Induction of Hair Growth by Fibroblast-Secreted WNTs. Gail Naughton and Frank Zeigler Histogen, Inc.

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1 Induction of Hair Growth by Fibroblast-Secreted WNTs Gail Naughton and Frank Zeigler Histogen, Inc.

2 Fibroblast Secreted Factors Formed during tissue-engineering manufacturing process by primary human foreskin fibroblasts in 3D The tissues are grown on a commercial scale, providing raw material for human extracellular matrix products, The fibroblast-secreted factors are collected as tissues grow, and do not require costly purification Contains a complex mixture of secreted factors and soluble activities with broad spectrum: angiogenesis, epithelial growth and differentiation, immuno-modulation

3 Growth Factors in 3D Fibroblast Conditioned Media PDGF-A Mitogen for fibroblasts, granulation tissue, chemotactic VEGF Angiogenesis HGF Angiogenesis IGF Mitogen for fibroblasts KGF Mitogen for keratinocytes HBEGF Mitogen for keratinocytes, fibroblasts TGF-β Mitogen for keratinocytes, fibroblasts TGF-β1 Stimulates matrix deposition TGF-β3 Stimulates matrix deposition, antiscarring SPARC Both anti- and proangiogenic IL3&IL6 Reduce local inflammation

4 Why Fibroblasts? Fibroblasts cooperate with epithelial cells during embryonic development to form skin and hair follicles Secreted signals from fibroblasts instruct epithelial stem cells to form hair follicles or epidermis, depending on the signal WNT proteins have been shown to be the primary inductive signal for hair follicle formation Fibroblasts are easy to obtain and grow, and we have successfully developed commercial-scale production processes which are economically viable in the past We have intellectual property (US and PCT patent applications) and significant expertise in our founders in fibroblast tissue-engineering There are no ethical hurdles to commercialization of fibroblasts, as there are with other cell types, such as embryonic stem cells or fetal-derived neural stem cells

5 Hair Follicle Development Occurs During Fetal Skin Morphogenesis Fibroblasts (called dermal papilla cells) secrete WNT proteins which initiate epithelial stem cells to form hair follicles Hair growth and loss occur through cyclical phases of existing follicles Alopecia androgenetica displays shortened anagen followed by premature catagen VEGF and other angiogenic factors are also secreted by fibroblasts New follicles are not normally formed after fetal development- skin wounds are an exception to this rule

6 Fibroblasts Increase WNT Gene Expression in 3-dimensional Cultures Within a Natural ECM Primary human neonatal foreskin fibroblasts were cultured as: Standard monolayers in tissue culture flasks for comparison to 3-dimensional cultures, within a naturally deposited, fetal-like ECM Samples of total RNA were compared using Agilent whole human genome microarrays for global gene expression (>40,000 genes)

7 Fibroblasts Regulate Collagen and ECM Gene Expression 3-Dimensional Cultures Within a Natural ECM FOLD INCREASE FOLD DECREASE COL4A COL20A COL19A COL9A COL10A COL6A COL9A COL14A1 2 SPARC 2.74 COL1A COL13A1 4 COL18A COL1A2 7.14

8 Fibroblasts Regulate Gene Expression of WNT Pathway Genes in 3-dimensional Cultures Within a Natural ECM FOLD INCREASE FOLD DECREASE WNT WNT7a 5.43 WNT 7b 4.05 WNT 2b 3.95 WNT 10a 3.86 WNT 8b 3.48 WNT WNT 3a 3.19 WNT 9b 3.06 WNT 9a 3.02 WNT WNT 5a 8.33 WNT WNT 5b 5.26

9 Fibroblasts Regulate Gene Expression of WNT Pathway Genes in 3-dimensional Cultures Within a Natural ECM FOLD INCREASE FOLD DECREASE LRP LRP LRP11 10 LRP DKK1 50 DKK FSZD FRZ FRZB 3.36 FRZD SFRP FRZD FRZD AXIN2 4.4 KREMEN KREMEN b-catenin 4.76 GSK3b 11.1 GSK3a 6.67 bfgf 50

