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.

Improved Production of Induced Pluripotent Stem Cells Using Dot Pattern Culture Plates.

The rate of cell proliferation is a crucial factor in cell production under good manufacturing practice (GMP) control. In this study, we identified a culture system for induced pluripotent cells (iPSCs) that supports cell proliferation and viability and maintains the cells in an undifferentiated state even at 8 days after seeding. This system involves the use of dot pattern culture plates that have been coated with a chemically defined scaffold which has high biocompatibility. Under cell starvation conditions, where medium exchange was not performed for 7 days or where the amount of medium exchange was reduced to half or a quarter, iPSC viability and lack of differentiation were maintained. The rate of cell viability in this culture system was greater than generally obtained by standard culture methods. The cells in this compartmentalized culture system could be induced to differentiate in a controlled and consistent manner: differentiation of endoderm occurred in a controlled and consistent manner: endoderm, mesoderm, and ectoderm could be consistently induced to differentiate in the cultures. In conclusion, we have developed a culture system that supports high viability in iPSCs and allows their controlled differentiation. This system has the potential for use in GMP-based production of iPSCs for clinical purposes.

Adhesion Characteristics of Human Pancreatic Islets, Duct Epithelial Cells, and Acinar Cells to a Polymer Scaffold.

We reported in 2018 that among several extracellular matrices, fibronectin, type I collagen, type IV collagen, laminin I, fibrinogen, and bovine serum albumin, fibronectin is particularly useful for adhesion of porcine pancreatic tissue. Subsequently, we developed a technology that enables the chemical coating of the constituent motifs of fibronectin onto cell culture dishes. In this experiment, we used islets (purity ≥ 90%), duct epithelial cells (purity ≥ 60%), and acinar cells (purity ≥ 99%) isolated from human pancreas according to the Edmonton protocol published in 2000 and achieved adhesion to the constituent motifs of fibronectin. A solution including cGMP Prodo Islet Media was used as the assay solution. In islets, adhesion was enhanced with the constitutive motifs of fibronectin compared with uncoated islets. In the functional evaluation of islets, insulin mRNA expression and insulin secretion were enhanced by the constitutive motif of fibronectin compared with non-coated islets. The stimulation index was comparable between non-coated islets and fibronectin motifs. In duct epithelial cells, adhesion was mildly promoted by the fibronectin component compared with non-coated component, while in acinar cells, adhesion was inhibited by the fibronectin component compared with the non-coated component. These data suggest that the constitutive motifs of fibronectin are useful for the adhesion of islets and duct epithelial cells.

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.

Semi-3D cultures using Laminin 221 as a coating material for human induced pluripotent stem cells.

It was previously believed that human induced pluripotent stem cells (hiPSCs) did not show adhesion to the coating material Laminin 221, which is known to have specific affinity for cardiomyocytes. In this study, we report that human mononuclear cell-derived hiPSCs, established with Sendai virus vector, form peninsular-like colonies rather than embryonic stem cell-like colonies; these peninsular-like colonies can be passaged more than 10 times after establishment. Additionally, initialization-deficient cells with residual Sendai virus vector adhered to the coating material Laminin 511 but not to Laminin 221. Therefore, the expression of undifferentiated markers tended to be higher in hiPSCs established on Laminin 221 than on Laminin 511. On Laminin 221, hiPSCs15M66 showed a semi-floating colony morphology. The expression of various markers of cell polarity was significantly lower in hiPSCs cultured on Laminin 221 than in hiPSCs cultured on Laminin 511. Furthermore, 201B7 and 15M66 hiPSCs showed 3D cardiomyocyte differentiation on Laminin 221. Thus, the coating material Laminin 221 provides semi-floating culture conditions for the establishment, culture and induced differentiation of hiPSCs.

Laminin-511 Activates the Human Induced Pluripotent Stem Cell Survival via α6ß1 Integrin-Fyn-RhoA-ROCK Signaling.

