Potency test Potency test Fractional betaFractional beta--cell Viabilitycell Viability

Cellular composition assessmentCellular 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 canbe 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 compositionwithout dissociation. However, it is very difficult to assess the viabilityof each cell subset individually in a short time before transplantation.

Assessment of Human Islet Cell Assessment of Human Islet Cell Composition and ViabilityComposition and Viabilityat the University of Miamiat the University of Miami

We have developed methods for the specific assessment of betabeta--cell contentcell content and viabilityviability 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 procedureIslets

DissociationSingle Cells

NG, TMRE, 7AAD staining

Flow Cytometer

Beta cell fractional viabilityBeta cell fractional viability

Immunostaining

Fixation

Laser Scanning Cytometer

Cellular compositionCellular composition

Assessment of Islet PreparationAssessment of Islet Preparation

Hum

an p

ancr

eas

Hum

an is

lets

Mou

se p

ancr

eas

Beta (insulin)Alpha (Glucagon)

Nuclei (DAPI))

Laser Scanning CytometerLaser Scanning Cytometer

The instrument consists of a base unit containing fluorescent microscope, optics/electronics unit coupled to an argon, HeNeand 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.

00 2020 4040 6060 8080 100100(%)(%)DTZ purityDTZ purity

1010

2020

3030

4040

5050(%)

Bet

a ce

ll %

Bet

a ce

ll %

ß-cell percentage and purity

00

55

1010

1515

2020(%)(%)

PPPP--cellcellδδ--cellcell00

2020

4040

6060

8080(%)(%)

ββ--cellcell αα--cellcell

Cellular Composition in IsletCellular 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 nonbetween dissociated and non--dissociated islet cells dissociated islet cells

Glu

c ago

n

PP

Somatostatin

Insu

l in

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

57β (%)

103432Nashville

10335Miamiδ (%)α (%)#

Whole Islet

57

54β (%)

102369Edmonton

113563Miamiδ (%)α (%)#

Dissociated Islet

Experimental procedureIslets

DissociationSingle Cells

NG, TMRE, 7AAD staining

Flow Cytometer

Beta cell fractional viabilityBeta cell fractional viability

Immunostaining

Fixation

Laser Scanning Cytometer

Cellular compositionCellular composition

Fractional beta cell viability assessmentFractional beta cell viability assessment

7AAD

dead cells (%)dead cells (%)

Beta cell viability (%)Beta cell viability (%)

TMRE

TMRE

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

NG

DNADNA--binding dyebinding dye

ZincZinc--binding dyebinding dye

Mitochondorial Mitochondorial Membrane Membrane PotentialPotential

indicating dyeindicating dye

Beta cell content (%)Beta cell content (%)

7-AAD

Newport green

TMRE

Rel

ativ

e ce

ll nu

mbe

r

NonNon--treatedtreated

6 hours6 hourspelletpellet

18 hours18 hourspelletpellet

TMRE7AAD

Living cellLiving cell BetaBeta--cell viabilitycell viability In vivo FunctionIn vivo Function

Days

Comparative analysis of cell viability, β-cell apoptosis and in vivo islet function.

1257

2971

73 49

Control

SNP 0.5 mM

Control

H2O2 200 μM

Control

IL-1β

Control

IL-1βTNF-αIFN-γ

TMRE

Beta cells

Rel

ativ

e ce

ll nu

mbe

r

Beta cells

Rel

ativ

e ce

ll nu

mbe

r

TMRE

Beta cells

Rel

ativ

e ce

ll nu

mbe

r

TMRE

TMRE

Beta cells

Rel

ativ

e ce

ll nu

mbe

r

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

ββ--cell viability (%)cell viability (%)

ββ --ce

ll ce

ll co

nten

tco

nten

t (%

)(%

)

Predictive Value of β-cell Content/Viability on in vivo Islet Function.

0

50

100

Reversal rate (%)Reversal rate (%)33%33%

82%82%

100%100%

0%0%

n=23n=23

n=28n=28

n=33n=33

n=18n=18

90.1%

Beta cell contentBeta cell contentx viabilityx viability 0.10 0.20 0.30 0.40 0.50 0.60

NonNon--ReversalReversal

ReversalReversal

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

Preparation II Preparation IIIPreparation I

6761 50

BetaBeta--cell cell contentcontent

80 49 82

42 32 22

Rel

ativ

e ce

ll nu

mbe

r

TMRE

NG

TMRE

WholeWholePreparationPreparation

BetaBeta--cell cell ViabilityViability

Preparation 1Preparation 1 Preparation 2Preparation 2450,000 IEQ 380,000 IEQPrePre--cultureculture

7-AAD

NG

TMRE

23

32

49

11

42

80

170,000 IEQ 380,000 IEQPostPost--cultureculture

TransplantedNot 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