10 Fibroblasts Regulate Gene Expression of BMP Pathway Genes in 3-dimensional Cultures Within a Natural ECM LRP4 FOLD INCREASE FOLD DECREASE BMP7/OP BMP BMP BMP BMPrecIb 3.37 BMP8b 3.36 BMP 8a 3.15 BMP BMP BMPrecIa 2.5 Osteocalcin 2.5 Osteopontin 6.25 BMPrecII 6.25

11 WNT Proteins Can Cause New Follicle Formation in Aged Adult Mice Wnt-dependent de novo hair follicle regeneration in adult mouse skin after wounding, M Ito, Z Yang, T Andl, C Cui, N Kim, SE Millar & G Cotsarelis, Nature, 447, 17 May 2007 Showed that regenerating epidermal stem cells responding to wounds can be coaxed into forming new follicles by WNT proteins expressed in transgenic mice Provides proof-of-concept for a product which contains soluble WNTs and wound-healing associated growth factors to induce new hair follicle formation in alopecias in adult humans

12 WNT Proteins in Hair Follicle Development and Growth Control WNTs are a group of developmental intercellular signaling proteins first identified in Drosphila as wingless mutation There are currently19 different human wnt genes They function to specify cell fate during embryogenesis but are also expressed in adult tissues Gene knockouts have tissue-specific defects ( e.g. neurogenesis, endoderm formation, blood, bone, cartilage, skin and hair) WNT signaling maintains the hair-inducing activity of the dermal papilla. J. Kishimoto et al, 2000 Genes and Dev, 14: Neonatal dermal fibroblasts expresses mrna for at least three WNT-7a, WNT-5a, and WNT-11

13 WNT Signal Transduction Frizzled family of 7-transmembrane pass receptors functions as wnt transducer when ligand-bound Receptor binding reduces GSK-3b phosphorylation of β- catenin which allows accumulation β- catenin stabilization leads to nuclear translocation and complexing with TCF/Lef family of transcription factors Nuclear localization of β-catenin indicates wnt signaling Hypothesis: fibroblasts secreted soluble WNT proteins

14 Wnt Signalling in Human Keratinocytes In Vitro: Nuclear Translocation of β-catenin is Induced by FCM A: Negative Control for β-catenin staining B: Untreated cultures C: Vehicle Negative Control D: Fibroblast-Secreted Factors No WNT activity detected in control cultures (B and C): WNT activity detected only in Fibroblast-Secreted WNT exposed culture (D): Demonstrates also that human skin cells are competent to respond In vitro data warranted in vivo studies

15 Additional In Vitro Data Show Candidate Soluble WNT Fibroblast-Secreted Factors also enhance human hematopoietic stem/ progenitor cells in colony-forming assays in vitro, which is an activity demonstrated for WNTs Effects of Fibroblast-Secreted Factors on HumanBone Marrow CD34 Cells in Colony Assays: CFU/8,000 cells plated (n=3) Blank 10X Fibroblast- Secreted Factors Myeloid/blast Erythroid KL, IL-3, G-CSF, GM- CSF, SplCM

16 WNT Signaling is Required for Hair Follicle Induction in Transgenic Mice WT WNTs WNTs BLOCKED Hair follicles β-catenin controls Hair Follicle Morphogenesis and Stem Cell Differentiation in the Skin, 2001 Cell, 105: , J Huelsken

17 Fibroblast-Secreted Factors Induce New Hair Follicle Formation in Adult SCID Mice: Preliminary Data in Mice Adult SCID mice were injected once S.Q. with either a Vehicle Control or Fibroblast-secreted factors. After 10 days histological biopsies were processed Hair growth activity detected in SCID mice (which have hair follicle defects) and so the standard model for hair growth was evaluated next (black-haired, C57/Bl6 mice) VEHICLE CONTROL 10x FIBROBLAST- SECRETED FACTORS

