Evaluation of Islet Purification Methods for Making a Continuous Density Gradient and Loading Tissue.

Islet purification is one of the most important steps of 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 porcine pancreas. In this study, we evaluated the methods for making a continuous density gradient and loading tissue. One method involved loading digested tissue on top of a continuous density gradient (top loading). The other method involved mixing digested tissue with low-density solution and then making a continuous gradient (mixed loading). There were no significant differences between the 2 purification methods in terms of the islet yield, rate of viability or purity, score, or in the stimulation index after purification. Furthermore, there were no marked differences in the attainability or suitability of posttransplantation normoglycemia. Our study shows the equivalency of these 2 methods of islet purification.

The Development of Cancer through the Transient Overexpression of Reprogramming Factors.

Although induced pluripotent stem (iPS) cells have significant implications for overcoming most of the ethical issues associated with embryonic stem cells, several issues related to the use of iPS cells in clinical applications remain unresolved, including the issue of teratoma formation. We previously reported that the induction of induced tissue-specific stem (iTS) cells from the pancreas (iTS-P) or liver (iTS-L) by the transient overexpression of reprogramming factors, combined with tissue-specific selection and the generation of iTS cells, could have important implications for the clinical application of stem cells. At the same time, we also generated "induced fibroblast-like (iF) cells" that were capable of self-renewal, which had a similar morphology to fibroblast cells. In this study, we evaluated iF cells. iF cells are unlikely to show adipogenic/osteogenic differentiation. Moreover, iF cells have the ability to form tumors and behave similarly to pancreatic cancer cells. The technology used in the generation of iPS/iTS cells is also associated with the risk of generating cancer-like cells.

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.

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.

Evaluation of Serum-Free, Xeno-Free Cryopreservation Solutions for Human Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (ASCs) have the potential to differentiate into cells of mesodermal origin, such as osteoblasts, adipocytes, myocytes, and chondrocytes, and cryopreservation is currently performed as a routine method for preserving ASCs to safely acquire large numbers of cells. For clinical application of ASCs, serum-free, xeno-free cryopreservation solutions should be used. This study determined the viability and adipo-osteogenic potential of cryopreserved ASCs using four cryopreservation solutions: 10% DMSO, Cell Banker 2 (serum free), Stem Cell Banker (=Cell Banker 3: serum free, xeno free), and TC protector (serum free, xeno free). The viability of the cryopreserved ASCs was over 80% with all cryopreservation solutions. No difference in the adipo-osteogenic potential was found between the cells that did or did not undergo cryopreservation in these cryopreservation solutions. These data suggest that Cell Banker 3 and TC protector are comparable with 10% DMSO and Cell Banker 2 for ASCs, and cryopreserved as well as noncryopreserved ASCs could be applied for regenerative medicine.

The Evaluation of Islet Purification Methods That Use Large Bottles to Create a Continuous Density Gradient.

Islet purification is one of the most important steps of islet isolation for pancreatic islet transplantation. The most common method of islet purification is density gradient centrifugation using a COBE 2991 cell processor. However, this method can damage islets mechanically through its high shearing force. We recently reported that a new purification method using large plastic bottles effectively achieves a high yield of islets from the porcine pancreas. In the present study, we evaluated the methods of making a continuous density gradient. The gradient was produced with a gradient maker and two types of candy cane-shaped stainless steel pipes. One method was to use a "bent-tipped" stainless steel pipe and to load from a high-density solution to a low-density solution, uploading the stainless steel pipe. The other method was to use a regular stainless steel pipe and to load from a low-density solution to a high-density solution, leaving the stainless steel pipe in place. There were no significant differences between the two solutions in terms of the islet yield, rate of viability or purity, score, or the stimulation index after purification. Furthermore, there were no differences in the attainability or suitability of posttransplantation normoglycemia. Our study shows the equivalency of these two methods of islet purification.

Comparison of Purification Solutions With Different Osmolality for Porcine Islet Purification.

