Preservation of pancreas in the University of Wisconsin solution supplemented with AP39 reduces reactive oxygen species production and improves islet graft function.

Ischemia-reperfusion injury (IRI) results in increased rates of delayed graft function and early graft loss. It has recently been reported that hydrogen sulfide (H2 S) protects organ grafts against prolonged IRI. Here, we investigated whether the preservation of pancreas in University of Wisconsin (UW) solution supplemented with AP39, which is a mitochondrial-targeted H2 S donor, protected pancreatic islets against IRI and improved islet function. Porcine pancreata were preserved in the UW solution with AP39 (UW + AP39) or the vehicle (UW) for 18 h, followed by islet isolation. The islet yields before and after purification were significantly higher in the UW + AP39 group than in the UW group. The islets isolated from the pancreas preserved in UW + AP39 exhibited significantly decreased levels of reactive oxygen species (ROS) production and a significantly increased mitochondrial membrane potential as compared to the islets isolated from the pancreas preserved in the vehicle. We found that the pancreas preserved in UW + AP39 improved the outcome of islet transplantation in streptozotocin-induced diabetic mice. These results suggest that the preservation of pancreas in UW + AP39 protects the islet grafts against IRI and could thus serve as a novel clinical strategy for improving islet transplantation outcomes.

Pancreas preservation with amphotericin B deteriorates islet yield for porcine islet isolation.

BACKGROUND: Amphotericin B is a crucial agent in the management of serious systemic fungal infections. It is also known to be cytotoxic. In this study, we evaluated the effect of amphotericin B added to the preservation solution on islet yield during islet isolation. METHODS: Porcine pancreata were preserved in the preservation solution with or without amphotericin B (0.25 µg/mL) for approximately 18 hours at 4°C, and then islet isolation was performed. An optimized number (1750 IE) of isolated islets from each group were transplanted into streptozotocin-induced diabetic mice. The culture of isolated islets and acinar tissue with amphotericin B was also evaluated. RESULTS: The islet yield before and after purification in the amphotericin B (-) group was significantly higher than that in the amphotericin B (+) group. After islet transplantation into diabetic mice, blood glucose levels reached the normoglycemic range, with 50% and 0% of that of the diabetic mice in the amphotericin B (-) and amphotericin B (+) groups, respectively. In the culture study, amphotericin B was found to be cytotoxic to porcine islets and acinar tissue. CONCLUSIONS: Amphotericin B added to the preservation solution deteriorates islet yield during porcine islet isolation. Thus, the use of amphotericin B should be considered carefully for the preservation of the pancreas for islet isolation and islet culture before islet transplantation.

Pancreas preservation in extracellular-type p38 inhibitor-containing solution improves islet yield for porcine islet isolation.

BACKGROUND: For islet transplantation, pancreas preservation and islet isolation activate p38, which is a member of the stress-activated group of mitogen-activated protein kinases (MAPKs). In this study, we evaluated an extracellular-type p38 inhibitor-containing (EP) solution with University of Wisconsin (UW) solution, the gold standard for organ preservation. The EP solution has high sodium-low potassium composition with low viscosity compared to UW solution. Moreover, EP solution contains a recently developed p38 inhibitor (11R-p38I110 ) from our laboratory. METHODS: Porcine pancreata were preserved in UW, EP, or EP-P solution (EP solution without 11R-p38I110 ), and then islet isolation was performed. An optimized number (1500 IE) of isolated islets from each group were transplanted into streptozotocin-induced diabetic mice. RESULTS: The islet yield before and after purification was significantly higher in the EP group than in the UW group. The islet yield before and after purification was not significantly different between the EP and EP-P groups; however, the EP solution prevented a reduction in the number of islets during culture. Western blot analysis showed that p38 activation was attenuated by EP solution. For islet transplantation into streptozotocin-induced diabetic mice, pancreas preservation in EP solution improved the outcome of islet transplantation. CONCLUSIONS: Pancreas preservation with EP solution preserved islet function better than with UW solution. The advantages of EP solution over UW solution may include the inhibition of p38 activity as well as the composition of the solution.

Novel cell-permeable p38-MAPK inhibitor efficiently prevents porcine islet apoptosis and improves islet graft function.

