WO2012035309A1

WO2012035309A1 - Methods

Info

Publication number: WO2012035309A1
Application number: PCT/GB2011/001372
Authority: WO
Inventors: Carrie Ann Ambler; Fiona Mary Watt
Original assignee: University Of Durham
Priority date: 2010-09-17
Filing date: 2011-09-19
Publication date: 2012-03-22
Also published as: GB201015567D0

Abstract

The present invention relates one or more activators of Notch signalling for use in methods of preparing cells for autologous cell replacement therapies, methods of promoting cell differentiation, methods of isolating differentiated cells, and methods using such isolated cells.

Description

Methods

In recent years, there has been much focus on the development of autologous cell replacement therapies. Autologous cell replacement therapies use cells derived from the patient to be treated, thus reducing risks from systemic immunological reactions, bio-incompatibility, and disease transmission associated with grafts or cells not cultivated from the individual. In such strategies, typically stem or progenitor cells are prepared from an individual; the stem or progenitor cells then differentiate in to the specific cells required for that individual; the differentiated cells then are transplanted into the individual.

Stem cell and progenitor cells hold tremendous promise as a source of functional differentiated cell types for regenerative medicine. In addition, stem cell and progenitor cells have great potential as an in vitro system for the study of developmental biology, allowing the effective isolation of distinct populations of cells that normally exist very close together in both space and time during embryonic development. However, given that stem cell and progenitor cells have the potential to differentiate into a variety of different cell types, it is necessary to identify controlled, scalable and directed methods to enable the development of specific cells and hence arrive at a predictable means of preparation.

The present inventors have investigated cell differentiation in the skin.

The skin is a bi-compartmental organ. The outer layer is maintained by stem cells and comprises a stratified epithelium, the interfollicular epidermis, with associated hair follicles, sebaceous glands and sweat glands. The sub-epidermal compartment comprises dermal fibroblasts, peripheral nerves, blood vessels, muscle and fat. Subpopulations of dermal cells, located in the dermal papilla and dermal sheath, regulate epidermal stem cell properties, most notably by controlling the hair growth cycle. The dermal skin layer includes a number multipotent stem and progenitor cells that are capable of providing cells that differentiate into a wide range of different cell types and tissues, including blood, fat, various types of skin tissue, neurons and glial cells. One such type of stem and progenitor cells is skin-derived precursor cells (SKPs). Skin- derived precursor cells are believed to migrate to the skin during embryogenesis, where they retain their multipotent capacity into adulthood.

When using cells prepared from an individual it is clearly important to be able to regulate the differentiation of the cells, both to the desired cell types and in a controllable manner.

The present inventors have investigated the role of the Notch signalling pathway in regulating embryonic and postnatal skin development. Notch signalling is activated when ligand binding initiates cleavage of the Notch receptor, which releases the Notch intracellular domain (NICD) from the plasma membrane. The NICD translocates to the nucleus to activate transcription of downstream targets.

Recent studies have tended to focus on the epidermal consequences of modulating Notch, however the inventors have now demonstrated that epidermal Notch signalling causes changes in the dermis. They show that Jagged 1 , a ligand for Notch, mediates many of these effects. As part of this work, they now have demonstrated that the Notch signalling pathway can promote the differentiation cells, including stem or progenitor cells, in the dermis to specific cell fates. This surprising and unexpected finding lead the inventors to develop methods of promoting the differentiation of cells in the dermis using activators of Notch signalling. Clearly these methods have much utility in the development of cell populations for cell replacement therapies.

A first aspect of the invention provides one or more activators of Notch for use in a method of preparing a population of cells for use in autologous cell replacement therapy, the method comprising contacting skin tissue of a patient in need of said therapy with one or more activators of Notch signalling, and subsequently isolating the required differentiated cells from the skin tissue. As discussed above, the Notch signalling pathway is a highly conserved cell signalling system present in most multicellular organisms. The Notch signaling pathway is important for cell-cell communication, which involves gene regulation mechanisms that control multiple cell differentiation processes during embryonic and adult life. Notch signaling also has a role in a wide range of crucial developmental processes. Notch signaling is dysregulated in many cancers, and faulty notch signaling is implicated in many diseases.

The Notch proteins (Notch 1-Notch4 in vertebrates) are single-pass receptors that are activated by the Delta (or Delta-like) and Jagged/Serrate families of membrane-bound ligands. Interaction with ligand leads to two additional proteolytic cleavages that liberate the Notch intracellular domain (NICD) from the plasma membrane. The NICD translocates to the nucleus, and interacts with its binding partners RBP-JK and Mastermind 1 to activate transcription of downstream targets, including members of the Hes and Hey families of transcriptional repressors.

However, it is important to point out that until the present invention, it was not known or suggested that activating Notch signalling in the epidermis leads to the differentiation of cells in the dermis.

The inventors have developed a method of preparing populations of cells for use in autologous cell replacement therapy by activating the Notch signalling pathway in skin tissue. In one embodiment of the invention, the epidermal layer of the skin tissue is contacted with the one or more activators of Notch signalling, and the required differentiated cells are isolated from the dermal layer of the skin tissue.

The method of the first aspect of the invention can also be reformulated so as not to require direct interaction with a patient.

Hence an alternative first aspect of the invention provides a method of preparing a population of cells for use in autologous cell replacement therapy, the method comprising isolating the required differentiated cells from a sample of skin tissue (previously obtained from a patient in need of said therapy), wherein the skin tissue has been contacted with one or more activators of Notch signalling. Here the method is applied to a skin sample that has been contacted with or more activators of Notch signalling. The isolated cells are then used in autologous cell replacement therapy. Hence the key features of the invention are present in the alternative first aspect of the invention. The embodiments of the first aspect of the invention described herein also apply to the alternative first aspect of the invention.

The method of the invention has a number of advantages over existing methods of preparing cells for autologous cell replacement therapies. Firstly, the skin is a readily accessible organ, and the isolation of skin tissue and skin regeneration methods are well established. Hence the activator of the Notch signalling pathway can be easily applied to the skin, and those differentiated cells can be easily isolated. Moreover, since the method of the invention can involve the isolation of skin tissue from a patient to be treated, then skin regeneration methods can be used to minimise the effect of this biopsy on the patient.

The method of the invention can be applied to skin tissue isolated from the patient to be treated. In this way the skin tissue can be cultured using standard well known techniques in the art, the activator of Notch signalling applied to the tissue, and those differentiated cells then isolated. In this way the patient to be treated is not exposed directly to the activator of Notch signalling.

However a preferred method of the invention is where the one or more activators of Notch signalling are applied to the skin tissue in situ.

In this preferred embodiment of the invention, the one or more activators of Notch signalling are applied to the skin of the patient in need of autologous cell replacement therapy. After a suitable period of time, the skin tissue is removed from the patient, and the required differentiated cells isoalted from the skin tissue. As stated above, preferably the one or more activators of Notch signalling is applied to the epidermal layer of the skin, and the differentiated cells are subsequently isolated from the dermal layer of the skin.

