study, we focused on the coat material and examined the adhesive properties of porcine pancreatic islets and exocrine tissue after isolation. Porcine islet isolation was performed, and isolated islets (purity ≥95%) and exocrine tissue (purity ≥99%) were used to achieve adhesion to several extracellular matrixes, fibronectin, collagen type I, collagen type IV, laminin I, fibrinogen, and bovine serum albumin (BSA). DMEM with 0.5% FBS was used as the assay medium. For exocrine tissue, the adhesion was promoted in fibronectin, collagen type I, laminin I, and fibrinogen. The adhesive ability to fibronectin was more than twice that to BSA, while the adhesive ability to collagen type I, laminin I, and fibrinogen was less than twice that to BSA. For islets, the adhesive ability to fibronectin was weaker than that of exocrine tissue. Furthermore, the adhesion effect in fibronectin was obtained within 30 minutes and in medium containing little serum for both islets and exocrine tissues. These data suggest that fibronectin may be useful for the adhesion of pancreatic tissue.

A Liquid Chromatography with Tandem Mass Spectrometry-Based Proteomic Analysis of Cells Cultured in DMEM 10% FBS and Chemically Defined Medium Using Human Adipose-Derived Mesenchymal Stem Cells

Human adipose-derived mesenchymal stem cells (hADSCs) are representative cell sources for cell therapy. Classically, Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) has been used as culture medium for hADSCs. A chemically defined medium (CDM) containing no heterologous animal components has recently been used to produce therapeutic hADSCs. However, how the culture environment using a medium without FBS affects the protein expression of hADSC is unclear. We subjected hADSCs cultured in CDM and DMEM (10% FBS) to a protein expression analysis by tandem mass spectrometry liquid chromatography and noted 98.2% agreement in the proteins expressed by the CDM and DMEM groups. We classified 761 proteins expressed in both groups by their function in a gene ontology analysis. Thirty-one groups of proteins were classified as growth-related proteins in the CDM and DMEM groups, 16 were classified as antioxidant activity-related, 147 were classified as immune system process-related, 557 were involved in biological regulation, 493 were classified as metabolic process-related, and 407 were classified as related to stimulus responses. These results show that the trend in the expression of major proteins related to the therapeutic effect of hADSCs correlated strongly in both groups.

Comparison of Tissue Loading Before and After the Creation of a Continuous Density Gradient in Porcine Islet Purification

The purification step is one of the most important and difficult procedures in islet isolation for pancreatic islet transplantation. We previously reported that a purification method using large plastic bottles effectively achieved a high yield of islets from the porcine pancreas. In this study, we evaluated the impact of the timing of tissue loading on porcine islet purification using large plastic bottles. One method involved loading digested tissue after creating a continuous density gradient (tissue after gradient [TAG]). The other method involved loading digested tissue before creating a continuous density gradient (tissue before gradient [TBG]). There were no significant differences between TAG and TBG in terms of the islet yield, rates of viability and purity, score, and in the stimulation index after purification. Furthermore, there were no marked differences in the attainability or suitability of post-transplantation normoglycemia. Our study shows the equivalency of these two methods of islet purification.

Role of Egr1 on Pancreatic Endoderm Differentiation

The low efficiency of in vitro differentiation of human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (iPSCs) into insulin-producing cells is a crucial hurdle for the clinical implementation of human pluripotent stem cells (PSCs). Our previous investigation into the key factors for the differentiation of PSCs into insulin-producing cells suggested that the expression of GATA binding protein 6 (GATA6) and Gremlin 1 (GREM1) and inhibition of early growth response protein 1 (Egr1) may be important factors. In this study, we investigated the role of Egr1 in pancreas development. The transfection of small interfering RNA (siRNA) of Egr1 in the early phase induced the differentiation of iPSCs derived from fibroblasts (FiPSCs) into pancreatic endoderm and insulin-producing cells. In contrast, the downregulation of Egr1 in the late phase suppressed the differentiation of FiPSCs into pancreatic endoderm and insulin-producing cells. In addition, the overexpression of Egr1 suppressed the differentiation of iPSCs derived from pancreatic cells into pancreatic endoderm and insulin-producing cells. These data suggest that the downregulation of Egr1 in the early phase can efficiently induce the differentiation of iPSCs into insulin-producing cells.