1. Humanized Mice in Dermatology Research Russell L. Griffin, BS, Thomas S. Kupper, MD, Sherrie J. Divito, MD, PhD Department of Dermatology, Brigham and Women’s Hospital, Harvard Medical School Boston, MA 02115

2. What are humanized mice? A humanized mouse expresses/contains functional human proteins, cells, tissues or organs They can be generated by transplanting human cells, tissues or organs into a mouse, or by genetically modifying the mouse to express human genes

3. Benefits of humanized mice Humanized mice better recapitulate human disease than traditional mouse models Genetic modifications can be employed to further “humanize” mice and overcome model limitations Humanized mice can serve as preclinical models to test novel therapeutics; results may better reflect human drug metabolism, side effect profiles and efficacy

4. Autoimmune/Inflammatory Diseases Psoriasis Alopecia Areata Atopic Dermatitis Graft-versus-Host-Disease Pemphigus Vulgaris Delayed-type Hypersensitivity Example dermatologic diseases studied using humanized mice Cutaneous Malignancies Melanoma Squamous Cell Carcinoma Genodermatoses Pachyonychia Congenita Dystrophic Epidermolysis Bullosa Lamellar Ichthyosis Wound healing Keloids & Hypertrophic Scars

5. Example: Humanized mouse model of melanoma to study a therapeutic b (a)Immunodeficient mice were sublethally irradiated and then injected intravenously with CD34+ hematopoietic stem cells (HPCs) which includes plasmacytoid dendritic cells (pDCs). Melanoma cells from a cell line were injected subcutaneously into the mouse ’ s skin. Mice were then treated (or not, control) with imiquimod, a TLR 7/8 agonist. (b) Tumor growth was measured and compared between mice that received imiquimod and those that did not (control). Figures were reproduced from Aspord et al., J Invest Dermatol, 2014

6. (a)Gross image and (b) histology image (stained with hematoxylin and eosin) demonstrating clinical and pathologic characteristics, respectively, of pachyonychia congenita. Bioengineered skin equivalents created from keratinocytes and fibroblasts taken from skin of patients with pachyonychia congenita were surgically grafted onto the backs of immunodeficient mice to generate this mouse model. Figures were reproduced from Garcia et al., J Invest Dermatol, 2011. Example: Humanized mouse model of pachyonychia congenita to serve in preclinical studies

7. a b c Mice were genetically modified to transgenically express human Desmoglein 3 (hDsg3 Tg ) in place of mouse Dsg3, because human anti-Dsg3 immunoglobulin does not recognize mouse Dsg3. Transgenic mice were then exposed to anti-Dsg3 immunoglobulin from human patients with PV. (a)Humanized mouse mucosa demonstrates clinical erosions characteristic of PV. (b)Direct immunofluorescence staining shows positive staining (c)Histology depicts classic intra-epidermal blistering Images were reproduced from Culton et al., J Invest Dermatol, 2015 Example: Humanized mouse model of pemphigus vulgaris (PV) to study pathobiology

8. Limitations of humanized mice: Practical issues Some models require advanced technical expertise Human cell/tissues—for example, fetal tissue—may be difficult to obtain Purchasing, housing and/or breeding host mice can be expensive

9. Limitations of humanized mice: Scientific issues Complete multi-lineage engraftment of human hematopoietic system is difficult to obtain – Although human neutrophil, red blood cell and platelet precursors engraft in mouse bone marrow, they do not circulate in mouse blood in any substantial number. – Memory T cell and humoral responses are difficult to consistently develop Cross reaction between co-expressed mouse and human factors can confound experimental results Absence of additional human factors (e.g., cytokines, homing ligands and receptors) that may be necessary to recapitulate human physiology/pathology