1 Department of Ophthalmology, University of Erlangen-Nürnberg, Erlangen, Germany

Therapeutic use of hair follicle-derived epithelial stem cells using a murine stem

cell deficiency model

Ewa Anna Meyer-Blazejewska, Hongshan Liu, Mindy K. Call, Ursula Schlötzer-Schrehardt,

Winston W-Y. Kao and Friedrich E. Kruse

2 Department of Ophthalmology, University of Cincinnati, OH, USA

The authors have no financial interest in the subject matter of this poster

Slide 1

Introduction: Hair follicle stem cells

The bulge region of the hair follicle (HF) is a major reservoir of multipotent adult stem cells (SC). (Cotsarelis et al. 1990)

bulge

Murine HF

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)

Slide 2

K12-Pax6-

Hair follicle

Cornea

outer root sheath

inner root sheath

bulge

Sebaceous gland

No expression of the corneal epithelial markers (K12, Pax6) in hair follicle

K12+

Pax6+/K12+

Introduction: Hair follicle markersSlide 3

Blazejewska et al.; Stem Cells, 2008

Induction of K12 and Pax6 expression in hair follicle SC in vitro using conditioned medium (CM) derived from limbal stroma

fibroblasts

Cytokeratin 12

mo

lecu

les

K12

/mo

lecu

les

ß-a

ctin

x10

3

n=5 n=5

**

**

Pax 6

central cornealfibroblast CM

peripheral corneal fibroblastCM

limbal fibroblastCM

3t3fibroblastCM

mo

lecu

les

Pa

x6/m

ole

cule

s ß

-act

in x

103

central cornealfibroblast CM

limbal fibroblastCM

3t3fibroblastCM

K12 K12/Pax6

peripheral corneal fibroblastCM

**

**

Introduction: previous work Slide 4

Purpose

To explore the therapeutic potential of murine hair follicle-derived stem cells to treat limbal stem cell deficiency and

replenish corneal epitheliumusing an in vivo animal model.

Slide 5

Method:Tri-Transgenic Mouse Model

Inducible K12 driven expression of EGFP

K12 IRES rtTA

DoxrtTA

rtTA

tet-O

pminCMV cre

cre

rtTA

pCA mT mG

pCA mG

DoxDoxDox

lox P lox P

Slide 6

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.

Method:Tri-Transgenic Mouse Model Slide 7

Method: Clonal expansion of hair follicle SCStem cell clones grown on a 3T3 feeder layer

Z-stack, 3D

Epithelial cells

3T3 cells

SC clone

SC clone

K15

SC clones

Red fluorescence: no K12 expression in HF-derived epithelial SC clones

SC clone

K12rtTA/Tet-O-Cre/ROSAmTmG

Slide 8

Method: Transplantation of SC on a fibrin gel

SC clones subcultured on a fibrin gel as carrierAfter limbal SC debridement

Directly after SC transplantation

Red: SC and progenitor cells

No Green: no K12 expression

Fibrin gel

K12rtTA/Tet-O-Cre/ROSAmTmG

Slide 9

Results: K12 induction post-transplantation

3 days postoperative

WT C57/Black6

K12+ cells

Regular light

Fluorescein staining

14 days postoperativeFibrin gel remains

Mouse eye

21 days postoperative

Slide 10

Results: K12 induction post-transplantation

DAPI EGFP

tdTomato Merge

Corneal epithelium: 7days postoperative

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.

K12+ (green)

no K12 (red)

Slide 11

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.

Slide 12