Presentation by Michael A. Brehm, Ph.D., Assistant Professor, Diabetes Center of Excellence, Program in Molecular Medicine, University of Massachusetts Medical School, at the JDRF New England Chapter's 14th Annual Spring Research Briefing on Tuesday, April 9, 2013.
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Michael Brehm, Ph.D.
1. Development of Humanized Mouse Models to
Study Diabetes
Michael A. Brehm
Human cells
& tissues
Diabetes Center of Excellence
Dale Greiner
David Harlan
Rita Bortell
Philip DiIorio
The Jackson Laboratory
Leonard Shultz
2. Why Do We Need Humanized
Mouse Models?
– Most experimental studies done in rodents
– Outcomes predicted by murine studies are not always
representative of actual outcomes in humans
• Seok, 2013. PNAS, Vol:110, p3507
– Permits study of human-specific infections, therapies
and immune responses
• Goal
– Enable clinically relevant in vivo studies of human cells,
tissues, and immune systems without putting patients at
risk
3. Application of Humanized Mice for
Biomedical Research
-Cancer biology
-Human hematopoiesis
-Infectious diseases
-Immunity
-Transplantation
-Regenerative medicine
-Beta cell function and homeostasis
-Autoimmunity
6. NOD-scid IL2rgnull (NSG) Mouse
Shultz, 2005. J. Immunol.; Ishikawa, 2005. Blood.
•Complete absence of IL2rg gene
-long life span
-further impairment of innate immunity
-complete absence of NK cells
-NOD-Rag1null IL2rgnull (NRG)
•NSG and NRG mice engraft at high levels with
human cells and immune systems:
-Tumors
-Skin
-Islets
-ES cells and iPS cells
-Hematopoietic stem cells
-Mature immune cells
7. Humanized Mice in Diabetes
• Study Human Beta Cell Biology In Vivo:
-Function
-Proliferation
-Beta stem and progenitor cells for replacement therapies
-Islet transplantation and allograft rejection
This will ultimately permit the analysis of human beta cell function in a
“real-life” situation where they will be transplanted into diabetic
individuals with intact immune systems
• Development of Models to Recreate T1D:
-Injection of PBMC or spleen cells from T1D donors
-Engraftment of HSC obtained from T1D donors
-Use of iPS cells derived from T1D donors
8. Novel Murine Models To Study
Human Beta-cell Function
Normoglycemic Models:
NSG -lacks T cells, B cells and NK cells
NRG -lacks T cells, B cells and NK cells
Hyperglycemic Models:
NSG Akita -develops spontaneous hyperglycemia
NRG Akita -develops spontaneous hyperglycemia
NSG RIP-DTR -induced hyperglycemica by treatment with diphtheria toxin
NSG-Tg(Ins-rtTA) Tg(TETO-
DTA)
-induced hyperglycemica by treatment with doxycycline
Insulin Resistant Models:
NSG ob/ob leptin deficient, obese
NSG db/db leptin receptor deficient, obese
NSG Glut4null altered glucose metabolism, lean
9. The “Akita” Model of Spontaneous Hyperglycemia
Without Autoimmunity
NOD-Rag1null IL2rgnull
NOD-Ins2Akita
NOD-Rag1null IL2rgnull Ins2Akita
This non-autoimmune diabetes develops as the mouse ages in the absence of toxic
chemical treatment that could harm the transplanted cells or humans immune system
Akita mutation is an
insulin-2 gene defect
that leads to
generation of mis-
folded insulin protein,
induction of ER
stress, beta cell
apoptosis, and
hyperglycemia
10. Islet Structure in NOD-Rag1null IL2rgnull Ins2Akita Mice
21 days 222 days
NRG NRGNRG-Akita NRG-Akita
Brehm et al, 2010. Diabetes
11. Human Beta Cells Proliferate in Response to Hyperglycemia
Human islets (1,500) from a 24-year-old female were transplanted into a
normoglycemic NRG (WT) mouse and into a hyperglycemic NRG-Akita mouse. At
3 wk the mice were pulsed with BrdU. The grafts were removed 1-wk later.
DiIorio, 2011. Pancreas
13. Recreating Type-1 Diabetes in Humanized Mice-I
• Immunodeficient Recipient
– HLA-Tg NSG mice
• HLA class I and class II Tg that cover >80% of T1D
• Cell and Tissue Source
– PBL or Spleen from T1D donors
– Autoantigen reactive T cell lines
– CD4 and CD8
– Autoantigen reactive T cell clones
– CD4 and CD8
– Hematopoietic stem cells
• nPOD (bone marrow from JDRF program)
• G-CSF mobilized hematopoietic stem cells
• TCR retrogenic
14. Recreating Human T1D in NSG Mice
Characteristic Strain Timeline
HLA class I transgenics
NSG-HLA-A2 Available—being characterized
NSG-HLA-A24 Available—being characterized
NSG-HLA-A11 ~1 year (NOD-Tg made, currently
backcrossing)
NSG-HLA-B7 Available—being characterized
NSG-HLA-Cw3 Available—being characterized
HLA class II transgenics
NSG-HLA-DR3 Available—being characterized
NSG-HLA-DR4 Available—being characterized
NSG-HLA-DQ8 Available—being characterized
Combinations of stocks NSG-HLA class I/II Tg Example: NSG HLA-A2 HLA-DR4
– HLA-Tg NSG mice: HLA class I and class II Tg that cover >80% of T1D
15. CD8 T cells from an HLA-A2 T1D Individual
Targets HLA-A2+ Islets In Vivo
10x106
Intrapancreatic injection
8 weeks
-Monitor Blood Glucose
-Flow Cytometry
-Pancreas Histology
-Serum
Autoantibodies
CD8-Enriched T cells from HLA-A2+ T1D donor spleen
NOD-scid IL2rgnull
HLA-A2 KDnull Tg
recipients
17. Recreating Type-1 Diabetes in Humanized Mice-II
T1D iPS Donor:
Humanized Mouse
• Functionality of cell populations
• Disease progression
• Treatments or cures
18. Summary
1. Generating humanized mouse models to study human
beta cell function and homeostasis
-normoglycemic, hyperglycemic or insulin-resistant conditions
-models to test beta stem or precursor cells for functionality
2. Recreating human type-1 diabetes in humanized mice
-Developing models to study the effector phase (T cells)
-Developing models to study initiation through the effector phase
(iPS cells)
3. Continuously improving the recipient mouse to optimize
human cell engraftment and function
-genetically introducing new human growth factors and cytokines
-further impairment of the mouse immune system
19. Acknowledgements
•UMass-Diabetes Center
of Excellence
– Phil Durost
– Darcy Reil
– Linda Paquin
– Amy Cuthbert
– Laurence Covassin
– Jamie Kady
– Meghan Dolan
– Pam St. Louis
– Mike Bates
•The Jackson Laboratory
– Leonard Shultz
• HSCI
– Doug Melton
– Derrick Rossi
NIDDK
NIAID
•UMass
– Rene Maehr
– Roger Davis
– Jack Leonard
– JeanMarie Houghton
– Michelle Kelliher
– Hardy Kornfeld
– Anuja Mathew
– Michael Czech
– Katherine Luzuriaga
– Rich Konz
– Flow Cytometry Core
Notas del editor
Revere tetracycline transactivator expressed in β cells Diphtheria toxin A subunit under a rtTA-responsive promoter