18 C57Bl/6 Mouse Model for Testing for Hair Growth Activity in Fibroblast-Secreted Factors Synchronized hair follicle cycling in rodents supports screening for hair growth activity by shaving mice (when follicles are resting) then applying test compounds and analyzing hair growth induction First anagen at 3 weeks, second at 7 weeks, tests conducted in between Follicular melanocytes migrate to epidermis only during anagen- so can visualize start of anagen (dark skin color) Mice only injected once subcutaneously at day 0 Hypothesis that WNT activity detected in in vitro assays will induce hair follicle cycling and growth in mice

19 Fibroblast-Secreted Factors Induced Hair Follicle Cycling and Growth in Adult C57Bl/6 Mice Vehicle Control 10x Fibroblast- Secreted Factors

20 Histological Comparison After 14 Days VEHICLE CONTROL 10x Fibroblast-Secreted Factors

21 Induction of Hair Follicle Growth and Cycling by 10x Fibroblast-Secreted Factors is Confined to the Injection Site SHAVED NORMAL SKIN INJECTION SITE

22 Fibroblast-Secreted Factors Induce Hair Follicle Cycling and Growth in Mice- Additional Studies : Injection site

23 Fibroblast-Secreted Factors Induce Hair Follicle Cycling and Growth in Mice- Additional Studies

24 Hair Density Increases After 30 days After a Single Injection in Mice: Evidence for New Follicle Formation VEHICLE CONTROL FIBROBLAST- SECRETED FACTORS

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26 Quantification of Hair Follicles Induced in Adult Mice by Fibroblast-Secreted WNTs: Combined Data (N=6) Follicle-like Structures/100x Histology Field Avg.+/-SEM P = 0.07 VEHICLE CONTROL INJECTED FIBROBLAST Blank Medium Control 10x NouriCel SECRETED WNT TREATMENT

27 Supplemental Studies Study repeated twice with monolayer conditioned media and conditioned media from 3D cultures of neonatal and adult fibroblasts 12/12 mice with 3D neonatal fibroblast conditioned media (FCM) had induced hair growth 1/12 mice had hair growth with adult cell conditioned media 0/6 mice had hair growth with monolayer FCM 0/6 control mice had hair growth Serum-free conditioned media from 3D neonatal fibroblasts has been developed with active wnt activity

28 Histochemical Detection of Alkaline Phosphatase Activity Localizes Dermal Papilla Cells: Increased Numbers of Dermal Papilla Are Induced by FCM BLANK MEDIUM CONTROL FIBROBLAST CONDITIONED MEDIUM

29 Further Characterization of Alkaline Phosphatase Activity In FCM Injected Skin cts

30 Other Potential Candidates in Fibroblast-Secreted Factors Could Also Play a Role in Hair Growth Activity Angiogenesis is associated with hair follicle cycling J Invest Dermatol 2000 May;114(5): VEGF is expressed during the growth phase (anagen) and neutralizing VEGF Abs reduce hair growth in mice J Clin Invest 2001 feb;107(4): Minoxidil mechanism of action involves up-regulation of VEGF in dermal papilla cells Br J Dermatol 1998 Mar;138(3): Fibroblast-Secreted Factors include bioactive VEGF at high levels in addition to soluble WNTs

31 Summary Evidence in adult mice demonstrates that injected Fibroblast- Secreted Factors (including WNTs and VEGF) can induce hair follicle cycling and growth WNT gene products and activity are reasonable candidates for effects on hair growth in adult mice Other factors, including VEGF, may also play a role. Fibroblast-Secreted Factors may offer a treatment for alopecias (androgenetic, chemotherapy, etc.) or even normal hair loss due to aging Will it work in humans? Evaluation in clinical trials is next step We are currently evaluating additional applications, including regeneration and protection of the neural and gastrointestinal systems