The osmolality of the purification solution is one of the most critical variables in human islet purification during islet isolation. We previously reported the effectiveness of a combined continuous density/osmolality gradient for the supplemental purification of human islets. We herein applied a combined continuous density/osmolality gradient for regular purification. The islets were purified with a continuous density gradient without osmolality preparation [continuous density/normal osmolality (CD/NO)] or continuous density/osmolality solution with osmolality preparation by 10× Hank's balanced salt solution (HBSS) [continuous density/continuous osmolality (CD/CO)]. The osmolality of the low-density solution was 400 mOsm/kg in both groups and that of the high-density solution was 410 mOsm/kg in the CD/NO group and 500 mOsm/kg in the CD/CO group. Unexpectedly, we noted no significant differences between the two solutions in terms of the islet yield, rate of viability and purity, score, stimulation index, or the attainability and suitability of posttransplantation normoglycemia. Despite reports that the endocrine and exocrine tissues of pancreata have distinct osmotic sensitivities and that high-osmolality solutions result in greater purification efficiency, the isolation and transplant outcomes did not markedly differ between the two purification solutions with different osmolalities in this study.

Potential Factors for the Differentiation of ESCs/iPSCs Into Insulin-Producing Cells.

The low efficiency of in vitro differentiation of human embryonic stem cells (ESCs) or human induced pluripotent stem cells (iPSCs) into insulin-producing cells thus creates a crucial hurdle for the clinical implementation of human pluripotent stem cells (PSCs). In this study, we investigated the key factors for the differentiation of PSCs into insulin-producing cells. We obtained microarray data of HUES8 and HUES6 from two GeneChips (GPL3921: Affymetrix HT Human Genome U133A Array, GPL570: Affymetrix Human Genome U133 Plus 2.0 Array) in a database of GEO (NCBI), since HUES8 can differentiate into pancreatic cells, while HUES6 hardly demonstrates any differentiation at all. The genes with more than fourfold higher expressions in HUES8 compared to HUES6 included RPS4Y1, DDX3Y, EIF1AY, GREM1, GATA6, and NLGN4Y. Since there were four genes, RPS4Y1, DDX3Y, EIF1AY, and NLGN4Y, on the Y chromosome and HUES8 was a male cell line and HUES6 was a female cell line, we excluded these genes in this study. On the other hand, genes with more than fourfold higher expressions in HUES6 compared to HUES8 included NLRP2, EGR1, and SMC3. We next compared iPSCs derived from pancreatic cells (PiPSCs) and iPSCs derived from fibroblasts (FiPSCs). PiPSCs differentiated into insulin-producing cells more easily than FiPSCs because of their epigenetic memory. The gene expressions of GREM1, GATA6, NLRP2, EGR1, and SMC3 in PiPSCs and FiPSCs were also investigated. The expression level of GREM1 and GATA6 in PiPSCs were higher than in FiPSCs. On the other hand, EGR1, which was lower in HUES8 than in HUES6, was predictably lower in PiPSCs than FiPSCs, while NLRP2 and SMC3 were higher in PiPSCs than FiPSCs. These data suggest that the expression of GATA6 and GREM1 and the inhibition of EGR1 may be important factors for the differentiation of PSCs into insulin-producing cells.

Cryopreservation of Adipose-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have the potential to differentiate into cells of mesodermal origin such as osteoblasts, adipocytes, myocytes, and chondrocytes. They possess an immunosuppressive effect, which makes them a viable cell population for the cell-based therapy of treatment-resistant immune diseases. Adipose-derived mesenchymal stem cells (ASCs) have been demonstrated to have the ability to acquire the properties of subcutaneous adipose tissue particularly easily, and cryopreservation is currently performed as a routine method for preserving ASCs to safely acquire large numbers of cells. However, many studies have reported that cellular activity after freezing and thawing may be affected by the solutions used for cryopreservation. Dimethyl sulfoxide (DMSO) is commonly used as a cryopreservation medium as it diffuses into the cell through the plasma membrane and protects the cells from the damage caused by freezing. As substitutes for DMSO or animal-derived serum, cell banker series, polyvinylpyrrolidone (PVP), sericin and maltose, and methyl cellulose (MC) have been investigated for their clinical applications. It is critical to develop a reliable cell cryopreservation protocol for regenerative medicine using MSCs.

