Mouse Models for Studying Human Mouse Models for Studying Human - PowerPoint PPT Presentation

Mouse Models for Studying Human Mouse Models for Studying Human Islet Transplantation Islet Transplantation Ronald G. Gill, Joshua Beilke Beilke, Nathan , Nathan Kuhl Kuhl, Michelle , Michelle Kerklo Kerklo, , Ronald G. Gill, Joshua and Mark M. Nicolls Nicolls and Mark M. 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

Rag1 -/- akita Weight Change Rag1 -/- akita Blood Glucose AK-1 AK-1 AK-2 AK-2 AK-3 35 26 AK-3 AK-4 30 AK-5 AK-4 24 ) AK-5 25 20 22 15 20 10 1 2 3 4 5 18 1 5 SZ Blood Glucose SZ Weight Change Streptozotocin 5 Day Weight Variability SZ-1 SZ-1 SZ-2 SZ-2 SZ-3 26 SZ-3 35 SZ-4 SZ-4 30 SZ-5 24 SZ-5 25 22 20 15 20 10 SZ-5 SZ-4 1 2 3 4 5 SZ-3 18 SZ-2 Animal SZ-1 1 5 Day

Utility of akita akita mice as islet transplant mice as islet transplant Utility of recipients recipients Mathews, CE et al. Transplantation. 73:1333, 2002

Islet Function in C57Bl/6 akita Mice Donor n Graft Function (Days) Donor n Graft Function (Days) ISOGRAFTS ISOGRAFTS 8 8 > 100 (x8) > 100 (x8) (C57Bl/6) (C57Bl/6) ALLOGRATS ALLOGRATS 3 9, 9, 12 3 9, 9, 12 (BALB/c) (BALB/c)

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 Non-Immune Injury Injury Islets

Isolated Islets Highly Express Proteins Isolated Islets Highly Express Proteins Associated with ER- -Distress Distress Associated with ER Nicolls , MR et al. J. Proteome. Res. 2:199, 2003

Function of Islet Grafts in Rag1-/- Recipients Donor Graft Function (days) Donor Graft Function (days) n n Mouse Mouse 8 8 >100 (x 8) >100 (x 8) Rat (WF) Rat (WF) 9 9 >100 (x 9) >100 (x 9) >100 (x 12) Porcine >100 (x 12) Porcine 12 12 47,65,74,91,94 >100 (x12) >100 (x12) 47,65,74,91,94 Human Human 17 17

Spontaneous Failure of Human Islets Rag1 -/-akita Mice Human Islet Tp#1 Blood Glucose (mM) Human Islet Tp#2 30 Normoglycemia 20 10 0 0 50 100 Day Post Transplantation

Pathology of Failed Human Islets Pathology of Failed Human Islets (day 70) (day 70) Amyloid Fibrosis Amyloid Fibrosis (Thyoflavin Thyoflavin S) S) (Tri- -Chrome) Chrome) ( (Tri

Failure of hIAPP hIAPP Transgenic Mouse Islets Transgenic Mouse Islets Failure of Plasma Glucose (mg/dl) 600 100 tg+ 100 tg- 500 400 300 200 100 -10 0 10 20 30 40 50 Day STZ Tx

Is le t C e ll A ttritio n in T ra n s p la n ta tio n β β δ α α δ α β β β β β α β β α α β 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 , β α in f u s io n a n d im p la n ta tio n in e c to p ic s ite . δ 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 ) D o n o r Is le t D o n o r Is le t 4 . A d a p tiv e im m u n e re s p o n s e (p re -tra n s p la n t ) (p o s t-tra n s p la n t) 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- -3 months prior to transplantation 3 months prior to transplantation 2 • Human islets can reverse diabetes in immune • Human islets can reverse diabetes in immune- - deficient akita akita mice ( mice ( Rag1 Rag1 - -/ /- -akita akita ) ) deficient • Human islets can spontaneously fail over time • Human islets can spontaneously fail over time from non- -immune factors (metabolic distress?) immune factors (metabolic distress?) from non

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