Pancreatic human islets and insulin-producing cells derived from embryonic stem cells are rapidly identified by a newly developed Dithizone

Khiatah, Bashar; Qi, Meirigeng; Wu, Youjun; Chen, Kuan-Tsen; Perez, Rachel; Valiente, Luis; Omori, Keiko; Isenberg, Jeffrey S.; Kandeel, Fouad; Yee, Jiing‐Kuan; Al-Abdullah, Ismail H.

doi:10.1038/s41598-019-45678-y

Cited by 12 publications

(11 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Islet viability was measured in duplicate using acridine orange (AO) and propidium iodide (PI) staining on day 1 of culture following established protocols 27 . Islet viability was significantly different between Collagenase P (82.7 ± 1.3%), Collagenase AF‐1 GMP grade (89.3 ± 1.21%), but we observed no significant difference in Collagenase AF‐1 GMP grade with Liberase MTF C/T GMP grade islet isolations (85.3 ± 3.42%) (Table 2)( P > .05).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Comparison of islet isolation result and clinical applicability according to GMP‐grade collagenase enzyme blend in adult porcine islet isolation and culture

Kwak

Park

Shim

et al. 2021

Xenotransplantation

View full text Add to dashboard Cite

Background Porcine islet xenotransplantation is a promising treatment for type 1 diabetes as an alternative to human pancreatic islet transplantation and long‐term insulin therapy. Several research groups have explored porcine islets as an alternative to the inconsistent and chronic shortage of pancreases from human organ donors. Studies have confirmed successful transplant of porcine islets into non‐human primate models of diabetes; however, in most cases, they require more than one adult porcine donor to achieve sufficient viable islet mass for sustained function. The importance of GMP‐grade reagents includes the following: specific enzymes utilized in the pancreatic isolation process were identified as a key factor in successful human clinical islet transplantation trials using cadaveric islets. As xenotransplantation clinical research progresses, isolation reagents and digestion enzymes play a key role in the consistency of the product and ultimately the outcome of the islet xenotransplant. In this study, we evaluated several commercially available enzyme blends that have been used for islet isolation. We evaluated their impact on islet isolation yield and subsequent islet function as part of our plan to bring xenotransplantation into clinical xenotransplantation trials. Methods Adult porcine islets were isolated from 16 to 17‐month‐old Yucatan miniature pigs following standard rapid procurement. Pigs weighed on average 48.71 ± 2.85 kg, and the produced pancreases were 39.51 ± 1.80 grams (mean ± SEM). After ductal cannulation, we evaluated both GMP‐grade enzymes (Collagenase AF‐1 GMP grade and Liberase MTF C/T GMP grade) and compared with standard non‐GMP enzyme blend (Collagenase P). Islet quality control assessments including islet yield, islet size (IEQ), membrane integrity (acridine orange/propidium iodide), and functional viability (GSIS) were evaluated in triplicate on day 1 post‐islet isolation culture. Results Islet yield was highest in the group of adult pigs where Collagenase AF‐1 GMP grade was utilized. The mean islet yield was 16 586 ± 1391 IEQ/g vs 8302 ± 986 IEQ/g from pancreases isolated using unpurified crude Collagenase P. The mean islet size was higher in Collagenase AF‐1 GMP grade with neutral protease than in Collagenase P and Liberase MTF C/T GMP grade. We observed no significant difference between the experimental groups, but in vitro islet function after overnight tissue culture was significantly higher in Collagenase AF‐1 GMP grade with neutral protease and Liberase MTF C/T GMP grade than the crude control enzyme group. As expected, the GMP‐grade enzyme has significantly lower endotoxin levels than the crude control enzyme group when measured. Conclusions This study validates the importance of using specifically blended GMP grade for adult pig islet isolation for xenotransplantation trials and the ability to isolate a sufficient number of viable islets from one adult pig to provide a sufficient number for islets for a clinical islet transplantation. GMP‐grade enzymes are highl...

show abstract

Section: Resultsmentioning

confidence: 99%

“…Aliquots were made before and after purification and 24‐hour tissue culture. The islets were counted using an islet number and islet equivalent number (IEQ) after staining with DTZ (Sigma‐Aldrich) 27 . Islet sizes were determined by calculating the average size of islets.…”

Section: Methodsmentioning

confidence: 99%

Comparison of islet isolation result and clinical applicability according to GMP‐grade collagenase enzyme blend in adult porcine islet isolation and culture

Kwak

Park

Shim

et al. 2021

Xenotransplantation

View full text Add to dashboard Cite

show abstract

“…Accordingly, the multiparametric islet assessment showed advantage over other assays including stimulation index in OCR assay and semi-automated viability for assessing islet transplantation outcomes. The multiparametric assessment requires only DTZ staining of islets 31 and should be performed immediately after islet isolation using light microscopy 25 . As the technical requirements for morphologic assessment are modest, this method can be applied widely.…”