An advantage of the in situ method of the invention is that the skin tissue is not subjected to cell or tissue culturing techniques, which thus minimises the likelihood of cells in the tissue growing poorly, developing abnormal morphologies, or developing and inheriting genetic mutations. Also, this minimises the exposure of the materials to be transplanted to non-human contaminants and minimises the likelihood of pathogen infection. As the cells used for treatment are the patient's own, there is no need to suppress the immune system.

As demonstrated in the accompanying examples, and further discussed below, the inventors have determined that activating Notch signalling, preferably in epidermal layer, promotes cell differentiation in the skin, specifically in the dermal skin layer. A wide range of different cell types can be prepared, and indeed the cells differentiate as a heterogeneous mixture of cells. Such cell types include neural progenitor cells, melanocytes, and dermal papilla cells; the neural progenitor cells are capable of subsequently differentiating in to neurons and Schwann cells.

As expanded on below, it can be appreciated that for specific autologous cell replacement therapies, specific cell types should be isolated from the skin tissue of the patient in need of said therapy; example protocols for such isolation methods are provided.

Hence, by way of example, the following protocol is offered as an embodiment of the invention.

A patient is presented as being in need of autologous cell replacement therapy; for example, the patient may be in need of treatment of a condition characterised by degeneration, damage to, the loss of, or the disorder in nervous tissue; or skin pigmentation; or hair formation and/or growth. A section of skin tissue of the patient, preferably the epidermal layer, is contacted with one or more activators of Notch signalling, for example JAGGED 1. The activator may be administered to the patient as a topical formulation, or as an injectable composition. The skin tissue is contacted with the activator for a period of time, for example several days to a week, then that region of skin tissue is removed from the patient. After treatment with a suitable protocol, cells are released from the skin tissue, preferably from the dermal layer. Those cells released will be a heterogeneous population of existing cells from the skin tissue, and different cell types whose differentiation was induced by the activator. Specific cell types are then isolated from the heterogeneous population using known methods in the art, for example using FACS with cell markers that can be reliably utilised to prepare such populations; clearly the markers to be used will vary from cell type to cell type. Once a suitable quantity of specific cells has been prepared, then they can be administered to the patient as part of routine methods of autonomous cell replacement therapies. Hence it can be seen that the method of the invention not only is of great use in the preparation of cells for therapeutic purposes (and therefore has evident industrial applicability), but also that method of the invention provides a significant advantage in that the tissue type used for preparing a suitable quantity of specific cells is readily accessible.

An optional further step can be included in the protocol provided above as an embodiment of the invention. This optional further step involves the cuituring of the cells isolated from the heterogeneous population, before those cells are administered to the patient as an autonomous cell replacement therapy. The step of cuituring the isolated cells is performed using standard cell cuituring techniques commonly known and used in the art. This additional cuituring step can be used to expand the number of cells prior to administration. In this way, a greater population of cells can be prepared for the autonomous cell replacement therapy. It can be appreciated that this optional further step can be included as appropriate in the further aspects of the invention disclosed herein.

The inventors have determined that activating Notch signalling in the skin epidermal layer leads to the differentiation of cells, including stem or progenitor cells, in the skin dermal layer.

For the purposes of the present invention, "stem cells" are taken to comprise nullipotent, totipotent, multipotent or pluripotent cells.

Stem cells are cells that have the potential to differentiate into a number of cell types in the body. Theoretically, stem cells may divide without limit to replenish other cells for as long as the organism is alive. Upon differentiation, the daughter cell has the potential to remain a stem cell or become another cell type, for example a lung cell and display its characteristics, thus holding promise for many diseases by replacing damaged tissues. These phenomena may be induced under specific physiological and experimental conditions. In general, stem cell therapy/regenerative medicine represents a therapeutic method by which degenerative and/progressive diseases (such as those caused by premature death or malfunction of cell types that the body is unable to replace) may be treated. It is hoped that addition of stem cells may help nucleate and promote the development of functional cells and/or tissues to replace those lost, thereby restoring normal healthy activity/function.

Totipotent cells are those cells capable of giving rise to all extraembryonic, embryonic and adult cells of the embryo. Accordingly it can be seen that totipotent cells may ultimately give rise to any type of differentiated cell found in an embryo or adult. By comparison, pluripotent cells are cells capable of giving rise to some extraembryonic and all embryonic and adult cells. Thus it can be seen that pluripotent cells are able to give rise to a more limited range of cell types than are totipotent cells. Nullipotent cells are those that will not undergo differentiation without the action of an exogenous cue to differentiation. ultipotent cells are cells able to give rise to diverse cell types in response to appropriate environmental cues (such as action of soluble growth factors or the substrate on which the cell, or its progeny, is located), but are more restricted in their potential lineage formation than are pluripotent, nullipotent or totipotent cells.

While not wishing to be bound by any particular theory, the inventors of the present invention consider that activating the Notch signaling pathway in the epidermal layer acts to induce cells present in the dermal layer to differentiate to a range of different cell types. These cells may be the progeny of stem or progenitor cells or may arise from direct differentiation or transdifferentiation.

By "activator of Notch signalling" we include those agents which promote the activation of Notch target genes.

In one embodiment of the invention, the activator is a ligand for Notch receptor proteins.

There are a number of ligands for Notch receptor proteins known in the art; for example, Delta 1 , Jagged 2 and JAGGED 1. Information concerning the Delta-like 1 and Jagged 2 ligands can be obtained from a number of sources; for example representative polypeptide sequences can be obtained from GenBank as can be appreciated by the skilled person: Delta-like 1 , http://www.uniprot.org/uniprot/O00548; Jagged 2, http://www.uniprot.ora/uniprot/Q9Y219. Hence in one embodiment of the first aspect of the invention the ligand is Delta 1 , Jagged 2 and/or Jagged 1 , or a fragment, variant or derivative of said ligands capable of activating Notch signalling. Preferably the activator of Notch signalling is JAGGED 1 , or a fragment, variant or derivative of JAGGED 1 capable of activating Notch signalling.

JAGGED 1 is a membrane bound protein. Human JAG 1 is the ligand for the receptor NOTCH 1.

JAGGED 1 protein encoded by JAG1 is the human homolog of the Drosophila jagged protein. Binding of JAGGED 1 with Notch initiates a series of signalling reactions - the Notch signalling pathway. Hence JAGGED 1 is an activator of Notch signalling.

Information concerning the amino acid sequence, and encoding nucleic acid sequence, of JAGGED 1 polypeptide can be readily obtained from, for example, GenBank or UniProt, and can be easily obtained from those sources by a person skilled in the art. An example of an amino acid sequence of the JAGGED 1 polypeptide is provided herein. This also includes a URL for the UniProt entry (obtained by searching the database with the name of the polypeptide).