During islet transplantation, mitogen-activated protein kinase (MAPK) p38 is preferentially activated in response to the isolation of islets and the associated inflammation. Although therapeutic effects of p38 inhibitors are expected, the clinical application of small-molecule inhibitors of p38 is not recommended because of their serious adverse effects on the liver and central nervous system. Here we designed peptides to inhibit p38, which were derived from the sites on p38 that mediate binding to proteins such as MAPK kinases. Peptide 11R-p38I110 significantly inhibited the activation of p38. To evaluate the effects of 11R-p38I110 , porcine islets were incubated with 10 µmol/L 11R-p38I110 or a mutant form designated 11R-mp38I110 . After islet transplantation, blood glucose levels reached the normoglycemic range in 58.3% and 0% of diabetic mice treated with 11R-p38I110 or 11R-mp38I110 , respectively. These data suggest that 11R-p38I110 inhibited islet apoptosis and improved islet function. Peptide p38I110 is a noncompetitive inhibitor of ATP and targets a unique docking site. Therefore, 11R-p38I110 specifically inhibits p38 activation, which may avoid the adverse effects that have discouraged the clinical use of small-molecule inhibitors of p38. Moreover, our methodology to design "peptide inhibitors" could be used to design other inhibitors derived from the binding sites of proteins.

Atorvastatin Inhibits the HIF1α-PPAR Axis, Which Is Essential for Maintaining the Function of Human Induced Pluripotent Stem Cells.

We herein report a novel mechanism of action of statin preparations using a new drug discovery method. Milk fat globule-EGF factor 8 protein (MFG-E8) was identified from the secretory component of mouse embryonic fibroblast (MEF) as a cell adhesion-promoting factor effective for screening active cellular agents of human induced pluripotent stem cells (hiPSCs) in vitro using electrochemical impedance. Our analyses showed that atorvastatin did not cause death in myocardial cells differentiated from hiPSCs but reduced the pluripotent cell survival in vitro when using serum- and albumin-free media, and inhibited the ability to form teratomas in mice. This result could have been already the cytopathic effect of atorvastatin, and complete elimination of hiPSCs was confirmed in the xenotransplantation assay. The administration of atorvastatin to hiPSCs caused the expression of hypoxia inducible factor (HIF)1α mRNA to be unchanged at 6 hr and downregulated at 24 hr. In addition, the inhibition of the survival of hiPSCs was confirmed by HIF1α-peroxisome proliferator-activated receptor (PPAR) axis inhibition. These results suggest that the addition of atorvastatin to hiPSC cultures reduces the survival of pluripotent cells by suppressing the HIF1α-PPAR axis. In summary, the HIF1α-PPAR axis has an important role in maintaining the survival of pluripotent hiPSCs.

Identification of Proteins Differentially Expressed by Adipose-derived Mesenchymal Stem Cells Isolated from Immunodeficient Mice.

Although cell therapy using adipose-derived mesenchymal stem cells (AdMSCs) regulates immunity, the degree to which cell quality and function are affected by differences in immunodeficiency of donors is unknown. We used liquid chromatography tandem-mass spectrometry (LC MS/MS) to identify the proteins expressed by mouse AdMSCs (mAsMSCs) isolated from normal (C57BL/6) mice and mice with severe combined immunodeficiency (SCID). The protein expression profiles of each strain were 98%-100% identical, indicating that the expression levels of major proteins potentially associated with the therapeutic effects of mAdMSCs were highly similar. Further, comparable levels of cell surface markers (CD44, CD90.2) were detected using flow cytometry or LC MS/MS. MYH9, ACTN1, CANX, GPI, TPM1, EPRS, ITGB1, ANXA3, CNN2, MAPK1, PSME2, CTPS1, OTUB1, PSMB6, HMGB1, RPS19, SEC61A1, CTNNB1, GLO1, RPL22, PSMA2, SYNCRIP, PRDX3, SAMHD1, TCAF2, MAPK3, RPS24, and MYO1E, which are associated with immunity, were expressed at higher levels by the SCID mAdMSCs compared with the C57BL/6 mAdMSCs. In contrast, ANXA9, PCBP2, LGALS3, PPP1R14B, and PSMA6, which are also associated with immunity, were more highly expressed by C57BL/6 mAdMSCs than SCID mAdMSCs. These findings implicate these two sets of proteins in the pathogenesis and maintenance of immunodeficiency.

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 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.

Induction of Expandable Adipose-Derived Mesenchymal Stem Cells from Aged Mesenchymal Stem Cells by a Synthetic Self-Replicating RNA.

