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FROM BENCH TO BESIDE: A BUMPY ROAD

YANN BARRANDON LABORATORY OF STEM CELL DYNAMICS SWISS FEDERAL INSTITUTE OF TECHNOLOGY AND DEPARTMENT OF EXPERIMENTAL SURGERY LAUSANNE MEDICAL SCHOOL

yann.barrandon@epfl.ch

REGENERATIVE MEDICINE
Stem cells as a tool to improve tissue renewal, repair and regeneration

In situ: design new drugs targeting stem cells Ex vivo: expansion of stem cells in culture and transplantation

STEM CELLS: THERAPY FOR THE FUTURE

Bone marrow Nervous system Skeletal muscle, cartilage, bone Cardiac muscle Pancreas, liver Skin and related epithelia (ocular surface)

FIRST TRANSPLANTATION OF AUTOLOGOUS CULTIVATED KERATINOCYTE STEM CELLS: 1983

CHALLENGE
1 - UNDERSTAND MORPHOGENESIS - REPAIR PROLIFERATION MIGRATION DIFFERENTIATION DEATH 2 - MANIPULATE LOCALISE ISOLATE EXPAND DIFFERENTIATE

3 - TRANSPLANT
ANATOMY ART ( GUNTER von HAGEN )

RENEWAL

CELL THERAPY EXTENSIVE THIRD DEGREE BURN WOUNDS

GENE THERAPY

DYSTROPHIC EPIDERMOLYSIS BULLOSA

COLLAGEN VII (COL7A1) DEFICIENCY ANCHORING FIBERS

Different strategies

Acute extensive wounds - no or little time Chronic wounds - plenty of time

Permanent coverage - transplantable autologous stem cells Improve healing - allogeneic cells

STRUCTURE OF HUMAN SKIN

EPIDERMIS

EPITHELIAL STEM CELLS IN EPIDERMIS, HAIR FOLLICLES AND SWEAT GLANDS

DERMIS

MESENCHYMAL STEM CELLS IN DERMIS AND HAIR FOLLICLES

MELANOCYTE STEM CELLS

SUBCUTIS

THE RENEWAL OF EPIDERMIS

SUPRABASAL CELLS (POST MITOTIC)

BASAL CELLS (MITOSIS)

STEM CELL

TRANSIT AMPLIFYING CELL (4 DIVISIONS MAXIMUM)

COLUMNAR ORGANIZATION OF THE EPIDERMIS (MOUSE)

FROM GAMBARDELLA AND BARRANDON CURR OPIN CELL BIOL 2003

CHALLENGE

Centre des brls, Hpital Percy, France Photo Raphal Gaillarde - Agence Gamma

Howard Green

PIONEERING REGENERATIVE MEDICINE BRINGING STEM CELLS FROM THE BENCH TO THE BEDSIDE

EX VIVO EXPANSION OF ADULT AUTOLOGOUS EPIDERMAL STEM CELLS

1 - 5 cm2

2 - 3 weeks 1 m2

FROM HOWARD GREEN AND COLLEAGUES ( Rheinwald and Green, 1975, Gallico et al., N. Engl. J. Med. 1984 )

9 YEAR OLD 95% BODY AREA THIRD DEGREE BURNS EXCISED TO FASCIA

NO SPONTANEOUS HEALING

AUTOLOGOUS SKIN TRANSPLANTATION

SPLIT THICKNESS GRAFTS and CULTURED EPITHELIUM GRAFTS

REGENERATION OF EPIDERMIS

NORMAL SKIN

EPIDERMIS GENERATED FROM TRANSPLANTED STEM CELLS (3.5 years after transplantation)
Ronfard et al., TRANSPLANTATION 2000

SPONTANEOUS HEALING

AUTOLOGOUS CUTANEOUS CELL THERAPY

REGENERATION OF EPIDERMIS
NORMAL STRATIFIED KERATINIZED EPITHELIUM PRESENCE OF HOLOCLONES

REGENERATION OF SURPERFICIAL DERMIS


ONDULATED DERMO-EPIDERMAL JUNCTION PRESENCE OF SUBEPIDERMAL VASCULAR ARCADES PRESENCE OF ELASTIC FIBERS OBSERVED IN FETAL WOUND HEALING NEVER OBSERVED IN NORMAL POST-NATAL WOUND HEALING

ABSENCE OF EPIDERMAL APPENDAGES


SWEAT GLANDS, SEBACEOUS GLANDS, HAIR FOLLICLES

1 - WHY SO LITTLE INFORMATION ON SHORT-TERM AND LONG-TERM BEHAVIOR OF TRANSPLANTED STEM CELLS? 2 - WHY HAS THE TECHNOLOGY NOT EVOLVED? 3 - WHY DO APPENDAGES (SWEAT GLANDS, SEBACEOUS GLANDS, HAIR FOLLICLES) NOT REGENERATE?