As way of guidance, an example of an amino acid sequence of JAGGED 1 is provided in Swiss-Prot entry P78504 (http://www.uniprot.org/uniprot/P78504), and given below:

JAGGED 1 : (SEQ ID N0.1 ) RSPRTRGRSGRPLSLLLALLCALRAKVCGASGQFELEILSMQNV GELQNGNCCGGARNPGDRKCTRDE CDTYFKVCLKEYQSRVTAGGPCSFGSGSTPVIGGNTFNLKASRGNDRNRIVLPFSFAWPRSYTLLVEA D SSNDTVQPDSIIEKASHSGMINPSRQWQTLKQNTGVAHFEYQIRVTCDDYYYGFGCNKFCRPRDDFFGHY ACDQNGNKTCMEG MGPECNRAICRQGCSP HGSC LPGDCRCQYG QGLYCDKCIPHPGCVHGICNEP QCLCET WGGQLCDKDLNYCGTHQPCLNGGTCSNTGPDKYQCSCPEGYSGPNCEIAEHACLSDPCHNRGS CKETSLGFECECSPGWTGPTCSTNIDDCSPNNCSHGGTCQDLVNGFKCVCPPQ TG TCQLDANECEAKP CVNAKSCK LIASYYCDCLPGWMGQNCDININDCLGQCQNDASCRDLVNGYRCICPPGYAGDHCERDIDE CASNPCLNGGHCQNEINRFQCLCPTGFSGNLCQLDIDYCEPNPCQNGAQCYNRASDYFCKCPEDYEG NC SHLKDHCRTTPCEVIDSCTVAMASNDTPEGVRYISSNVCGPHGKCKSQSGGKFTCDCNKGFTGTYCHENI NDCESNPCRNGGTCIDGV SYKCICSDG EGAYCETNINDCSQNPCHNGGTCRDLVNDFYCDCKNGWKGK TCHSRDSQCDEATCNNGGTCYDEGDAFKCMCPGGWEGTTCNIARNSSCLPNPCHNGGTCWNGESFTCVC KEG EGPICAQNTNDCSPHPCYNSGTCVDGD WYRCECAPGFAGPDCRININECQSSPCAFGATCVDEIN GYRCVCPPGHSGAKCQEVSGRPCITMGSVIPDGAK DDDCNTCQCLNGRIACSKV CGPRPCLLHKGHSE CPSGQSCIPILDDQCFVHPCTGVGECRSSSLQPVKTKCTSDSYYQDNCA ITFTFNKE MSPGLTTEHIC SELRNLNILKNVSAEYSIYIACEPSPSANNEIHVAISAEDIRDDGNPIKEITDKIIDLVSKRDGNSSLIA AVAEVRVQRRPLKNRTDFLVPLLSSVLTVAWICCLVTAFY CLRKRRKPGSHTHSASEDNTTNNVREQLN QIK PIEKHGANTVPIKDYE KNSK SKIRTHNSEVEEDD^4DKHQQKARFAKQPAYTL·VDREEKPP G P TKHPN NKQDNRDLESAQSLNRMEYIV

Note that amino acids 1 to 33 is a signal peptide, and the mature protein starts from reside 34.

As way of guidance, an example of the nucleotide sequence encoding human JAGGED 1 is provided in NM_000214

(http://www.ncbi.nlm.nih.gov/nuccore/168480146).

By "JAGGED 1" we include fragments or variants of this polypeptide, and further homologues, orthologues or paralogues of JAGGED 1 family members which have the necessary biological function.

A "fragment" of said polypeptide will preferably comprise less than the total amino acid sequence of the full native polypeptide; preferably the fragment retains its biological activity, e.g. its capacity to function as a Notch signalling molecule, preferably by functioning as a NOTCH 1 ligand.

A "variant" of the polypeptide also refers to a polypeptide wherein at one or more positions there have been amino acid insertions, deletions, or substitutions, either conservative or non-conservative, provided that such changes result in a protein whose basic properties, for example protein interaction, thermostability, activity in a certain pH- range (pH-stability) have not significantly been changed. "Significantly" in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein. Preferably the variant retains its biological activity, e.g. its capacity to function as a Notch signalling molecule, preferably by functioning as a NOTCH 1 ligand. By "conservative substitutions" is intended combinations such as Gly, Ala; Val, lie, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.

Such variants may be made using the methods of protein engineering and site-directed mutagenesis as would be well known to those skilled in the art.

A further embodiment of the method of the invention is wherein the fragment or variant of JAGGED 1 is Soluble JAGGED 1.

It is known that RNA transcripts prepared from the JAGGED 1 gene can be subject to 'alternative splicing'; in other words, pre-mRNA molecules can be differentially processed by the cellular splicosome to produce more than one mRNA molecule for translation. As well as the "normal" mRNA transcript which encodes the polypeptide provided above as JAGGED 1 , JAGGED 1 RNA can also be "alternatively spliced" to produce a mRNA transcript encoding Soluble JAGGED 1 polypeptide. As expected from its name, while 'normal' JAGGED 1 is often found in cells as a 'membrane bound' protein, the soluble form of JAGGED 1 is not bound to membrane and is secreted into the extracellular matrix. It is likely that this form of JAGGED 1 has specific biological functions.

An example of a polypeptide sequence for Soluble JAGGED 1 is provided below: Soluble JAGGED 1 : (SEQ ID NO.2)

MRSPRTRGRS GRPLSLLLAL LCALRAKVCG ASGQFELEIL SMQNVNGELQ NGNCCGGARN

PGDR CTRDE CDTYFKVCLK EYQSRVTAGG PCSFGSGSTP VIGGNTFNLK ASRGNDRN I

VLPFSFAWPR SYTLLVEAWD SSNDTVQPDS IIEKASHSGM INPSRQWQTL KQNTGVAHFE

YQIRVTCDDY YYGFGCNKFC RPRDDFFGHY ACDQNGNKTC MEGWMGPECN RAICRQGCSP

KHGSCKLPGD CRCQYGWQGL YCDKCIPHPG CVHGICNEPW QCLCETNWGG QLCDKDLNYC

GTHQPCLNGG TCSNTGPDKY QCSCPEGYSG PNCEIAEHAC LSDPCHNRGS CKETSLGFEC

ECSPGWTGPT CSTNIDDCSP NNCSHGGTCQ DLVNGFKCVC PPQWTGKTCQ LDA ECEA P

CVNA SCK L IASYYCDCLP G MGQNCDIN INDCLGQCQ DASCRDLVNG YRCICPPGYA

GDHCERDIDE CASNPCLNGG HCQNEINRFQ CLCPTGFSGN LCQLDIDYCE PNPCQNGAQC

Y RASDYFCK CPEDYEGKNC SHLKDHCRTT PCEVIDSCTV AMASND PEG VRYISSNVCG

PHGKCKSQSG GKFTCDCNKG FTGTYCHENI NDCESNPCR GGTCIDGVNS YKCICSDG E

GAYCETNIND CSQNPCHNGG TCRDLVNDFY CDCKNGWKGK TCHSRDSQCD EATC NGGTC

YDEGDAFKCM CPGGWEGTTC NIAR SSCLP NPCHNGGTCV VNGESFTCVC KEGWEGPICA QNTNDCSPHP CYNSGTCVDG DNWYRCECAP GFAGPDCRIN INECQSSPCA FGATCVDEIN GYRCVCPPGH SGAKCQEVSG RPCITMGSVI PDGAKWDDDC NTCQCLNGRI ACSKVWCGPR PCLLHKGHSE CPSGQSCIPI LDDQCFVHPC TGVGECRSSS LQPVKTKCTS DSYYQDNCAN ITFTFNKEM SPGLTTEHIC SELRNLNILK NVSAEYSIYI ACEPSPSANN EIHVAISAED IRDDGNPI E ITD IIDLVS KRDGNSSLIA AVAEVRVQRR PLK RTS

We also include "fusions" of the JAGGED 1 polypeptide in which said polypeptide is fused to any other polypeptide. For example, the said polypeptide may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said polypeptide. Examples of such fusions are well known to those skilled in the art. Similarly, the said polypeptide may be fused to an oligo-histidine tag such as His6 or to an epitope recognised by an antibody such as the well known Myc tag epitope.

