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SSEA-Positive Myeloid Cells are Involved in Hair Loss in the Alopecia Areata Mouse Model

Novel induction of alopecia areata in C3H/HeJ mice shows a potential role of previously unrecognized endogenous SSEA-positive myeloid cells in driving inflammatory cascade and hair loss mechanisms.

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SSEA-Positive Myeloid Cells are Involved in Hair Loss in the Alopecia Areata Mouse Model

  1. 1. SSEA-positive myeloid cells are involved in hair loss in AA mouse model Aziz Ghahary, Professor and Director: of Burn And Wound Healing Research Group Department of Surgery / Plastic Surgery, UBC December 5,, 2018
  2. 2. No conflict interest to declare Conflict of Interest:
  3. 3. Patchy Alopecia Areata Alopecia Universalis Amos Gilhar, Ralf Paus, Richard S. Kalish , JCI, 2007Immuno privilege of hair follicles
  4. 4. AA Skin Normal Skin CD4+ Cells in AA and Control
  5. 5. Question What is the reason(s) that inflammation persists in AA affected skin?
  6. 6. Establish a Novel AA Mouse Model To Address this question we:
  7. 7. Either graft induced or spontaneously developed AA C3H/HeJ Wang etal. : JID, 2015, Pages 2530-2532 - Single-LNC suspensions - cultured with IL-2 + IL-7 + IL-15 + Mouse T-Activator CD3/-- - CD28 Dynabeads - 6 days to increase LNC numbers 8–10-fold. - one donor/ 80–100 recipients 10 Million Cells
  8. 8. CD4+: 3.6% CD8+: 9.8% CD90+:22.9% Remove AA affected skin Skin cell suspensions ( 66 - 88 10 6 cells / mouse ) Intra-Dermal Injection of Cells Isolated from AA Affected Skin
  9. 9. FACS analysis of CD4+ and CD8+ T cells in the cultured mixture of skin cells isolated from AA affected mice Suspended cells CD4+ (3.7%) CD8+ (14.7%) Adherent cells 0.4% 2% Adherent cellsIntra-dermal Injection of isolated cells 3 Million Cells/ mouse
  10. 10. Alopecia Universalis and patchy alopecia were induced by injection of suspended and adherent AA affected skin cells Note: Injection of suspended cells mainly induced alopecia universalis; while adherent cells mainly induces patchy alopecia. Suspended Cells Adherent Cells
  11. 11. Untreated Control Skin Cell Mixture Intra-dermal Injection of Mixture of AAAffected Skin Cells Induced Alopecia
  12. 12. CD3 Normal Skin AA-Skin H&E CD11b-FIFC CD11c-APC 1.4 % 3.9 % 1.0 % 1.9 % (1.0%) (1.9%) (1.4%) (3.9%) Sub-types of Immune Cells in AAAffected Skin Normal AA CD4-FIFCCD3-PE CD8-PE 7.1 % 23.2% 1.8 % 2.5 % 1.2 % 14.2% (7.1%) (23.2%) (1.8%) (2.5%) (1.2%) (4.3 %) Normal AA Normal AA FITC: Fluorescein isothiocyanate
  13. 13. Fibroblast conditioned medium increases the number of splenocytes M-CSF (or CSF-1): is a hematopoietic GF which Increases the: 1. proliferation, 2. differentiation, 3. and survival of Macrophages & monocytes Li et al. J Cell Physiol. 2016
  14. 14. Identifying M‐CSF in Fibroblast Conditioned Medium FCM fractionation Identifying M-CSF in FCM Li et al. J Cell Physiol. 2016
  15. 15. Normal skin AA-affected skin M-CSF -Actin Normal Skin AA Skin 1 2 1 2 0 0.1 0.2 0.3 0.4 Normal skin AA skin RatioofM-CSF/-actin Expression of macrophage colony-stimulating factor (M-CSF) in AA-affected skin
  16. 16. Proliferation of syngeneic splenocytes increases when co-cultured with M-CSF-treated splenocytes cells Splenocytes M-CSF treated cell CM 2 chamber Co-culture Mix Co-Culture CFSE
  17. 17. SSEA-1 CD45 Merge M-CSF-Treated Adherent Splenocytes Cells Are positive for Stem Cell Marker (SSEA 1and 3) SSEA-3 CD45 Merge SSEA : Stage Specific Embryonic Antigen
  18. 18. M-SCF Treated Splenocytes Are Positive for CD11b and SSEA 1and 3 CD11b SSEA-1 Merge Cells from AA- mice CD11b SSEA-3 Merge Cells from AA- mice
  19. 19. Normal skin Presence of SSEA-1 and -3 Cells in Mice received M-CSF-Treated Adherent Splenocytes SSEA-1 MergeSSEA-3
  20. 20. Co-expression of SSEA -3 with M-CSF receptor on AA affected skin SSEA-3 M-CSF Receptor Merge
  21. 21. Normal skin AA–affected skin C3H/HeJ Normal AA Normal skin AA-affected skin CD3 (red) Ki67 (red) High Level of Ki67 Expressing Cells in AA-affected Mouse Skin
  22. 22. Mice received one million M-CSF treated cells + 3 millions of splenocytes for 10 weeks Intra-dermal injection of M-CSF-treated splenocytes causes hair loss in C3H/HeJ mice NumberofAA 0 1 2 3 4 5 0 1 2 3 4 5 6 7 8 9 10 Untreated 3 million Splenocytes 1 million Myeloid cells 1 million Myeloid cells + 3 million Splenocytes Weeks
  23. 23. Untreated Splenocytes M-CSF-Splenocytes Combination H & E staining CD3 CD19 T and B cells in AA affected skin in Mice Received M-CSF-Treated Splenocytes
  24. 24. Weeks NumberofAA 0 1 2 3 4 5 0 2 4 6 8 10 Untreated Myeloid cells Myeloid cells +Splenocytes Untreated M-CSF cells + splenocytes Intradermal Injection of a Combination of M-CSF-Treated Splenocytes and non- Treated Splenocytes induces AA in B6 mice
  25. 25. Triggers Fibroblasts Proliferation M-CSF Fibroblasts Express & release Attached cells (resident and infiltrated monocytes/ macrophages and myeloid cells ) SSEA-positive cells Inflammation B & T cell activation ? Antibodies &cytokines Skin Alopecia Areata Hair follicles Hair loss Conclusion
  26. 26. Supervisor: Dr Aziz Ghahary Abdi Ghaffari Dr. Claudia Chavez Dr. Elham Rahmani Darya Habibi Dr. Reza B. Jalili Dr . Farshad Forouzandeh Dr Abelardo Medina Dr. Ruhangiz Kilani Dr. Yuayan Li Mali poormasjedi Amir Akbari Amy Lai Matthew Carr Dr. Azadeh Tabatabai Dr. Terry-Ann Curie Ryan Hartwell Dr. Jun Zhang Sanam Salimi M. Bahar Members of Wound Healing Research Group
  27. 27. Confidential and Proprietary. Copyright (c). All Rights Reserved THANK YOU THANK YOU
  28. 28. Thank You View of Vancouver Over the Clouds

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Novel induction of alopecia areata in C3H/HeJ mice shows a potential role of previously unrecognized endogenous SSEA-positive myeloid cells in driving inflammatory cascade and hair loss mechanisms.

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