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.

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.

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.

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.

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.

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.

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.

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 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.

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.

Mutations in the C1 element of the insulin promoter lead to diabetic phenotypes in homozygous mice.

Genome editing technologies such as CRISPR-Cas9 are widely used to establish causal associations between mutations and phenotypes. However, CRISPR-Cas9 is rarely used to analyze promoter regions. The insulin promoter region (approximately 1,000 bp) directs ß cell-specific expression of insulin, which in vitro studies show is regulated by ubiquitous, as well as pancreatic, ß cell-specific transcription factors. However, we are unaware of any confirmatory in vivo studies. Here, we used CRISPR-Cas9 technology to generate mice with mutations in the promoter regions of the insulin I (Ins1) and II (Ins2) genes. We generated 4 homozygous diabetic mice with 2 distinct mutations in the highly conserved C1 elements in each of the Ins1 and Ins2 promoters (3 deletions and 1 replacement in total). Remarkably, all mice with homozygous or heterozygous mutations in other loci were not diabetic. Thus, the C1 element in mice is required for Ins transcription in vivo.

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.

Excellent Islet Yields after 18-h Porcine Pancreas Preservation by Ductal Injection, Pancreas Preservation with MK Solution, Bottle Purification, and Islet Purification Using Iodixanol with UW Solution and Iodixanol with MK Solution.

Successful islet isolation is the key to successful islet transplantation. Our group recently modified the islet isolation protocol to include pancreatic ductal injection of the preservation solution, pancreas storage in modified extracellular-type trehalose-containing Kyoto (MK) solution, and use of an iodixanol-based purification solution and bottle purification. In this study, we applied these methods to porcine islet isolation after 18-h pancreas preservation and compared two solutions with different compositions in bottle purification. Islet yield before purification was 651,661 ± 157,719 islet equivalents (IE) and 5576 ± 1538 IE/g pancreas weight. An IU solution was made by adding iodixanol to University of Wisconsin solution and an IK solution was made by adding iodixanol to MK solution. The efficacy of the two solutions for islet isolation was compared. There were no significant differences between the two purification methods with regard to islet yield, survival rate, purity, score, or stimulation index. These results indicate that our isolation protocol produces efficient islet yields from prolonged cold-stored pancreas and that IU and IK solutions are equally useful for islet purification.

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.

Induction of Expandable Tissue-Specific Progenitor Cells from Human Pancreatic Tissue through Transient Expression of Defined Factors.

We recently demonstrated the generation of mouse induced tissue-specific stem (iTS) cells through transient overexpression of reprogramming factors combined with tissue-specific selection. Here we induced expandable tissue-specific progenitor (iTP) cells from human pancreatic tissue through transient expression of genes encoding the reprogramming factors OCT4 (octamer-binding transcription factor 4), p53 small hairpin RNA (shRNA), SOX2 (sex-determining region Y-box 2), KLF4 (Kruppel-like factor 4), L-MYC, and LIN28. Transfection of episomal plasmid vectors into human pancreatic tissue efficiently generated iTP cells expressing genetic markers of endoderm and pancreatic progenitors. The iTP cells differentiated into insulin-producing cells more efficiently than human induced pluripotent stem cells (iPSCs). iTP cells continued to proliferate faster than pancreatic tissue cells until days 100-120 (passages 15-20). iTP cells subcutaneously inoculated into immunodeficient mice did not form teratomas. Genomic bisulfite nucleotide sequence analysis demonstrated that the OCT4 and NANOG promoters remained partially methylated in iTP cells. We compared the global gene expression profiles of iPSCs, iTP cells, and pancreatic cells (islets >80%). Microarray analyses revealed that the gene expression profiles of iTP cells were similar, but not identical, to those of iPSCs but different from those of pancreatic cells. The generation of human iTP cells may have important implications for the clinical application of stem/progenitor cells.

A Liquid Chromatography with Tandem Mass Spectrometry-Based Proteomic Analysis of Primary Cultured Cells and Subcultured Cells Using Mouse Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (MSC-ATs) are representative cell sources for cell therapy. However, how cell stress resulting from passage influences the MSC-AT protein expression has been unclear. In this study, a protein expression analysis was performed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) using mouse primary cultured cells (P0) and cells passaged three times (P3) as samples. A total of 256 proteins were classified as cellular process-related proteins, while 179 were classified as metabolic process-related proteins in P0. These were considered to be adaptive responses of the cells to an in vitro environment. However, seven proteins of growth were identified (Csf1, App, Adam15, Alcam, Tbl1xr1, Ninj1, and Sbds) in P0. In addition, four proteins of antioxidant activity were also identified (Srxn1, Txndc17, Fam213b, and Apoe) in P0. We identified 1139 proteins expressed in both P0 and P3 cells that had their expression decreased to 69.4% in P3 cells compared with P0 cells, but 1139 proteins are very likely proteins that are derived from MSC-AT. The function of MSC-ATs was maintained after three passages. However, the LC-MS/MS analysis data showed that the protein expression was degraded after three passages. MSC-ATs retained about 70% of their protein expression ability in P3 cells.

A Novel Preservation Solution Containing a JNK Inhibitory Peptide Efficiently Improves Islet Yield for Porcine Islet Isolation.

BACKGROUND: For islet transplantation, pancreas preservation in University of Wisconsin (UW) solution is associated with disadvantages, such as collagenase inhibition, resulting in poor islet yield and islets with poor viability. In this study, we evaluated a novel preservation solution, the extracellular-type c-Jun N-terminal kinase (JNK) inhibitor-containing (EJ) solution. METHODS: The EJ solution has high sodium-low potassium composition with low viscosity compared to UW solution. Moreover, EJ solution contains a recently developed JNK inhibitor from our laboratory. RESULTS: We first compared the performance of EJ solution with that of UW solution. Islet yield before and after purification was significantly higher in the EJ group than in the UW group. Second, we compared the performance of EJ solution with that of EJ solution without the JNK inhibitor (EJ-J solution). After pancreas preservation in EJ solution, JNK activity was maintained at a relatively low level during islet isolation. Islet yield before and after purification was significantly higher in the EJ group than in the EJ-J group. After islet transplantation into streptozotocin-induced diabetic mice, blood glucose levels reached the normoglycemic range in 61.5% and 7.7% of diabetic mice in the EJ and EJ-J groups, respectively. Moreover, EJ solution exhibited reduced inhibition of collagenase digestion compared with UW solution. CONCLUSIONS: Advantages of EJ solution over UW solution were inhibition of JNK activity and reduced collagenase inhibition. EJ solution may therefore be more suitable for islet isolation than UW solution.