It will be recognised by those skilled in the art that the amino acid sequence of the JAGGED 1 polypeptide can be used to identify homologues to that polypeptide (or nucleic acid encoding the polypeptide).

Methods by which homologues (or orthologues or paralogues) of polypeptides can be identified are well known to those skilled in the art: for example, in silico screening or database mining. Preferably, such polypeptides have at least 40% sequence identity, preferably at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to the JAGGED 1 polypeptide.

Methods of determining the percent sequence identity between two polypeptides are well known in the art. For example, the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally.

The polypeptide, or fragments, variants or homologues thereof, may originate from any organism. However, a preferred embodiment of the first aspect of the invention is wherein the JAGGED 1 polypeptide components, or fragments, variants or homologues thereof, are mammalian; more preferably they are human. A further type of activator which can be used in the method of the invention is where the activator is a member of the Notch signalling pathway and can function to activate that pathway. A preferred embodiment is where the activator is fragment of NOTCH. More preferably the activator is Notch intracellular domain (NICD).

As mentioned above, once the extracellular domain interacts with a ligand, an ADAM- family metalloprotease cleaves the notch protein just outside the membrane. This releases the extracellular portion of notch, which continues to interact with the ligand. The ligand plus the notch extracellular domain is then endocytosed by the ligand- expressing cell. After this first cleavage, an enzyme called γ-secretase cleaves the remaining part of the notch protein just inside the inner leaflet of the cell membrane of the notch-expressing cell. This releases the intracellular domain of the notch protein - termed the Notch intracellular domain (NICD), which then moves to the nucleus, where it can regulate gene expression thus activating the Notch signaling pathway.

Further information on NOTCH 1 (a member of the Notch signaling pathway) is provided in Swiss-Prot entry P46531 (http://www.uniprot.org/uniprot/P46531 ). A reference nucleotide sequence for NOTCH 1 is provided as a link from this accession.

The Notch intracellular domain (NICD) comprises amino acid residues 1754 to 2555 of the amino acid sequence of NOTCH 1 provided in Swiss-Prot entry P46531.

NICD (SEQ ID N0.3)

VLLSRKRRRQHGQLWFPEGFKVSEASKKKRREPLGEDSVGLKPLKNASDGALMDDNQNEW GDEDLET KFRFEEPWLPDLDDQTDHRQWTQQHLDAADLRMSAMAPTPPQGEVDADCMD VNVRGPDGFTPLMIASCSGGGLETGNSEEEEDAPAVISDFIYQGASLHNQTDRTGETALH LAARYSRSDAAKRLLEASADANIQDNMGRTPLHAAVSADAQGVFQILIRNRATDLDARMH DGTTPLIIAARLAVEGMLEDLINSHAD AVDDLG SALHWAAAVN VDAAVVIJLKNGA KDMQNNREETPLFLAAREGSYETAKVLLDHFANRDITDHMDRLPRDIAQERMHHDIVRLL DEY LVRSPQLHGAPLGGTPTLSPPLCSPNGYLGSLKPGVQGK VRKPSSKGliACGSKEA

KDLKARRKKSQDGKGCLLDSSGMLSPVDSLESPHGYLSDVASPPLLPSPFQQSPSVPLNH LPGMPDTHLGIGHLNVAAKPEMAALGGGGRLAFETGPPRLSHLPVASGTSTVLGSSSGGA LNFTVGGSTSLNGQCEWLSRLQSGMVPNQYNPLRGSVAPGPLSTQAPSLQHGMVGPLHSS LAASALSQMMSYQGLPSTRLATQPHLVQTQQVQPQNLQMQQQNLQPANIQQQQSLQPPPP PPQPHLGVSSAASGHLGRSFLSGEPSQADVQPLGPSSLAVHTILPQESPALPTSLPSSLV PPVTAAQFLTPPSQHSYSSPVDNTPSHQLQVPEHPFLTPSPESPDQWSSSSPHSNVSDWS EGVSSPPTSMQSQIARIPEAFK

As way of guidance, an example of the nucleotide sequence encoding human NOTCH1 is provided in NM_017617 (http://www.ncbi.nlm.nih.gov/nuccore/148833507).

By "NICD" we include fragments or variants of this polypeptide, and further homologues, orthoiogues or paralogues of NICD family members which have the necessary biological function.

A discussion is provided above on the terms "fragments or variants of this polypeptide, and further homologues, orthoiogues or paralogues", and the information provided therein also applies to NICD.

By "activator of Notch signalling", we also include those agents which activate those members of the Notch signalling pathway; for example, agents that interact with the NOTCH polypeptide to activate the signalling pathway.

As way of example, it can be envisaged that molecules that specifically interact with NOTCH, particularly the Notch extracellular domain, to induce NOTCH activity can function as activators of Notch signalling. For example, it can be expected that some antibodies, particularly monoclonal antibodies, may interact with this domain of NOTCH to activate the pathway.

Monoclonal antibodies that bind to the extracellular domain of NOTCH can be obtained from a wide variety of commercial sources, for example,

By "activator of Notch signalling", we also include those agents which function as members of the Notch signalling pathway, or activate those members of the Notch signalling pathway.

As way of example, the metalloprotease enzyme ADAM 10 may be used as an activator of Notch signalling. As well as ADAM 10, we also include fragments or variants of this polypeptide, and further homologues, orthoiogues or paralogues of ADAM 10 family members which have the necessary biological function. Further information on ADAM 10 is provided at GenBank accession number NP_001101.1 (http://www.ncbi.nlm.nih.gOv/protein/NP_001101.1 ). A reference nucleotide sequence for ADAM 10 is provided as a link from this accession.

