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.

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. No conflict interest to declare
Conflict of Interest:

4. Patchy Alopecia Areata Alopecia Universalis
Amos Gilhar, Ralf Paus,
Richard S. Kalish , JCI, 2007Immuno privilege of
hair follicles

5. AA Skin
Normal Skin
CD4+ Cells in AA and Control

6. Question
What is the reason(s) that
inflammation persists in AA
affected skin?

7. Establish a Novel
AA Mouse Model
To Address this question we:

8. 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

9. 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

10. 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

11. 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

12. Untreated Control Skin Cell Mixture
Intra-dermal Injection of Mixture of AAAffected Skin Cells Induced Alopecia

13. 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

14. 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

15. Identifying M‐CSF in Fibroblast Conditioned Medium
FCM fractionation
Identifying M-CSF in FCM
Li et al. J Cell Physiol. 2016

16. 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

17. 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

18. 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

19. 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

20. Normal skin
Presence of SSEA-1 and -3 Cells in Mice received M-CSF-Treated Adherent
Splenocytes
SSEA-1
MergeSSEA-3

21. Co-expression of SSEA -3 with M-CSF receptor on AA affected skin
SSEA-3 M-CSF Receptor Merge

22. 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

23. 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

24. 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

25. 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

26. 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

27. 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

28. Confidential and Proprietary. Copyright (c). All Rights Reserved
THANK YOU
THANK YOU

29. Thank You
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