Islet Culture/Preservation Before Islet Transplantation.

Although islet culture prior to transplantation provides flexibility for the evaluation of isolated islets and the pretreatment of patients, it is well known that isolated islets deteriorate rapidly in culture. Human serum albumin (HSA) is used for medium supplementation instead of fetal bovine serum (FBS), which is typically used for islet culture research, to avoid the introduction of xenogeneic materials. However, FBS contains several factors that are beneficial to islet viability and which also neutralize the endogenous pancreatic enzymes or exogenous enzymes left over from the isolation process. Several groups have reported the comparison of cultures at 22°C and 37°C. Recent studies have demonstrated the superiority of 4°C preservation to 22°C and 37°C cultures. We herein review the current research on islet culture/preservation for clinical islet transplantation.

Establishment of a pancreatic stem cell line from fibroblast-derived induced pluripotent stem cells.

BACKGROUND: For cell therapies to treat diabetes, it is important to produce a sufficient number of pancreatic endocrine cells that function similarly to primary islets. Induced pluripotent stem (iPS) cells represent a potentially unlimited source of functional pancreatic endocrine cells. However, the use of iPS cells for laboratory studies and cell-based therapies is hampered by their high tumorigenic potential and limited ability to generate pure populations of differentiated cell types in vitro. The purpose of this study was to establish a pancreatic stem cell line from iPS cells derived from mouse fibroblasts. METHODS: Mouse iPS cells were induced to differentiate into insulin-producing cells by a multi-step differentiation protocol, which was conducted as described previously with minor modifications. Selection of the pancreatic stem cell was based on morphology and Pdx1 expression. The pancreatic potential of the pancreatic stem cells was evaluated using a reverse transcription PCR, real-time PCR, immunofluorescence, and a glucose challenge test. To assess potential tumorigenicity of the pancreatic stem cells, the cells were injected into the quadriceps femoris muscle of the left hindlimb of nude mice. RESULTS: The iPS-derived pancreatic stem cells expressed the transcription factor--Pdx1--a marker of pancreatic development, and continued to divide actively beyond passage 80. Endocrine cells derived from these pancreatic stem cells expressed insulin and pancreatic genes, and they released insulin in response to glucose stimulation. Mice injected with the pancreatic stem cells did not develop tumors, in contrast to mice injected with an equal number of iPS cells. CONCLUSION: This strategy provides a new approach for generation of insulin-producing cells that is more efficient and safer than using iPS cells. We believe that this approach will help to develop a patient-specific cell transplantation therapy for diabetes in the near future.

In vivo imaging of transplanted islets labeled with a novel cationic nanoparticle.

To monitor pancreatic islet transplantation efficiency, reliable noninvasive imaging methods, such as magnetic resonance imaging (MRI) are needed. Although an efficient uptake of MRI contrast agent is required for islet cell labeling, commercially-available magnetic nanoparticles are not efficiently transduced into cells. We herein report the in vivo detection of transplanted islets labeled with a novel cationic nanoparticle that allowed for noninvasive monitoring of islet grafts in diabetic mice in real time. The positively-charged nanoparticles were transduced into a ß-cell line, MIN6 cells, and into isolated islets for 1 hr. MRI showed a marked decrease in the signal intensity on T1- and T2-weighted images at the implantation site of the labeled MIN 6 cells or islets in the left kidneys of mice. These data suggest that the novel positively-charged nanoparticle could be useful to detect and monitor islet engraftment, which would greatly aid in the clinical management of islet transplant patients.

Culture Conditions for Mouse Pancreatic Stem Cells.