An example of a polypeptide sequence for ADAM 10 is provided below:

ADAM 10 (SEQ ID NO.4)

MVLLRVLILLLSWAAGMGGQYGNPLNKYIRHYEGLSY VDSLHQKHQRAKRAVSHEDQFLRLDFHAHGRH FNLRMKRDTSLFSDEFKVETSNKVLDYDTSHIYTGHIYGEEGSFSHGSVIDGRPEGFIQTRGGTFYVEPA ERYIKDRTLPFHSVIYHEDDI YPHKYGPQGGCADHSVFERMRKYQMTGVEEV QIPQEEHAA GPELLR KKRTTSAEKNTCQLYIQTDHLFFKYYGTREAVIAQISSHVKAIDTIYQTTDFSGIRNISFMVKRIRINTT ADEKDPTNPFRFPNIGVEKFLELNSEQNHDDYCLAYVFTDRDPDDGVLGIiAWVGAPSGSSGGICE SKLY SDGK KSLNTGIITVQNYGSHVPPKVSHITFAHEVGHNFGSPHDSGTECTPGESKNLGQKENG YIMYAR ATSGDKLN NKFSLCSIRNISQVLEKKRN CFVESGQPICGNGMVEQGEECDCGYSDQC DECCFDANQP EGRKCKLKPGKQCSPSQGPCCTAQCAFKSKSE CRDDSDCAREGICNGFTALCPASDPKPNFTDCNRHTQ VCINGQCAGSICE YGLEECTCASSDGKDDKELCHVCCMKK DPSTCASTGSVQ SRHFSGRTITLQPGS PCNDFRGYCDVFMRCRLVDADGPLARLKKAIFSPELYENIAEWIVAHWWAVLLMGIALIMLMAGFIKICS VHTPSSNPKLPPPKPLPGTLKRRRPPQPIQQPQRQRPRESYQMGHMRR

A discussion is provided above on the terms "fragments or variants of this polypeptide, and further homologues, orthologues or paralogues", and the information provided therein also applies to ADAM 10.

We also include "fusions" of the ADAM 10 polypeptide, as discussed above in relation to JAGGED 1.

As way of example, the metalloprotease enzyme ADAM 17 may be used as an activator of Notch signalling. As well as ADAM 17, we also include fragments or variants of this polypeptide, and further homologues, orthologues or paralogues of ADAM 17 family members which have the necessary biological function.

Further information on ADAM 17 is provided at GenBank accession number P78536 (http://www.ncbi.nlm.nih.goV/protein/P78536.1 ). A reference nucleotide sequence for ADAM 17 is provided as a link from this accession. An example of a polypeptide sequence for ADAM17 is provided below: ADAM17 (SEQ ID NO.5)

MRQSLLFLTS WPFVLAPRP PDDPGPGPHQ RLEKLDSLLS DYDILSLSNI QQHSVRKRDL QTSTHVETLL TFSALKRHFK LYLTSSTERF SQNFKWWD GK ESEYTVK WQDFFTGHW GEPDSRVLAH IRDDDVIIRI NTDGAEY IE PLWRFVNDTK DKRMLVYKSE DIKNVSRLQS PKVCGYLKVD NEELLPKGLV DREPPEELVH RVKRRADPDP MKNTCKLLW ADHRFYRYMG RGEESTTTNY LIELIDRVDD IYR TSWDNA GFKGYGIQIE QIRILKSPQE VKPGEKHYNM AKSYPNEEKD AWDV MLLEQ FSFDIAEEAS KVCLAHLFTY QDFDMGTLGL YVGSPRANS HGGVCPKAYY SPVGK NIYL NSGLTSTKNY GK ILT E D LVTTHELGHN FGAEHDPDGL AECAPNEDQG G YVMYPIAV SGDHENNKMF SNCSKQSIYK TIESKAQECF QERSNKVCGN SRVDEGEECD PGIMYLN DT CCNSDCTLKE GVQCSDRNSP CCKNCQFETA Q KCQEAINA TC GVSYCTG NSSECPPPGN AEDDTVCLDL GKCKDGKCIP FCEREQQLES CACNETDNSC KVCCRDLSGR CVPYVDAEQK NLFLRKGKPC TVGFCDMNGK CEKRVQDVIE RFWDFIDQLS INTFGKFLAD NIVGSVLVFS LIFWIPFSIL VHCVD KLDK QYESLSLFHP S VEMLSSMD SASVRIIKPF PAPQTPGRLQ PAPVIPSAPA APKLDHQRMD TIQEDPSTDS HMDEDGFEKD PFPNSSTAAK SFEDLTDHPV TRSEKAASFK LQRQNRVDSK ETEC

A discussion is provided above on the terms "fragments or variants of this polypeptide, and further homologues, orthologues or paralogues", and the information provided therein also applies to ADAM 17.

We also include "fusions" of the ADAM 17 polypeptide, as discussed above in relation to JAGGED 1.

The polypeptide to be used in the methods of the invention may be produced using a number of known techniques. Preferably such polypeptides are JAGGED 1 , Soluble JAGGED 1 , NICD, ADAM 10 or ADAM 17, as described above. For instance, the polypeptide may be isolated from naturally occurring sources. Indeed, such naturally occurring sources of the polypeptide may be induced to express increased levels of the polypeptide, which may then be purified using well-known conventional techniques. Alternatively cells that do not naturally express the polypeptide may be induced to express the polypeptide.

It is possible to isolate polypeptide using a molecule which can specifically bind to polypeptide, such as an antibody. Using such a binding molecule in conditions that preserve the integrity of the polypeptide, such as non-denaturing conditions, the polypeptide can be isolated substantially pure of any contaminants.

For example, a culture of cells that contain the polypeptide can be grown in vitro, the polypeptide extracted from the cells, and using an antibody to the polypeptide, preferably under non-denaturing conditions, the polypeptide can be isolated.

A further suitable technique to isolate the polypeptide involves cellular expression of a fusion between a gene encoding the polypeptide and a fusion tag or label, such as a his construct. The expressed polypeptide may subsequently be highly purified by virtue of the his "tag".

Cells may be induced to express increased levels of the polypeptide. This effect may be achieved either by manipulating the expression of endogenous polypeptide, or causing the cultured cells to express exogenous polypeptide. Expression of exogenous polypeptide may be induced by transformation of cells with well-known vectors into which cDNA encoding the polypeptide may be inserted. It may be preferred that exogenous polypeptide is expressed transiently by the cultured cell (for instance such that expression occurs only during ex vivo culture).

As discussed above, information concerning nucleic acid sequences encoding polypeptide can be obtained from, for example, GenBank or UniProt, and can be easily obtained from those sources by a person skilled in the art.

It will be appreciated that the nucleic acid encoding polypeptide may be delivered to the biological cell without the nucleic acid being incorporated in a vector. For instance, the nucleic acid encoding polypeptide may be incorporated within a liposome or virus particle. Alternatively a "naked" DNA molecule may be inserted into the biological cell by a suitable means e.g. direct endocytotic uptake.

The exogenous genes encoding polypeptide (contained within a vector or otherwise) may be transferred to the biological cells by transfection, infection, microinjection, cell fusion, protoplast fusion or ballistic bombardment. For example, transfer may be by ballistic transfection with coated gold particles, liposomes containing the exogenous gene, and means of providing direct DNA uptake (e.g. endocytosis). The method of the invention outlined above includes the step of the isolating the required differentiated cells from the skin tissue. Such isolation methods are well known and are commonly used in the art.

For example, differentiated cells can be isolated from the skin tissue population using FACS - fluorescent-activated cell sorting. Fluorescence-activated cell sorting is a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell.

The technique is of particular use in stem cell research. When cells are obtained from multicellular organisms, the DNA is naturally identical in each one. However, the proteins of each cell vary widely. Therefore, a method of separating cells based on their phenotype i.e. FACS is extremely useful. Cell subpopulations can be separated by tagging with an antibody conjugated with a fluorescent dye, targeted to a protein specific to those cells. When excited by the laser, the dye emits a particular wavelength of light, allowing the apparatus to identify and isolate the relevant cells.