Section: Discussionmentioning

confidence: 99%

“…Isolated islets were purified with continuous density gradient centrifugation using a COBE 2991 cell processor system (Terumo BCT, Lakewood, CO, USA) 24 . Islet fractions were pooled according to purity as determined by Dithizone (DTZ) staining (iDTZ, Gemini Bio-products, CA, USA) 25 . Islet particulate number (IPN) was determined, and total islet counts were then converted into islet equivalent (IEQ) which represent islets of 150 µm diameter IEQ 26 .…”

Section: Methodsmentioning

confidence: 99%

A Multiparametric Assessment of Human Islets Predicts Transplant Outcomes in Diabetic Mice

Komatsu

Gonzalez

et al. 2021

Cell Transplant

Self Cite

View full text Add to dashboard Cite

Prior to transplantation into individuals with type 1 diabetes, in vitro assays are used to evaluate the quality, function and survival of isolated human islets. In addition to the assessments of these parameters in islet, they can be evaluated by multiparametric morphological scoring (0–10 points) and grading (A, B, C, D, and F) based on islet characteristics (shape, border, integrity, single cells, and diameter). However, correlation between the multiparametric assessment and transplantation outcome has not been fully elucidated. In this study, 55 human islet isolations were scored using this multiparametric assessment. The results were correlated with outcomes after transplantation into immunodeficient diabetic mice. In addition, the multiparametric assessment was compared with oxygen consumption rate of isolated islets as a potential prediction factor for successful transplantations. All islet batches were assessed and found to score: 9 points ( n = 18, Grade A), 8 points ( n = 19, Grade B), and 7 points ( n = 18, Grade B). Islets that scored 9 (Grade A), scored 8 (Grade B) and scored 7 (Grade B) were transplanted into NOD/SCID mice and reversed diabetes in 81.2%, 59.4%, and 33.3% of animals, respectively ( P < 0.0001). Islet scoring and grading correlated well with glycemic control post-transplantation ( P < 0.0001) and reversal rate of diabetes ( P < 0.05). Notably, islet scoring and grading showed stronger correlation with transplantation outcome compared to oxygen consumption rate. Taken together, a multiparametric assessment of isolated human islets was highly predictive of transplantation outcome in diabetic mice.

show abstract

“…Islets were cultured in Connaught Medical Research Laboratories (CMRL)-1066 supplemented media for 24–48 hours prior to assessment. Islet purity was determined by estimating the proportion of dithizone (DTZ)-containing (red-stained) islets to all tissues present (islets, acinar, and ductal cells) as described [ 28 , 29 ]. In each isolation, duplicate islet samples were taken to determine islet size as described [ 29 ].…”

Section: Methodsmentioning

confidence: 99%

Chronic marijuana usage by human pancreas donors is associated with impaired islet function

Kaddis²,

Chen³

et al. 2021

PLoS ONE

Self Cite

View full text Add to dashboard Cite

We investigated the effect of chronic marijuana use, defined as 4 times weekly for more than 3 years, on human pancreatic islets. Pancreata from deceased donors who chronically used marijuana were compared to those from age, sex and ethnicity matched non-users. The islets from marijuana-users displayed reduced insulin secretion as compared to islets from non-users upon stimulation with high glucose (AUC, 3.41 ± 0.62 versus 5.14 ±0.47, p<0.05) and high glucose plus KCl (AUC, 4.48 ± 0.41 versus 7.69 ± 0.58, p<0.001). When human islets from chronic marijuana-users were transplanted into diabetic mice, the mean reversal rate of diabetes was 35% versus 77% in animals receiving islets from non-users (p<0.01). Immunofluorescent staining for cannabinoid receptor type 1 (CB1R) was shown to be colocalized with insulin and enhanced significantly in beta cells from marijuana-users vs. non-users (CB1R intensity/islet area, 14.95 ± 2.71 vs. 3.23 ± 0.87, p<0.001). In contrast, CB1R expression was not co-localized with glucagon or somatostatin. Furthermore, isolated islets from chronic marijuana-users appeared hypertrophic. In conclusion, excessive marijuana use affects islet endocrine phenotype and function in vitro and in vivo. Given the increasing use of marijuana, our results underline the importance of including lifestyle when evaluating human islets for transplantation or research.

show abstract

Pancreatic human islets and insulin-producing cells derived from embryonic stem cells are rapidly identified by a newly developed Dithizone

Cited by 12 publications

References 32 publications

Comparison of islet isolation result and clinical applicability according to GMP‐grade collagenase enzyme blend in adult porcine islet isolation and culture

Comparison of islet isolation result and clinical applicability according to GMP‐grade collagenase enzyme blend in adult porcine islet isolation and culture

A Multiparametric Assessment of Human Islets Predicts Transplant Outcomes in Diabetic Mice

Chronic marijuana usage by human pancreas donors is associated with impaired islet function

Contact Info

Product

Resources

About