1. Therapeutic use of hair follicle-derived epithelial stem cells using a murine stem cell deficiency model
Slide 1
The authors have no financial interest in the subject matter of this poster
Ewa Anna Meyer-Blazejewska, Hongshan Liu, Mindy K. Call,
Ursula Schlötzer-Schrehardt,
Winston W-Y. Kao and Friedrich E. Kruse
1 Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany
2 Department of Ophthalmology, University of Cincinnati, OH, USA

2. Introduction: Hair follicle stem cells
Slide 2
Introduction: Hair follicle stem cells
Murine HF
The bulge region of the hair follicle (HF) is a major reservoir of multipotent adult stem cells (SC). (Cotsarelis et al. 1990)
Cytokeratin 15 (K15), a marker for stem and progenitor cells in the bulge and outer root sheath of the hair follicle.
(Cotsarelis et al., 1990, 1999; Fiqueira et al., 2007)
bulge

3. Introduction: Hair follicle markers
Slide 3
Introduction: Hair follicle markers
No expression of the corneal epithelial markers
(K12, Pax6) in hair follicle
Hair follicle
K12-
Pax6-
Sebaceous gland
inner root sheath
Cornea
bulge
K12+
outer
root sheath
Pax6+/K12+

4. Introduction: previous work
Slide 4
Induction of K12 and Pax6 expression in hair follicle SC in vitro using conditioned medium (CM) derived from limbal stroma fibroblasts
n=5
n=5
Cytokeratin 12
Pax 6 * ** * ** * *
molecules K12/molecules ß-actin x103
molecules Pax6/molecules ß-actin x103
central
corneal
fibroblast CM
peripheral
corneal
fibroblast CM
limbal
fibroblast CM
3t3
fibroblast CM
central
corneal
fibroblast CM
peripheral
corneal
fibroblast CM
limbal
fibroblast CM
3t3
fibroblast CM
K12
K12/Pax6
Blazejewska et al.; Stem Cells, 2008

5. Purpose To explore the therapeutic potential
Slide 5
Purpose
To explore the therapeutic potential
of murine hair follicle-derived stem cells
to treat limbal stem cell deficiency and replenish corneal epithelium
using an in vivo animal model.

6. Inducible K12 driven expression of EGFP
Slide 6
Method:Tri-Transgenic Mouse Model
Inducible K12 driven expression of EGFP
cre
rtTA
K12
IRES
rtTA
pminCMV
cre
tet-O
Dox
Dox
Dox
Dox
rtTA
pCA mT mG
lox P
lox P
pCA mG

7. Method:Tri-Transgenic Mouse Model
Slide 7
Method:Tri-Transgenic Mouse Model
We have generated a tri-transgenic mouse model that is both tissue specific and inducible and allows for the detection of K12 expressing cells by the presence of green fluorescence. This transgenic mouse system is comprised of three parts the first of which is the K12 rtTA line that provides the tissue specificity. This line was generated via a knock-in strategy in which an IRES-rtTA (Internal Ribosome Entry Site-reverse tetracycline Transcriptional Activator) minigene was inserted directly after the stop codon of the mouse Krt12 gene. Thereby only differentiated corneal epithelial cells are able to express rtTA. The second component of the tri-transgenic mouse model is Tet-O-Cre. This line uses components of the Tet-On system and together with the K12 rtTA line provides the ability for induction. Specific Tetracycline operator (Tet-O) elements are followed by a CMVmin (CMV minimal) promotor and the Cre recombinase gene. In the absence of tetracycline or a tetracycline derivate such as doxycycline , rtTA is unable to bind to the promotor and therefore Cre is not produced. Once doxycycline is added to the system, it can bind with rtTA and together this complex can further bind to the Tet-O elements and drive the expression of Cre. The third component of the system is the ROSA26mTmG line (Jackson Laboratories) which serves as a dual reporter. This mouse line has loxP sites flanking a membrane-targeted tdTomato (mT) cassette and express red fluorescence in all cell types. Upon breeding to a Cre recombinase mouse line (Tet-O-Cre), the resulting offspring will have td Tomato cassette deleted only in the cells expressing Cre (only in K12 positive cells) allowing for expression of a membrane-targeted enhanced green fluorescent protein (mG). This system allows for the live visualization and tracking of K12 expressing cells.

8. Method: Clonal expansion of hair follicle SC
Slide 8
Method: Clonal expansion of hair follicle SC
Stem cell clones grown on a 3T3 feeder layer
SC clone
SC clones
3T3 cells
SC clone
K15
Epithelial
cells
Red fluorescence: no K12 expression in HF-derived epithelial SC clones
SC clone
K12rtTA/Tet-O-Cre/ROSAmTmG
Z-stack, 3D

9. Method: Transplantation of SC on a fibrin gel
Slide 9
Method: Transplantation of SC on a fibrin gel
After limbal SC debridement
SC clones subcultured on a fibrin gel as carrier
K12rtTA/Tet-O-Cre/ROSAmTmG
Directly after SC transplantation
Red:
SC and progenitor cells
No Green:
no K12 expression
Fibrin gel

10. Results: K12 induction post-transplantation
Slide 10
Results: K12 induction post-transplantation
3 days postoperative
Mouse eye
21 days postoperative
Fibrin gel
remains
14 days postoperative
Regular light
Fluorescein staining
K12+ cells
WT C57/Black6

11. Results: K12 induction post-transplantation
Slide 11
Corneal epithelium: 7days postoperative
DAPI
EGFP
K12+ (green)
tdTomato
Merge
no K12 (red)
The specimen was prepared by removing the cornea, treating with 0.2% sodium borohydride for 45 min at room temperature (helps in the reduction of background fluorescence), counterstaining with DAPI overnight, and imaging. The total thickness of the Z-stack is 37.5 µm with each slice having a thickness of 1.5 µm. All images are from slice 14 of the Z-stack.

12. Slide 12
Conclusions
Hair follicle bulge-derived epithelial SC possess the potential to differentiate into corneal epithelial-like phenotype in vivo.
Hair follicle SC express K12 (corneal epithelial differentiation marker) and regenerate the corneal epithelium up to 3 weeks post-transplantation when transplanted in a murine limbal SC deficiency model.