As can be appreciated by the skilled person, the antibodies used in the FACS will vary from specific cell type to cell type, since each specific type of cell has a particular "signature" of cell markers. Examples of particular antibodies that can be used are discussed further below.

Preferably the methods of the invention use a substantially pure population of the required differentiated cells. That population can be prepared according to the isolation methods described above, particularly FACS.

By "substantially pure" we include that the population of cells have at least 70% of the specific cell type specified. Preferably there are 75%, 80%, 85%, 90%, 95% or more specific cells in the population.

A second aspect of the invention provides a population of cells prepared according to the first aspect of the invention for use as a medicament for autologous cell replacement therapy. A third aspect of the invention provides a method of treating a subject having a disease condition and in need of autologous cell replacement therapy, the method comprising preparing a population of cells for use autologous cell replacement therapy according to the first aspect of the invention and then administering the cells to the said subject.

As discussed further above in relation to the first aspect of the invention, the inventors have determined that activating Notch signalling, preferably in epidermal layer, promotes cell differentiation in the skin, specifically in the dermal skin layer. A wide range of different cell types can be prepared, and indeed the cells differentiate as a heterogeneous mixture of cells. Such cell types include neural progenitor cells, melanocytes and dermal papilla cells; the neural progenitor cells are capable of subsequently differentiating in to neurons and Schwann cells.

Hence leading from this finding it can be understood that the differentiated cells prepared according to the method of the first aspect of the invention can be used in a medicament for the treatment of disorders requiring autologous cell replacement therapy.

As will be appreciated, the cells to be used will vary according to the condition to be treated.

In one embodiment of the aspects of the invention, the differentiated cells are neural progenitor cells and the autologous cell replacement therapy is for the treatment of a condition is characterised by degeneration, damage to, the loss of, or the disorder in nervous tissue.

"Neural cells" are the cells located within the nervous system. Neural cells come in a variety of different forms; however, the most common delineation between types stems from their function. Sensory neurons are responsible for the brain and nervous system's response mechanisms to stimuli such as light, sound and touch. Motor neurons cause muscle contractions and affect glands when signals are sent from the brain or spinal cord. In addition, inter-neurons are responsible for connecting each neural cell within the various regions of the nervous system. Other types of nerve cells also exist, each with its own unique characteristics and function. These types of neural cells are generally found in specific areas of the nervous system. Among these include anterior horn cells, basket cells, Betz cells, granule cells, medium spiny neurons, Pukinje cells, pyramidal cells and Renshaw cells.

Damage to or loss of nervous tissue, for example neuronal or glial cells, may occur within the CNS. The CNS includes the brain, the spinal cord, and neurons whose cell bodies lie within, or have a primary synapse in, the brain or spinal cord. Examples of such neurons are neurons of origin of the corticospinal tract, ruborospinal tract and retinal ganglion cells and the CNS branch of sensory axons. There are also neurons whose cell body lies within the CNS, but the axon is largely in the PNS, such as the neurons of the cranial nerves (damage to which can e.g. cause Bell's palsy) and motor neurons that innervate the musculature and whose cell bodies are in the ventral horn of the spinal cord.

A condition associated with neuronal cell loss or damage may be a spinal cord injury, for example an injury caused by assault, accident, tumour, intervertebral disc or bone abnormality, or surgery, e.g. surgery for spinal problems and/or surgery to remove tumours.

Hence a preferred embodiment of the aspects of the invention is where the condition is a spinal injury and the differentiated cells are neurons and/or Schwann cells.

The neuronal cell loss or damage may be CNS damage other than spinal cord injury, particularly CNS damage of the following kinds: hypoxic injury including stroke and perinatal hypoxia; brain injury, including (without limitation) injury caused by assault, accident, tumour (e.g. a brain tumour or a non-brain tumour that affects the brain, such as a bony tumour of the skull that impinges on the brain) or surgery, e.g. surgery to remove tumours or to treat epilepsy; immune-mediated disease including multiple sclerosis; CNS damage following infection, neurodegenerative diseases and genetic diseases affecting the nervous system, including metabolic disorders (e.g. Gaucher's) and leukodystrophies (e.g. Krabbe's).

Neurodegenerative diseases may include diseases characterised by the intracellular accumulation of protein aggregates. Aggregates may accumulate, for example, in the cytoplasm of a cell. Commonly, aggregates may form in neuronal cells, such as brain cells, for example in disorders such as Alzheimer's disease, Huntington's disease, Parkinson's disease, and motor neurone disease (amyotrophic lateral sclerosis).

As mentioned above, substantially pure populations of specific cell types can be isolated from the skin tissue, preferably the dermal layer, using methods routine in the art. For example FACS can be used to prepare neural progenitor cells using antibodies that specifically bind with the cell surface markers CD45, CD34, PDGFRa and p75 NTR. The neural progenitors do not bind CD45 and CD34, and hence those markers (which do bind immunocytes and dermal fibroblasts respectively) can be used to remove unwanted cells from the tissue sample.

Further cell surface markers can also be optionally used to isolate the neural cells; for example, integrin subunits Beta 1 and/or Alpha 2, 4, or 6 and also the EGFR receptor.

Antibodies to the specific cell markers can be obtained from a variety of commercial sources; for example, CD34 (BD biosciences), CD45 (BD biosciences), CD1 17 (c-kit; BD biosciences), PDGFRa (R and D systems), P75NTR (Alomone), EGFR (Sigma), Beta 1 integrin (CD29, eBiosciences), Integrins alpha 2, 4, 6, (CD49b, CD49d, CD49f respectively; antibodies from eBiosciences).

It can be appreciated that the neural progenitor cells can subsequently differentiate to further neural cells, including neurons and Schwann cells. Hence as used herein, by 'neural progenitor cells' we also include neurons and Schwann cells.

In a further embodiment of the aspects of the invention, the differentiated cells are melanocytes and the autologous cell replacement therapy is for the treatment of a condition is characterised by degeneration, damage to, the loss of, skin pigmentation.

A further preferred embodiment is where the disorder is vitiligo.

Vitiligo is quite a common skin disease which affects at least one person in every hundred in countries throughout the world including the UK. Anyone, male or female, irrespective of skin colour or ethnic origin can develop the condition. Vitiligo causes the skin, and sometimes the hair, to turn white in patches. This is because melanocytes, the cells which give the skin its colour, have either been damaged or destroyed. The disease can spread, rapidly or slowly, to cover the entire body surface (universal vitiligo) but this is not inevitable. The most common form of vitiligo appears in symmetrical form (generalized vitiligo) affecting both sides of the body. In some cases only one half of the body is affected (segmental vitiligo) and this type has limited progression and is more difficult to treat. Vitiligo can begin at any age, though about fifty percent of people develop it before the age of twenty.

Vitiligo is not infectious. Although there are no physical symptoms apart from sunburn in the white patches if they are not protected from the sun, it can cause severe psychological distress, especially when the face, neck, hands and genitals are affected. Although the disease is more noticeable on dark or tanned skin the degree of distress is not necessarily linked to skin colour or to the extent of the disease. However, people with dark skin from certain ethnic groups who develop vitiligo may feel particularly stigmatized and fear a loss of identity should the disease become widespread.