Adipose-derived mesenchymal stem cells (ADSCs) have attracted attention due to their potential for use in the treatment of various diseases. However, the self-renewal capacity of ADSCs is restricted and their function diminishes during passage. We previously generated induced tissue-specific stem cells from mouse pancreatic cells using a single synthetic self-replicating Venezuelan Equine Encephalitis (VEE)-reprogramming factor (RF) RNA replicon (SR-RNA) expressing the reprogramming factors POU class 5 homeobox 1 (OCT4), Krueppel-like factor 4 (KLF4), Sex determining region Y-box 2 (SOX2), and Glis Family Zinc Finger 1 (GLIS1). This vector was used to generate induced pluripotent stem (iPS) cells. Here, we applied this SR-RNA vector to generate human iTS cells from aged mesenchymal stem cells (hiTS-M cells) deficient in self-renewal that were derived from adipose tissue. These hiTS-M cells transfected with the SR-RNA vector survived for 15 passages. The hiTS-M cells expressed cell surface markers similar to those of human adipose-derived mesenchymal stem cells (hADSCs) and differentiated into fat cells and osteoblasts. Global gene expression profiling showed that hiTS-M cells were transcriptionally similar to hADSCs. These data suggest that the generation of iTS cells has important implications for the clinical application of autologous stem cell transplantation.

A Comparison of Proteins Expressed between Human and Mouse Adipose-Derived Mesenchymal Stem Cells by a Proteome Analysis through Liquid Chromatography with Tandem Mass Spectrometry.

Adipose-derived mesenchymal stem cells (ADSCs) have become a common cell source for cell transplantation therapy. Clinical studies have used ADSCs to develop treatments for tissue fibrosis, such as liver cirrhosis and pulmonary fibroma. The need to examine and compare basic research data using clinical research data derived from mice and humans is expected to increase in the future. Here, to better characterize the cells, the protein components expressed by human ADSCs used for treatment, and mouse ADSCs used for research, were comprehensively analyzed by liquid chromatography with tandem mass spectrometry. We found that 92% (401 type proteins) of the proteins expressed by ADSCs in humans and mice were consistent. When classified by the protein functions in a gene ontology analysis, the items that differed by >5% between human and mouse ADSCs were "biological adhesion, locomotion" in biological processes, "plasma membrane" in cellular components, and "antioxidant activity, molecular transducer activity" in molecular functions. Most of the listed proteins were sensitive to cell isolation processes. These results show that the proteins expressed by human and murine ADSCs showed a high degree of correlation.

Establishment of Induced Pancreatic Stem Cells by Yes-Associated Protein 1.

Recently, we and others generated induced tissue-specific stem/progenitor (iTS/iTP) cells. The advantages of iTS/iTP cells compared with induced pluripotent stem (iPS) cells are (1) easier generation, (2) efficient differentiation, and (3) no teratomas formation. In this study, we generated mouse induced pancreatic stem cells (iTS-P cells) by the plasmid vector expressing Yes-associated protein 1 (YAP). The iTS-P YAP9 cells expressed Foxa2 (endoderm marker) and Pdx1 (pancreatic marker) while the expressions of Oct3/4 and Nanog (marker of embryonic stem [ES] cells) in iTS-P YAP9 cells was significantly lower compared with those in ES cells. The iTS-P YAP9 cells efficiently differentiated into insulin-expressing cells compared with ES cells. The ability to generate autologous iTS cells may be applied to diverse applications of regenerative medicine.

Cytoprotective Effect of Pteryxin on Insulinoma MIN6 Cells Due to Antioxidant Enzymes Expression via Nrf2/ARE Activation.

The low-level antioxidant activity of pancreatic islets causes type 1 diabetes due to oxidative stress, which is also the cause of failure in the pancreatic islets' isolation and cell transplantation. In our previous study, pteryxin was found to be a natural product as a nuclear factor-erythroid-2-related factor (Nrf2) activator. This study focused on elucidation that the potentiality of pteryxin can activate the antioxidant enzymes, even under oxidative stress, by hydrogen peroxide (H2O2). Pteryxin treated with mouse insulinoma MIN6 cells was enhanced the antioxidant gene expressions in the ARE (antioxidant response element) region for HO-1 (Heme Oxygenase-1), GCLC (Glutamate-cysteine ligase catalytic subunit), SOD1 (Super Oxide dismutase1), and Trxr1 (Thioredoxin reductase1), and those enzymes were also expressed during the nuclei transference of cytoplasmic Nrf2. In fact, the cells exposed to H2O2 concentrations of a half-cell lethal in the presence of pteryxin were then induced main antioxidant enzymes, HO-1, GCLC, and Trxr1 in the ARE region. The increased glutathione (GSH) levels associated with the GCLC expression also suggested to be cytoprotective against oxidative stress by activating the redox-metabolizing enzymes involving their increased antioxidant activity in the cells. In addition, Akt is a modulator for Nrf2, which may be responsible for the Nrf2 activation. These results allowed us to consider whether pteryxin or its synthesized congeners, an Nrf2 activator, is a potential preservative agent against islet isolation during cell transplantation.

