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Mouse Models for Studying Human Mouse Models for Studying Human Islet Transplantation Islet Transplantation

Ronald G. Gill, Joshua Ronald G. Gill, Joshua Beilke Beilke, Nathan , Nathan Kuhl Kuhl, Michelle , Michelle Kerklo Kerklo, , and Mark M. and Mark M. Nicolls Nicolls Barbara Davis Center for Childhood Diabetes Barbara Davis Center for Childhood Diabetes University of Colorado Health Science Center University of Colorado Health Science Center

Can a mouse model form an in vivo ‘potency’ assay?

Mouse Models for Assessing Human Islet Function

• Immune

Immune-

• suppressed wild

suppressed wild-

• type mice (e.g. anti

type mice (e.g. anti-

• CD4)

CD4)

• T cell

T cell-

• deficient nude (

deficient nude (nu/nu nu/nu) mice ) mice

• Severe

Severe-

• combined immune

combined immune-

• deficient (

deficient (SCID SCID) )

• Recombinase

Recombinase activating gene 1,2 activating gene 1,2-

• deficient (

deficient (Rag Rag-

• /

/-

• )

)

Insulin akita Mutation

• Missense

Missense mutation (Cys96Tyr) in Insulin 2 (Ins 2) gene mutation (Cys96Tyr) in Insulin 2 (Ins 2) gene

• Prevents appropriate folding of pro

Prevents appropriate folding of pro-

• insulin

insulin

• Autosomal

Autosomal-

• dominant (chromosome 7)

dominant (chromosome 7)

• Functions as a

Functions as a ‘ ‘dominant dominant-

• negative

negative’ ’

• Durable and irreversible hyperglycemia (>450

Durable and irreversible hyperglycemia (>450-

• 500mg/dl)

500mg/dl)

• Males more severe than females

Males more severe than females

10 15 20 25 30 35

1 2 3 4 5 AK-1 AK-2 AK-3 AK-4 AK-5

10 15 20 25 30 35

1 2 3 4 5 SZ-1 SZ-2 SZ-3 SZ-4 SZ-5 18 20 22 24 26

)

1 5 AK-1 AK-2 AK-3 AK-4 AK-5

18 20 22 24 26 1 5

SZ-1 SZ-2 SZ-3 SZ-4 SZ-5

Day Animal Streptozotocin 5 Day Weight Variability SZ-1 SZ-2 SZ-3 SZ-4 SZ-5

Rag1-/- akita Blood Glucose Rag1-/- akita Weight Change SZ Blood Glucose SZ Weight Change

Utility of Utility of akita akita mice as islet transplant mice as islet transplant recipients recipients

Mathews, CE et al. Transplantation. 73:1333, 2002

Islet Function in C57Bl/6akita Mice

Donor Donor n n Graft Function (Days) Graft Function (Days) ISOGRAFTS ISOGRAFTS (C57Bl/6) (C57Bl/6)

8 8 > 100 (x8) > 100 (x8)

ALLOGRATS ALLOGRATS (BALB/c) (BALB/c)

3 3 9, 9, 12 9, 9, 12

Islet Transplantation in B6 Rag1-/-akita Mice

Donor Islets Transplant 2000 IEQ under the kidney capsule of B6 Rag1-/-akita

• Monitor blood glucose
• Nephrectomy – immunohistochemistry

Correlation between in vitro assays and in vivo function in Rag1-/-akita mice Purity Viability S.I. In vivo function (>30 days) 60 60 1.6 Yes 90 80 3.0 Yes 80 75 5.2 Yes 85 72 2.4 Yes 90 70 2.4 Yes 40 60 0.8 No 60 77 0.2 No 60 60 0.6 No 60 60 2.1 No 75 75 4.0 No 50 60 1.1 Yes

Immune Injury Non-Immune Injury Islets

Isolated Islets Highly Express Proteins Isolated Islets Highly Express Proteins Associated with ER Associated with ER-

• Distress

Distress

Nicolls, MR et al. J. Proteome. Res. 2:199, 2003

Function of Islet Grafts in Rag1-/- Recipients

Graft Function (days) Graft Function (days) Donor Donor n n Mouse Mouse 8 8 >100 (x 8) >100 (x 8) Rat (WF) Rat (WF) 9 9 Porcine Porcine Human Human 12 12 17 17 >100 (x 9) >100 (x 9) >100 (x 12) >100 (x 12) 47,65,74,91,94 47,65,74,91,94 >100 (x12) >100 (x12)

Spontaneous Failure of Human Islets Rag1-/-akita Mice

Blood Glucose (mM)

Human Islet Tp#1 Human Islet Tp#2 Normoglycemia

30 20 10 50 100 Day Post Transplantation

Pathology of Failed Human Islets Pathology of Failed Human Islets (day 70) (day 70)

Amyloid Amyloid

( (Thyoflavin Thyoflavin S) S)

Fibrosis Fibrosis

(Tri (Tri-

• Chrome)

Chrome)

Failure of Failure of hIAPP hIAPP Transgenic Mouse Islets Transgenic Mouse Islets

50 40 30 20 10

• 10

100 200 300 400 500 600 100 tg+ 100 tg-

Day Plasma Glucose (mg/dl)

STZ Tx

δ β α β β δ α α α β β β β β β β δ α α α β

Is le t C e ll A ttritio n in T ra n s p la n ta tio n

D o n o r Is le t (p re -tra n s p la n t) D o n o r Is le t (p o s t-tra n s p la n t) 1 . M e c h a n ic a l s tre s s f ro m is le t is o la tio n , in f u s io n a n d im p la n ta tio n in e c to p ic s ite . 2 . N o n - im m u n e re s p o n s e (S p e c if ic A im 1 ) 3 . In n a te im m u n e re s p o n s e (S p e c if ic A im 2 ) 4 . A d a p tiv e im m u n e re s p o n s e a . A u to im m u n e re s p o n s e b . A llo im m u n e re s p o n s e 5 . Is le t-to x ic im m u n o s u p p re s s io n 6 . F a ilu re o f is le t re v a s c u la riz a tio n

F ig u re 1 . F a c to rs in flu e n c in g lo s s o f is le t c e lls fo llo w in g tra n s p la n ta tio n .

Summary / Conclusions Summary / Conclusions

• Spontaneously diabetic

Spontaneously diabetic akita akita mice demonstrate a mice demonstrate a stable and irreversible model of hyperglycemia stable and irreversible model of hyperglycemia

• Diabetic

Diabetic akita akita mice can be readily maintained for mice can be readily maintained for 2 2-

• 3 months prior to transplantation

3 months prior to transplantation

• Human islets can reverse diabetes in immune

Human islets can reverse diabetes in immune-

• deficient

deficient akita akita mice ( mice (Rag1 Rag1-

• /

/-

• akita

akita)

)

• Human islets can spontaneously fail over time

Human islets can spontaneously fail over time from non from non-

• immune factors (metabolic distress?)

immune factors (metabolic distress?)

Collaborators Collaborators

Mark Mark Nicolls Nicolls Michelle Michelle Kerklo Kerklo Gina Gina Rayat Rayat Josh Josh Beilke Beilke Nathan Nathan Kuhl Kuhl