The course of vitiligo is unpredictable. Some people may not notice a change in their condition for many years, while for others it can spread quite rapidly. In some cases the white patches can spontaneously repigment, particularly in children, though it is rare for the disease to resolve completely without treatment.

In relation to the present invention, the inventors have determined that the method of the first aspect of the invention can be used to prepare melonocytes that can transplanted into a patient and hence can be used to treat skin pigmentation disorders including vitiligo. Clearly given the prevalence of vitiligo in the human population, the method of this aspect of the invention can be of great use in treating this distressing disorder.

As mentioned above, substantially pure populations of specific cell types can be isolated from the skin tissue, preferably the dermal layer, using methods routine in the art. For example FACS can be used to prepare melanocyte cells using antibodies that specifically bind with the cell surface markers CD117 (c-kit; Immunotech), and also be isolating cells that do not have the CD45 cell marker (BD Biosciences) and CD34 marker (BD Biosciences).

In cases where vitiligo is associated with inflammation, melanocytes could be combined with anti-inflammatory agents such as dexamethasome. Hence in this embodiment of the invention where disorder is vitiligo associated with inflammation, the autologous cell replacement therapy comprises melanocytes in combination with one or more anti-inflammatory agents.

In a further embodiment of the second and third aspects of the invention, the differentiated cells are dermal papilla cells and the autologous cell replacement therapy is for the treatment of a condition is characterised by degeneration, damage to, the loss of, hair follicle formation and/or growth.

Hair follicle development occurs only once in an animals' life during late gestational and early post-natal stages. In this short window of time, all of the hair follicles on the body are generated. It has been shown by recombining epidermis from hair-forming skin regions with dermis from non-hair-forming skin regions that the primary signals for hair follicle induction lie within the dermal population. The importance of reciprocal signalling between the epidermis and dermis is well-established in the context of the hair follicle, since both hair development and postnatal maintenance depend on reciprocal interactions between the epidermis and specialized dermal cells that constitute the dermal papilla (DP).

It is known that populations of stem cells in the dermis and epidermis supply the cells needed for normal tissue homeostasis and during wound repair. Adult epidermal stem cells retain their capacity to respond to hair inducing signals throughout life and when these cells are mixed with embryonic or neonatal dermal cells, but not adult dermal cells, and re-implanted subcutaneously new hair follicles arise.

Hair plays a vital role in thermoregulation, but importantly in modern society the contribution of hair to our physical appearance is tightly linked to the psychological well-being of humans. Hair loss can cause devastating feelings and negatively impact a person's self-esteem, self-image and confidence often leading to depression. Thus, not surprisingly billions of pounds are spent each year in the UK alone on hair loss solutions ranging from hair restoration surgery to hair growth-stimulating chemicals to prosthetics such as hair extensions and wigs.

There are a number of different forms of hair loss. Specific conditions include alopecia and male pattern baldness.

Different forms of hair loss are caused by structural and/or cyclical changes to the hair follicle, some of which are reversible, and others render the follicle incapable of producing new hair. Some forms of alopecia, such as androgenetic alopecia, occur in adults during normal healthy aging, but other forms of hair loss are often accompanied by trauma to the follicle either from physical or immunological damage.

The present inventors have determined that the method of the first aspect of the invention can be used to prepare dermal papilla cells that, when transplanted into a patient, can induce new hair follicle formation (neogenesis). Further information is provided in the accompanying example. Clearly given the prevalence of hair loss in the human population, for example particularly alopecia or male pattern baldness, the method of this aspect of the invention can be of great use in treating this distressing disorder.

As mentioned above, substantially pure populations of specific cell types can be isolated from the skin tissue, preferably the dermal layer, using methods routine in the art. For example FACS can be used to prepare dermal papilla cells using antibodies that specifically bind with the cell surface markers. For example, using FACS cells that do not have the CD117, CD45 and CD34 cell surface markers (antibodies as above); a example of a positive cell marker for dermal papilla cells is integrin subunit alpha 8.

A fourth aspect of the invention provides a method of treating a condition characterised by degeneration, damage to, the loss of, or the disorder in nervous tissue using autologous cell replacement therapy, said method comprising contacting skin tissue of a patient in need of therapy to one or more activators of Notch signalling, and subsequently isolating the differentiated neural progenitor cells from the skin tissue.

A fifth aspect of the invention provides a method of treating a condition characterised by degeneration, damage to, the loss of, or the disorder in skin pigmentation using autologous cell replacement therapy, said method comprising contacting skin tissue of a patient in need of therapy to one or more activators of Notch signalling, and subsequently isolating the differentiated melanocytes cells from the skin tissue.

A sixth aspect of the invention provides a method of treating a condition characterised by degeneration, damage to, the loss of, or the disorder in hair formation and/or growth using autologous cell replacement therapy, said method comprising contacting skin tissue of a patient in need of therapy to one or more activators of Notch signalling, and subsequently isolating the differentiated dermal papilla cells from the skin tissue.

An embodiment of the fourth, fifth, and sixth aspects of the invention is where the methods further comprising the step of administering the isolated differentiated cells to the patient.

An embodiment of the fourth, fifth and sixth aspects of the invention is where the one or more activators of Notch signalling are defined in relation to the first aspect of the invention.

A further embodiment of the fourth, fifth and sixth aspects of the invention is where the epidermal layer of the skin tissue is contacted with the one or more activators of Notch signalling, and the required differentiated cells are isolated from the dermal layer of the skin tissue.

A seventh aspect of the invention provides a method of prevention or treating a condition characterised by degeneration, damage to, the loss of, or the disorder in skin pigmentation said method comprising contacting skin tissue of a patient in need of therapy to one or more activators of Notch signalling.

The inventors have also determined that skin pigmentation conditions, such as vitiligo, can also be treated by contacting skin tissue of a patient in need of therapy to one or more activators of Notch signalling. Following exposure to the one or more activators of Notch signalling, cells in the skin, preferably dermal layer cells, differentiate to melanocytes thus alleviating the condition with out the need for autologous cell replacement therapy. Hence this method of the invention does not require the differentiated cells to be isolated from the skin tissue, since the differentiation of cells in situ caused by activating of Notch signalling are sufficient to alleviate the condition.

It can be appreciated that this aspect of the invention is based on the inventors' findings that activating Notch signalling in the epidermis leads to the differentiation of cells in the dermis, including stem or progenitor cells. Thus the seventh aspect of the invention has this feature in common with the further aspects of the invention provided herein.

An embodiment of the seventh aspect of the invention is where the one or more activators of Notch signalling are defined in relation to the first aspect of the invention.

An eighth aspect of the invention provides a method of promoting the differentiation of dermal, the method comprising contacting skin tissue to one or more activators of Notch signalling, and subsequently isolating the differentiated cells from the skin tissue.

The method of the eighth aspect of the invention can also be reformulated so as not to require direct interaction with a patient.

