Potency test Potency test Fractional beta- -cell Viability cell - PowerPoint PPT Presentation

Potency test Potency test Fractional beta- -cell Viability cell Viability Fractional beta Cellular composition assessment Cellular composition assessment Hirohito Ichii, M.D., Ph.D. Hirohito Ichii, M.D., Ph.D.

Why is difficult to assess islet potency? Why is difficult to assess islet potency? Current methodologies for the evaluation of islet cell viability are largely based on tests that rely on DNA- binding dyes. While these tests identify cells that have lost selective membrane permeability, they do not allow us to recognize apoptotic cells, which do not yet stain with DNA-binding dyes. Current methods of analysis do not discriminate between cell subsets in the preparation and they do not allow us to selectively define beta-cell viability.

Why is difficult to assess islet potency? Why is difficult to assess islet potency? 1. In clinical islet transplantation, 30-90% pure islets assessed by DTZ can be transplanted. We transplant not only islet cells, but also acinar and ductal cells. Average purity is 50-60% in Miami Beta 25% Alpha 15% Acinar 30% Ductal 15% 2. Each preparation has different cellular composition, different viability, and sensitivity to noxious stimuli. 3. It is not impossible to evaluate islet viability and cellular composition without dissociation. However, it is very difficult to assess the viability of each cell subset individually in a short time before transplantation.

Assessment of Human Islet Cell Assessment of Human Islet Cell Composition and Viability Composition and Viability at the University of Miami at the University of Miami We have developed methods for the specific assessment of beta- -cell content cell content and viability viability in human islets based on: beta • cellular composition analysis ( Laser Scanning Cytometry ; LSC) and • identification of beta cell-specific apoptosis at the mitochondrial level ( Flow Cytometry ) We have validated our methods using in vivo assessment of islet potency (transplantation into immunodeficient mice ). H. Ichii et al. Am J Transplant 2005; 5: 1635-45

Experimental procedure Islets Dissociation Single Cells Fixation NG, TMRE, 7AAD staining Immunostaining Flow Cytometer Laser Scanning Cytometer Cellular composition Cellular composition Beta cell fractional viability Beta cell fractional viability

Assessment of Islet Preparation Assessment of Islet Preparation Alpha (Glucagon) Human pancreas Beta (insulin) Nuclei (DAPI)) Human islets Mouse pancreas

Laser Scanning Cytometer Laser Scanning Cytometer The LSC is an instrument designed to enable fluoresence-based quantitative measurements on tissue sections or other cellular preparations at single-cell level. The instrument consists of a base unit containing fluorescent microscope, optics/electronics unit coupled to an argon, HeNe and Violet laser, a computer- controlled motorized stage.

ß-cell percentage and purity Cellular Composition in Islet Cellular Composition in Islet (%) (%) (%) (%) (%) 50 50 80 80 20 20 Beta cell % Beta cell % 40 40 60 60 15 15 30 30 40 40 10 10 20 20 20 5 20 5 10 10 0 0 0 0 β - cell α α - β δ - δ 80 (%) 0 20 40 60 80 100(%) -cell -cell cell 0 20 40 60 100 -cell cell PP- -cell cell PP DTZ purity DTZ purity β -cell (%) or α , δ , PP β + α + δ + PP-cell (%)

Comparison of cellular composition assessment Comparison of cellular composition assessment between dissociated and non- -dissociated islet cells dissociated islet cells between dissociated and non Insulin Somatostatin Glucagon Alpha (Glucagon) Beta- (insulin) Nuclei (DAPI) PP β -cells α -cells δ -cells 57.4 ± 19.6% 32.8 ± 16.3% 9.8 ± 2.9% Non-dissociated islet 54.4 ± 11.4% 34.7 ± 12.5% 10.9 ± 4.6% Dissociated islet cells No statistically significant differences are observed.