Induced hepatic stem cells are suitable for human hepatocyte production.

Human hepatocytes were transfected with Sendai virus vectors (SeV) expressing OCT3/4, SOX2, KLF4, and C-MYC to produce hepatocyte-derived induced pluripotent stem cells (iPSCs). The messenger RNA (mRNA) expression of undifferentiated markers (passage 19-21) and hepatocyte-specific markers (HSMs) (passage 0-20) in 48 established hepatocyte-derived iPSC-like colonies was examined. Among the 48 clones, 10 clones continuously expressed HSM mRNA (HNF1ß and HNF4α) in passage 0-20. The colonies which expressed HSMs (iTS-L cells: induced tissue-specific stem cells from liver) showed a different tendency in microarray and methylation analyses to fibroblast-derived iPSCs (strain: 201B7). iTS-L cells were less likely to form teratomas in mice than iPSCs (He). The iTS-L cells were differentiated into hepatocyte-like cells more efficiently than iPSCs (He) or iPSCs (201B7). These data suggest that SeV expressing OCT3/4, SOX2, KLF4, and C-MYC induce the generation of iPSCs and iTS-L cells.

Protocol for the generation of human induced hepatic stem cells using Sendai virus vectors.

Our recent study demonstrated the generation of induced tissue-specific stem/progenitor (iTS/iTP) cells by the transient overexpression of reprogramming factors combined with tissue-specific selection. Here, we present a protocol to reprogram human hepatocytes to generate human induced tissue-specific liver stem (iTS-L) cells. Human hepatocytes are transfected with Sendai virus vectors (SeV) expressing OCT3/4, SOX2, KLF4, and c-MYC. iTS-L cells continuously express mRNA of hepatocyte-specific markers (HNF1ß and HNF4α) and do not form teratomas. For complete details on the use and execution of this protocol, please refer to Nakashima et al. (2022).1.

Pancreas Preservation in Modified Histidine-lactobionate Solution Is Superior to That in University of Wisconsin Solution for Porcine Islet Isolation.

BACKGROUND: We previously reported that modified extracellular-type trehalose-containing Kyoto (MK) solution, which contains a trypsin inhibitor (ulinastatin), significantly improved the islet yield compared with University of Wisconsin (UW) preservation, which is the gold standard for organ preservation for islet isolation. In this study, we evaluated the efficiency of a modified histidine-lactobionate (MHL) solution in addition to UW or MK solution. The MHL solution has a high sodium-low potassium composition with low viscosity compared with the UW solution. Moreover, similar to MK solution, MHL solution also contains ulinastatin. METHODS: Porcine pancreata were preserved in UW, MK, or MHL solution, followed by islet isolation. An optimized number (1500 IE) of isolated islets from each group were then transplanted into streptozotocin-induced diabetic mice. RESULTS: The islet yield before and after purification was significantly higher in the MHL group than in the UW group. On the contrary, the islet yield before and after purification was not significantly different between the MHL and MK groups. Preserving the porcine pancreata in MHL solution improved the outcome of islet transplantation in streptozotocin-induced diabetic mice compared with that in UW solution. CONCLUSIONS: Pancreas preservation with MHL solution preserves islet function better than UW solution. The effect of MHL solution is similar to that of MK solution, suggesting that MHL solution can be used as an alternative to MK solution for pancreatic islet transplantation.

Pancreas Preservation with a Neutrophil Elastase Inhibitor, Alvelestat, Contributes to Improvement of Porcine Islet Isolation and Transplantation.

For pancreatic islet transplantation, pancreas procurement, preservation, and islet isolation destroy cellular and non-cellular components and activate components such as resident neutrophils, which play an important role in the impairment of islet survival. It has been reported that inhibitors of neutrophil elastase (NE), such as sivelestat and α1-antitrypsin, could contribute to improvement of islet isolation and transplantation. In this study, we investigated whether pancreatic preservation with alvelestat, a novel NE inhibitor, improves porcine islet yield and function. Porcine pancreata were preserved with or without 5 µM alvelestat for 18 h, and islet isolation was performed. The islet yields before and after purification were significantly higher in the alvelestat (+) group than in the alvelestat (-) group. After islet transplantation into streptozotocin-induced diabetic mice, blood glucose levels reached the normoglycemic range in 55% and 5% of diabetic mice in the alvelestat (+) and alvelestat (-) groups, respectively. These results suggest that pancreas preservation with alvelestat improves islet yield and graft function and could thus serve as a novel clinical strategy for improving the outcome of islet transplantation.

