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

Potency test Potency test

Fractional beta Fractional beta-

• cell Viability

cell Viability 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

• f 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 beta-

• cell content

cell content and viability viability in human islets based on:

• 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

NG, TMRE, 7AAD staining

Flow Cytometer

Beta cell fractional viability Beta cell fractional viability Immunostaining Fixation

Laser Scanning Cytometer

Cellular composition Cellular composition

Assessment of Islet Preparation Assessment of Islet Preparation

Human pancreas

Human islets

Mouse pancreas

Beta (insulin) Alpha (Glucagon) Nuclei (DAPI))

Laser Scanning Cytometer Laser Scanning Cytometer

The instrument consists of a base unit containing fluorescent microscope,

• ptics/electronics unit

coupled to an argon, HeNe and Violet laser, a computer- controlled motorized stage. The LSC is an instrument designed to enable fluoresence-based quantitative measurements on tissue sections or other cellular preparations at single-cell level.

20 20 40 40 60 60 80 80 100 100(%) (%) DTZ purity DTZ purity 10 10 20 20 30 30 40 40 50 50 (%) Beta cell % Beta cell %

ß-cell percentage and purity

5 5 10 10 15 15 20 20 (%) (%)

PP PP-

• cell

cell δ δ-

• cell

cell

20 20 40 40 60 60 80 80 (%) (%)

β β-

• cell

cell α α-

• cell

cell

Cellular Composition in Islet Cellular Composition in Islet

β+α+δ+ PP-cell (%) β-cell (%) or α, δ, PP

Comparison of cellular composition assessment Comparison of cellular composition assessment between dissociated and non between dissociated and non-

• dissociated islet cells

dissociated islet cells

Glucagon PP Somatostatin Insulin Beta- (insulin) Alpha (Glucagon) Nuclei (DAPI)

β -cells α -cells δ -cells

Non-dissociated islet

57.4±19.6% 32.8±16.3% 9.8±2.9%

Dissociated islet cells

54.4±11.4% 34.7±12.5% 10.9±4.6%

No statistically significant differences are observed.

Human Islet Cellular Composition

54 57

β (%)

10 34 32

Nashville

10 33 5

Miami δ (%) α (%) # Whole Islet

57 54

β (%)

10 23 69

Edmonton

11 35 63

Miami δ (%) α (%) # Dissociated Islet

Experimental procedure

Islets

Dissociation Single Cells

NG, TMRE, 7AAD staining

Flow Cytometer

Beta cell fractional viability Beta cell fractional viability Immunostaining Fixation

Laser Scanning Cytometer

Cellular composition Cellular composition

Fractional beta cell viability assessment Fractional beta cell viability assessment

7AAD

dead cells (%) dead cells (%) Beta cell viability (%) Beta cell viability (%)

TMRE TMRE

Other cell viability (%) Other cell viability (%)

NG

DNA DNA-

• binding dye

binding dye Zinc Zinc-

• binding dye

binding dye Mitochondorial Mitochondorial Membrane Membrane Potential Potential indicating dye indicating dye Beta cell content (%) Beta cell content (%) 7-AAD Newport green TMRE

Relative cell number

Non Non-

• treated

treated 6 hours 6 hours pellet pellet 18 hours 18 hours pellet pellet TMRE 7AAD Living cell Living cell Beta Beta-

• cell viability

cell viability In vivo Function In vivo Function Days

Comparative analysis of cell viability, β-cell apoptosis and in vivo islet function. 12 57 29 71 73 49

Control SNP 0.5 mM Control H2O2 200 μM Control IL-1β Control IL-1β TNF-α IFN-γ TMRE Beta cells

Relative cell number

Beta cells

Relative cell number

TMRE Beta cells

Relative cell number

TMRE TMRE Beta cells

Relative cell number

48 23 43 23 80 56 43 65

Analysis of β-cell Fractional Viability After Noxious Stimuli in vitro

Correlation of β-cell content and viability with in vivo Islet Function

Reversal Non-Reversal 2,000IEQ/mouse

20 30 40 50 60 70 80 20 30 40 50 60 70 80 90 100 β β-

• cell viability (%)

cell viability (%)

β β-

• cell

cell content

content (%)

(%)

Predictive Value of β-cell Content/Viability

• n in vivo Islet Function.

50 100

Reversal rate (%) Reversal rate (%)

33% 33% 82% 82% 100% 100% 0% 0% n=23 n=23 n=28 n=28 n=33 n=33 n=18 n=18

90.1%

Beta cell content Beta cell content x viability x viability

0.10 0.20 0.30 0.40 0.50 0.60

Non Non-

• Reversal

Reversal Reversal Reversal

β-cell-specific analysis of viability/apoptosis in human islet preparations

Preparation II Preparation III Preparation I

67 61 50

Beta Beta-

• cell

cell content content

80 49 82 42 32 22

Relative cell number

TMRE NG TMRE

Whole Whole Preparation Preparation Beta Beta-

• cell

cell Viability Viability

Preparation 1 Preparation 1 Preparation 2 Preparation 2

450,000 IEQ 380,000 IEQ

Pre Pre-

• culture

culture

7-AAD NG TMRE 23 32 49 11 42 80 170,000 IEQ 380,000 IEQ

Post Post-

• culture

culture

Transplanted

Not Transplanted

Islet Dissociation

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?

• Does it affect the cellular composition?

Does it affect the cellular composition? 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