Human Islet Cellular Composition Whole Islet β (%) α (%) δ (%) # 5 57 33 10 Miami 32 54 34 10 Nashville Dissociated Islet β (%) α (%) δ (%) # 63 54 35 11 Miami 69 57 23 10 Edmonton

Experimental procedure Islets Dissociation Single Cells Fixation NG, TMRE, 7AAD staining Immunostaining Flow Cytometer Laser Scanning Cytometer Cellular composition Cellular composition Beta cell fractional viability Beta cell fractional viability

Fractional beta cell viability assessment Fractional beta cell viability assessment DNA- DNA -binding dye binding dye dead cells (%) dead cells (%) 7-AAD 7AAD Beta cell content (%) Zinc- -binding dye binding dye Beta cell content (%) Zinc Newport green NG Beta cell viability (%) Beta cell viability (%) Mitochondorial Mitochondorial Membrane Membrane TMRE Potential Potential indicating dye indicating dye Other cell viability (%) Other cell viability (%) TMRE TMRE

Comparative analysis of cell viability, β -cell apoptosis and in vivo islet function. Beta- -cell viability cell viability In vivo Function In vivo Function Living cell Beta Living cell 73 Non- - Non 49 treated treated Relative cell number 71 29 6 hours 6 hours pellet pellet 12 57 18 hours 18 hours pellet pellet 7AAD TMRE Days

Analysis of β -cell Fractional Viability After Noxious Stimuli in vitro Beta cells Beta cells 43 65 Relative cell number Relative cell number Control Control 43 IL-1 β 23 SNP 0.5 mM TMRE TMRE Beta cells Beta cells 48 80 Relative cell number Relative cell number Control Control IL-1 β H 2 O 2 200 μ M 23 56 TNF- α IFN- γ TMRE TMRE

Correlation of β -cell content and viability with in vivo Islet Function 2,000IEQ/mouse 80 70 (%) content (%) 60 Reversal cell content Non-Reversal 50 - cell 40 β - β 30 20 20 30 40 50 60 70 80 90 100 β - β -cell viability (%) cell viability (%)

Predictive Value of β -cell Content/Viability on in vivo Islet Function. n=23 n=23 100% 100% 100 n=28 n=28 82% 82% Reversal rate (%) n=33 Reversal rate (%) n=33 50 33% 33% n=18 n=18 0% 0% 0 Non- -Reversal Reversal Non Reversal Reversal Beta cell content Beta cell content 0.10 0.20 0.30 0.40 0.50 0.60 x viability x viability 90.1%

β -cell-specific analysis of viability/apoptosis in human islet preparations Preparation I Preparation II Preparation III 61 Whole 67 50 Whole Preparation Preparation Relative cell number TMRE 42 32 22 Beta- -cell cell Beta content content NG 82 Beta- -cell cell 80 49 Beta Viability Viability TMRE

Preparation 1 Preparation 2 Preparation 2 Preparation 1 Pre- -culture culture Pre 450,000 IEQ 380,000 IEQ 7-AAD 23 11 NG 32 42 TMRE 49 80 Post- -culture culture 170,000 IEQ 380,000 IEQ Post Transplanted Not Transplanted

Islet Dissociation • Does it affect the cellular composition? Does it affect the cellular composition? NO. The cellular composition of dissociated islets is comparable to that of whole islets by IHC. If cell loss occurs, this is not selective for any specific cell subset and the overall proportion is maintained. • Does it affect cell viability? Does it affect cell viability? • MAYBE. However, it may not matter! Good correlation between Viable Beta Index and in vivo function suggests that this approach can PREDICT the potency of isolated islets and that it is REPRESENTATIVE of the quality of the preparation

Ongoing Studies • Evaluation of the correlation between Viable Beta-Index and islet engraftment in the clinical setting • Develop a method to assess the viability of multiple islet cell subsets (i.e., ductal, alpha…) • Identify more sensitive marker than MMP for islet cell potency assessment