AP39, a Mitochondrial-Targeted H2S Donor, Improves Porcine Islet Survival in Culture.

The rapid deterioration of transplanted islets in culture is a well-established phenomenon. We recently reported that pancreas preservation with AP39 reduces reactive oxygen species (ROS) production and improves islet graft function. In this study, we investigated whether the addition of AP39 to the culture medium could reduce isolated islet deterioration and improve islet function. Isolated islets from porcine pancreata were cultured with 400 nM AP39 or without AP39 at 37 °C. After culturing for 6-72 h, the islet equivalents of porcine islets in the AP39(+) group were significantly higher than those in the AP39(-) group. The islets in the AP39(+) group exhibited significantly decreased levels of ROS production compared to the islets in the AP39(-) group. The islets in the AP39(+) group exhibited significantly increased mitochondrial membrane potential compared to the islets in the AP39(-) group. A marginal number (1500 IEs) of cultured islets from each group was then transplanted into streptozotocin-induced diabetic mice. Culturing isolated islets with AP39 improved islet transplantation outcomes in streptozotocin-induced diabetic mice. The addition of AP39 in culture medium reduces islet deterioration and furthers the advancements in ß-cell replacement therapy.

In vivo evaluation of GG2-GG1/A2 element activity in the insulin promoter region using the CRISPR-Cas9 system.

The insulin promoter is regulated by ubiquitous as well as pancreatic ß-cell-specific transcription factors. In the insulin promoter, GG2-GG1/A2-C1 (bases - 149 to - 116 in the human insulin promoter) play important roles in regulating ß-cell-specific expression of the insulin gene. However, these events were identified through in vitro studies, and we are unaware of comparable in vivo studies. In this study, we evaluated the activity of GG2-GG1/A2 elements in the insulin promoter region in vivo. We generated homozygous mice with mutations in the GG2-GG1/A2 elements in each of the Ins1 and Ins2 promoters by CRISPR-Cas9 technology. The mice with homozygous mutations in the GG2-GG1/A2 elements in both Ins1 and Ins2 were diabetic. These data suggest that the GG2-GG1/A2 element in mice is important for Ins transcription in vivo.

Gene Expression in Pancreatic Cancer-Like Cells and Induced Pancreatic Stem Cells Generated by Transient Overexpression of Reprogramming Factors.

We previously reported that transient overexpression of reprogramming factors can be used to generate induced pluripotent stem (iPS) cells, induced tissue-specific stem (iTS) cells, and fibroblast-like (iF) cells from pancreatic tissue. iF cells have tumorigenic ability and behave similarly to pancreatic cancer cells. In this study, we analyzed gene expression in iF cells and iTS-P cells (iTS cells from pancreatic tissue) via microarray analysis and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The expression levels of the Mybl2 and Lyn genes, which are reported to be oncogenes, were significantly higher in iF cells than in iTS-P cells. The expression level of Nestin, which is expressed in not only pancreatic progenitor cells but also pancreatic ductal adenocarcinomas, was also higher in iF cells than in iTS-P cells. Itgb6 and Fgf13, which are involved in the pathogenesis of diseases such as cancer, exhibited higher expression levels in iF cells than in iTS-P cells. Unexpectedly, the expression levels of genes related to epithelial-mesenchymal transition (EMT), except Bmp4, were lower in iF cells than in iTS-P cells. These data suggest that the Mybl2, Lyn, Nestin, Itgb6, and Fgf13 genes could be important biomarkers to distinguish iTS-P cells from iF cells.

Kyoto probe-1 reveals phenotypic differences between mouse ES cells and iTS-P cells.

Kyoto probe 1 (KP-1) rapidly distinguishes between human ES/iPS (hES/iPS) cells and their differentiated cells. Recently, we generated induced tissue-specific stem cells from pancreas (iTS-P cells) using reprogramming factors and tissue-specific selection. The iTS-P cells have self-renewal potential, and subcutaneously transplanting them into immunodeficient mice did not generate teratomas. In this study, we applied KP-1 to analyze mouse ES (mES) cells and mouse iTS-P (miTS-P) cells. KP-1 completely stained mES cells in colonies, but only miTS-P cells at the edge of a colony. This difference was caused by cell type-specific expression of different ABC transporters. These finding suggest that KP-1 will be useful for distinguishing between iPS and iTS-P cells.