1 - WHY SO LITTLE INFORMATION ON SHORT TERM AND LONG-TERM BEHAVIOR OF TRANSPLANTED STEM CELLS? Difficult patient follow-up (fed up with doctors..) - Poor communication between basic and medical research laboratories Difficult to experiment on human, obvious ethical reasons, regulatory rules (GMP), cost Difficulties to assay stemness No control of stem cell engraftment

Necessity of a reliable and predictable animal model

CREATING FOUNDATIONS FOR REGENERATIVE MEDICINE

RECAPUTILATING THE HUMAN IN THE PIG TO UNDERSTAND STEM CELL ENGRAFTMENT

Transplantation of a single cell derived graft

WHY NO EPIDERMAL APPENDAGES?


(HAIR FOLLICLES, SEBACEOUS GLANDS, SWEAT GLANDS)

ABSENCE OF MULTIPOTENT EPIDERMAL STEM CELLS


NO MULTIPOTENT STEM CELLS IN ADULT SKIN MULTIPOTENT STEM CELLS DO NOT SURVIVE IN CULTURE CURRENT CULTURE CONDITIONS FAVOR EPIDERMAL DIFFERENTIATION

ABSENCE OF INDUCTIVE SIGNALS (AND/OR)

LONG TERM REGENERATION OF HAIR FOLLICLES FROM CULTIVATED MULTIPOTENT EPITHELIAL STEM CELLS

1 cell

in vitro expansion

grafting

1 cell

in vitro expansion

grafting

0
In vitro self-renewal

50
In vivo self-renewal

175
In vitro self-renewal

196

313 days
In vivo self-renewal

Oshima et al., Cell 2001 Claudinot et al., PNAS 2005

THOUSANDS OF HAIR FOLLICLES CAN BE GENERATED FROM A SINGLE MULTIPOTENT STEM CELL

Karyotype
1 cell 1 cell

Histology

0
In vitro self-renewal

50
In vivo self-renewal

175
In vitro self-renewal

196

313 days
In vivo self-renewal

NO FUSION DIPLOID

STRATIFIED SQUAMOUS EPITHELIA


SKIN (EPIDERMIS, HAIR FOLLICLE) OCULAR SURFACE (CONJUNCTIVA, CORNEA) ORAL CAVITY EOSOPHAGUS VAGINA KERATINIZED (epidermis)

PROTECTIVE FUNCTION SELF RENEWING STEM CELLS


NON KERATINIZED (cornea)

TRANSPLANTATION OF SIMPLE AND STRATIFIED EPITHELIA OF THE ADULT MOUSE

FOOT PAD SOFT PALATE VAGINA LIMBUS CORNEA CONJUNCTIVA EOSOPHAGUS TRACHEA BLADDER GUT
Oshima et al., Cell 2001

FORMATION OF HAIR FOLLICLES BY NON SKIN STEM CELLS

BEFORE

AFTER

FOOT PAD

SOFT PALATE

VAGINA

LIMBUS CONJUNCTIVA CORNEA

STRUCTURE OF HUMAN SKIN

EPIDERMIS

DERMIS

SUBCUTIS

STEM CELLS THERAPY

Feasibility (procedures - rules - ethics) Safety (balance benefit/risk) Traceability Reproducibility Efficacy Cost

Analysis
NATURE BIOTECHNOLOGY December 2002 Volume 20 Number 12 pp 1178 - 1179

Tissue engineering firms go under


Aaron Bouchie
New York, NY

Two leading tissue engineering companies, Organogenesis (Canton, MA) and Advanced Tissue Sciences (ATS; La Jolla, CA), filed for bankruptcy on September 25 and October 10, respectively (Nat. Biotechnol. 20, 1072, 2002). These two firms were the first and only firms to bring mass-produced engineered skin products to market for the treatment of chronic wounds, such as diabetic foot ulcers and veinous leg ulcers. But sales were far too dismal to coverlet alone outpacethe enormous operating costs. As the firms ran out of money, investors were unwilling to give them more funds in the currently harsh economic climate. The main reason for the demise of Organogenesis and ATS was a poor business model: Lack of communication with the targeted medical community led to a gross overestimation of the market for artificially engineered skin and overreaching promises to investors.

THERAPEUTICAL USE OF SKIN STEM CELLS


AUTOLOGOUS CELL THERAPY (PERMANENT) EXTENSIVE SKIN DEFECTS (BURNS) DEFECTS OF THE OCULAR SURFACE PLASTIC AND RECONSTRUCTIVE SURGERY AUTOLOGOUS GENE THERAPY (PERMANENT) INVALIDATING GENODERMATOSES (EPIDERMOLYSIS BULLOSA) PRODUCTION OF PROTEINS OF MEDICAL INTEREST (FACTOR IX, GH) ALLOGENIC CELL THERAPY (TEMPORARY) WOUND HEALING ALLOGENIC GENE THERAPY (TEMPORARY) WOUND HEALING

TRANSPLANTATION OF RELATED STEM CELLS LIMBAL STEM CELLS, TOOTH STEM CELLS

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