Recently, mouse pancreatic stem cells have been isolated from adult mouse pancreata. However, these pancreatic stem cells could be maintained only under specific culture conditions with lot-limited fetal bovine serum (FBS). For the efficient isolation and maintenance of mouse pancreatic stem cells, it is important to identify culture conditions that can be used independent of the FBS lot. In this study, we evaluated the culture conditions required to maintain mouse pancreatic stem cells. The mouse pancreatic stem cells derived from the pancreas of a newborn mouse, HN#101, were cultured under the following conditions: 1) Dulbecco's modified Eagle's medium (DMEM) with 20% lot-limited FBS, in which mouse pancreatic stem cells could be cultured without changes in morphology and growth activity; 2) complete embryonic stem (ES) cell media; and 3) complete ES cell media on feeder layers of mitomycin C-treated STO cells, which were the same culture conditions used for mouse ES cells. Under culture conditions #1 and #3, the HN#101 cells continued to form a flat "cobblestone" monolayer and continued to divide actively beyond the population doubling level (PDL) 100 without growth inhibition, but this did not occur under culture condition #2. The gene expression profile and differentiated capacity of the HN#101 cells cultured for 2 months under culture condition #3 were similar to those of HN#101 cells at PDL 50. These data suggest that complete ES cell media on feeder layers could be useful for maintaining the undifferentiated state of pancreatic stem cells.

Isolation Efficiency of Mouse Pancreatic Stem Cells Is Age Dependent.

Mouse pancreatic stem cells have been isolated from mouse pancreata. This study evaluated the efficacy of isolating mouse pancreatic stem cells using mice of different ages. The pancreata of newborn mice, 8-week-old mice, and 24-week-old mice were harvested and digested by using collagenase. The "duct-like" cells in the digested pancreatic tissue were then inoculated into 96-well plates, cloned by limiting dilution, and cultured in DMEM with 20% FBS. Pancreatic stem cells were isolated from the pancreata of all newborn mice, while cells could only be isolated from 10% of the pancreata of 8-week-old mice and could not be isolated from the pancreata of any 24-week-old mice. These data suggest that young mice may have some pancreatic stem cells and that older mice may only have a few pancreatic stem cells. These data also indicate that it is extremely difficult to isolate pancreatic stem cells from older mice, suggesting that future research focus its efforts on finding methods of isolating pancreatic stem cells from adult mice.

Comparison of Incubation Solutions Prior to the Purification of Porcine Islet Cells.

For pancreatic islet transplantation, one of the most important steps of islet isolation is islet purification. The most common method of islet purification is density gradient centrifugation because there are differences in density between islets and acinar tissue. However, the density of islets/acinar tissue depends on several conditions, such as the incubation time before purification and the osmolality of the preincubation solution. In this study, we evaluated the impact of using two different preincubation solutions before purification. We used the University of Wisconsin (UW) solution and a new preservation solution (HN-1), which we recently developed. There were no significant differences between the two solutions in terms of the islet yield, rate of viability, and purity or stimulation index after purification. There were also no differences in the attainability and suitability of posttransplantation normoglycemia. Our study shows that the HN-1 solution is equivalent to the UW solution for preincubation before islet purification.

Comparison of New Preservation Solutions, HN-1 and University of Wisconsin Solution, in Pancreas Preservation for Porcine Islet Isolation.

For pancreatic islet transplantation, maintaining organ viability after pancreas procurement is critical and a major determinant for better graft function and survival. University of Wisconsin (UW) solution is currently the gold standard for abdominal organ preservation and the pancreas in particular. However, in the use of UW preservation solution for islet transplantation, there are disadvantages to be overcome, such as the inhibition of collagenase activity during pancreatic digestion. In this study, we compared UW solution with HN-1 solution in pancreas preservation for islet isolation. Islet yield was significantly greater in the HN-1 group than the UW group both before and after purification. In the in vitro assay, the adenosine triphosphate content in cultured islets was significantly higher in the HN-1 group than in the UW group. Furthermore, in streptozotocin-induced diabetic nude mice, the islet graft function of the HN-1 group was superior to that of the UW group. We concluded that the use of HN-1 solution is a promising approach for optimal pancreas preservation in islet transplantation.