In human induced pluripotent stem cells (hiPSCs), laminin-511/α6ß1 integrin interacts with E-cadherin, an intercellular adhesion molecule, to induce the activation of the phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathway. The interaction between laminin-511/α6ß1 integrin and E-cadherin, an intercellular adhesion molecule, results in protection against apoptosis through the proto-oncogene tyrosine-protein kinase Fyn(Fyn)-RhoA-ROCK signaling pathway and the Ras homolog gene family member A (RhoA)/Rho kinase (ROCK) signaling pathway (the major pathway for cell death). In this article, the impact of laminin-511 on hiPSC on α6ß1 integrin-Fyn-RhoA-ROCK signaling is discussed and explored along with validation experiments. PIK3CA mRNA (mean [standard deviation {SD}]: iMatrix-511, 1.00 [0.61]; collagen+MFGE8, 0.023 [0.02]; **P < 0.01; n = 6) and PIK3R1 mRNA (mean [SD]: iMatrix-511, 1.00 [0.79]; collagen+MFGE8, 0.040 [0.06]; *P < 0.05; n = 6) were upregulated by iMatrix-511 resulting from an increased expression of Integrin α6 mRNA (mean [SD]: iMatrix-511, 1.00 [0.42]; collagen+MFGE8, 0.23 [0.05]; **P < 0.01; n = 6). The iMatrix-511 increased the expression of p120-Catenin mRNA (mean [SD]: iMatrix-511, 1.00 [0.71]; collagen+MFGE8, 0.025 [0.03]; **P < 0.01; n = 6) and RAC1 mRNA (mean [SD]: iMatrix-511, 1.00 [0.28]; collagen+MFGE8, 0.39 [0.15]; **P < 0.01; n = 6) by increasing the expression of E-cadherin mRNA (mean [SD]: iMatrix-511, 1.00 [0.38]; collagen+MFGE8, 0.16 [0.11]; **P < 0.01; n = 6). As a result, iMatrix-511 increased the expression of P190 RhoGAP (GTPase-activating proteins) mRNA, such as ARHGAP1 mRNA (mean [SD]: iMatrix-511, 1.00 [0.57]; collagen+MFGE8, 0.032 [0.03]; **P < 0.01; n = 6), ARHGAP4 mRNA (mean [SD]: iMatrix-511, 1.00 [0.56]; collagen+MFGE8, 0.039 [0.049]; **P < 0.01; n = 6), and ARHGAP5 mRNA (mean [SD]: iMatrix-511, 1.00 [0.39]; collagen+MFGE8, 0.063 [0.043]; **P < 0.01; n = 6). Western blotting showed that phospho-Rac1 remained in the cytoplasm and phospho-Fyn showed nuclear transition in iPSCs cultured on iMatrix-511. Proteome analysis showed that PI3K signaling was enhanced and cytoskeletal actin was activated in iPSCs cultured on iMatrix-511. In conclusion, laminin-511 strongly activated the cell survival by promoting α6ß1 integrin-Fyn-RhoA-ROCK signaling in hiPSCs.

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.

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.

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.

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.

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. Hank's 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.

A Liquid Chromatography with Tandem Mass Spectrometry-Based Proteomic Analysis of the Proteins Secreted by Human Adipose-Derived Mesenchymal Stem Cells.

Liquid chromatography using a tandem mass spectrometer (LC-MS/MS) is a method of proteomic analysis. A shotgun analysis by LC-MS/MS comprehensively identifies proteins from tissues and cells with high resolution. The hepatic function of mice with acute hepatitis following the intraperitoneal administration of CCL4 was improved by the tail vein administration of the culture conditional medium (CM) of human mesenchymal stem cells from adipose tissue (hMSC-AT). In this study, a secreted protein expression analysis of hMSC-AT was performed using LC-MS/MS; 128 proteins were identified. LC-MS/MS showed that 106 new functional proteins and 22 proteins (FINC, PAI1, POSTN, PGS2, TIMP1, AMPN, CFAH, VIME, PEDF, SPRC, LEG1, ITGBL, ENOA, CSPG2, CLUS, IBP4, IBP7, PGS1, IBP2, STC2, CTHR1, CD9) were previously reported in hMSC-AT-CMs. In addition, various proteins associated with growth (SAP, SEM7A, PTK7); immune system processes (CO1A2, CO1A1, CATB, TSP1, GAS6, PTX3, C1 S, SEM7A, G3P, PXDN, SRCRL, CD248, SPON2, ENPP2, CD109, CFAB, CATL1, MFAP5, MIF, CXCL5, ADAM9, CATK); and reproduction (MMP2, CATB, FBLN1, SAP, MFGM, GDN, CYTC) were identified in hMSC-AT-CMs. These results indicate that a comprehensive expression analysis of proteins by LC-MS/MS is useful for investigating new factors associated with cellular components, biological processes, and molecular functions.

The Healing Effect of Human Milk Fat Globule-EGF Factor 8 Protein (MFG-E8) in A Rat Model of Parkinson's Disease.

We searched for drugs that alleviate the reduction of dopaminergic neurons caused by the administration of lipopolysaccharide (LPS) to the substantia nigra of the rat brain. Human milk fat globule-EGF factor 8 protein (MFG-E8) is similar to MFG-E8-S, a short isoform, of the mouse MFG-E8. However, the function of MFG-E8-S was not clear. Rats with LPS-induced Parkinson's disease were prepared and the effects of human MFG-E8 were examined. MFG-E8 improved the significant reduction in mesencephalic dopamine neurons induced by the administration of LPS. LPS was administered to human induced pluripotent stem cell (iPSC)-derived dopaminergic neurons to induce inflammation and the effect of MFG-E8 was examined. MFG-E8 showed no toxicity toward neurons. We reanalyzed the results using public microarray data. MFG-E8 mRNA was found to be expressed in all parts of the body, particularly by adipose-derived stem cells (ADSCs). Furthermore, we investigated the culture supernatant of ADSCs using the liquid chromatography-tandem mass spectrometry (LC⁻MS/MS) analysis method and successfully identified the peptide of the MFG-E8 F5/8 type C domain. The results suggested that MFG-E8-S may have a preventive effect against Parkinson's disease.