Hence an alternative eighth aspect of the invention provides a method of promoting the differentiation of dermal cells, the method comprising isolating the required differentiated cells from skin tissue, wherein skin tissue has been contacted with one or more activators of Notch signalling.

An embodiment of the eighth aspect of the invention is where the differentiated cells are neural progenitor cells, melanocytes or dermal papilla cells; the progenitor cells can further differentiate to neural and/or Schwann cells.

A ninth aspect of the invention provides a method of preparing a substantially pure population of neural progenitor cells, melanocytes or dermal papilla cells, the method comprising contacting skin tissue to one or more activators of Notch signalling, and subsequently isolating the differentiated cells from skin tissue.

The method of the ninth aspect of the invention can also be reformulated so as not to require direct interaction with a patient. Hence an alternative ninth aspect of the invention provides a method of preparing a substantially pure population of neural progenitor cells, melanocytes or dermal papilla cells, the method comprising isolating the required differentiated cells from skin tissue, wherein skin tissue has been contacted with one or more activators of Notch signalling.

As way of example, there now follows details of a method which can be used according to the aspects of the invention.

Skin tissue is contacted with one or more activators of Notch signalling for a suitable period of time. The skin tissue is then collected. Following dissection, the fat and muscle was removed from the skin by cutting or gently scraping the inside surface with a scalpel. The skin was then treated with dispase then digested with a mixture of Collagenase Type I, Collagenase Type II, Collagenase Type IV and/or Hyaluronidase (full details are provided in the accompanying examples).

Following digestion, the mixture is filtered to isolate the cells. The specific cells to be used for the autologous cell replacement therapy are then isolated using the FACS procedure outlined above.

The aspects of the invention described above can relate to the use of cells as therapeutic agents in medicaments.

Medicaments should comprise a therapeutically effective amount of the cells and a pharmaceutically acceptable vehicle.

A "therapeutically effective amount" is any amount of the cells which can be used for the particular autologous cell replacement therapy.

A "subject" or "patient" may be a vertebrate, mammal, domestic animal or human being. It is preferred that the subject or patient to be treated is human.

A "pharmaceutically acceptable vehicle" as referred to herein is any physiological vehicle known to those skilled in the art as useful in formulating pharmaceutical compositions. Where the medicament or method of the invention involves the use of biological cells, preferably the formulation for comprises biological cells in a suitable liquid carrier.

Such a liquid carrier is preferably non-immunogenic, and may comprise a saline solution, cell culture medium, or distilled water. Formulations for injection may be as described above, or may also be provided in the form of a gel, which may preferably be capable of resolution by the body of the subject treated. Formulations suitable for implantation may take the forms described for injection or inhalation, and may also comprise biological cells provided in a scaffold or matrix capable of providing a foundation for new tissue development.

Methods of administering medicaments containing cells to a patient are well known in the art. Hence once a suitable population of the required differentiated cells is prepared according to the methods set out above, then the medicament can be used according to usual practice.

In particular, methods of implanting cells in to a patient are well known in the art. Hence where the cells are to be used for an autologous cell replacement therapy, for example, a condition is characterised by degeneration, damage to, the loss of, or the disorder in nervous tissue; skin pigmentation; hair follicle formation and/or growth, then well known methods in the art can be used to administer the cells to the patient or subject.

A tenth aspect of the invention provides a composition formulated for administration to the skin comprising one or more activator of Notch signalling.

"Activator of Notch signalling" is defined above in relation to the first aspect of the invention, and those particular activators discussed above are included in this aspect of the invention. Preferably the composition comprises Delta 1 , Jagged 2, JAGGED 1 , Soluble JAGGED 1 , NICD, ADAM 10 and/or ADAM 17, or a fragment, variant or derivative of said activators capable of activating Notch signalling.

In this aspect of the invention the activator of Notch signalling is formulated for administration to the skin. In one embodiment of this aspect, the activator is formulated for subcutaneous injection.

Hence in this embodiment the activator of Notch signalling is formulated as a liquid medicament for subcutaneous injection, or as a lyophilised formulation which is subsequently presented in a liquid form shortly before being administered to the patient or subject in need of autologous cell replacement therapy. Methods of preparing liquid formulations suitable for subcutaneous administration are well known in the art. Devices may be provided, so that the formulation may be delivered subcutaneously to the epidermis or epidermis/dermis boundary of the skin. The formulations may be administered by biolistic means known in the art, or by injection. When injected a device may be, for example, an automated delivery device which is designed to cause delivery to the desired layer of the skin. Thus, a device may be provided in which, in use, a needle only penetrates the skin to the desired depth, in order that the one or more activators of Notch signalling may be administered appropriately.

However, preferably the activator is formulated for topical application to the skin.

For application topically to the skin, the activator of Notch signalling can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

It will be appreciated that the amount of activator of Notch signalling to be used in the invention and thus formulated into a medicament, is determined by its biological activity and bioavailability which, in turn, depends on the mode of administration and the physicochemical properties of the agents employed. The frequency of administration will also be influenced by the abovementioned factors, and particularly the half-life of the cells or agents within the subject being treated. Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular agents in use, the strength of the preparation, the mode of administration, and the advancement of the disease condition that is to be treated. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration. Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials etc), may be used to establish specific formulations of compositions and precise therapeutic regimes (such as daily doses of the agents and the frequency of administration).

Daily doses may be given as a single administration (e.g. a daily tablet for oral consumption or as a single daily injection). Alternatively agents used may require administration twice or more times during a day, dependent of pharmacological, toxicological or efficacy studies.

All of the features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

The invention will now be further described with reference to the following examples and Figures.

Figure 1. Characterisation of K14NICDER transgenic mice. (A) H & E stained back skin sections of wild type (WT) and K14NICDER transgenic littermate mice treated with 40HT for 21 days. Insert shows higher magnification view of epidermis and underlying dermis. Epidermal layers are indicated, b: basal; s: spinous; g: granular; c: cornified. (B) Back skin of 40HT-treated wild type (WT) and K14NICDER transgenic littermate mice stained with antibodies to Hes1 (red) and Keratin 14 (green). Asterisk marks Hes1 positive dermal cell. (C) RNA in situ hybridization using radiolabeled antisense probes to Hes1 on back skin sections from 40HT-treated wild type (WT) and K14NICDER transgenic mice. Epidermal-dermal boundary is marked by dashed line (red). (D-G) Back skin of 40HT-treated wild type (WT) and K14NICDER transgenic littermate mice stained with antibodies to Keratin 10 (green, D), keratin 17 (red, E), Ki67 (green, F), a6 integrin subunit (green, G) and laminin 5 (red, G). (H) Transmission electron micrographs of the epidermal basement membrane zone in wild type (WT) and K14NICDER transgenic littermate mice treated with 40HT for 14 days. Arrows mark hemidesmosomes. B : basement membrane; Coll: collagen fibres. (B, D, G) Sections were DAPI counterstained (blue). Mice were 40HT-treated for 14 days (C-F), 15 days (B) or 21 days (A, G-H). Scale bars: 1 m (H), 10 μηι (B), 25 μιη (D, right panel; G, left panel; and G, right panel insert) or 50 pm (A, C, D, left panel, E, F).

Figure 2. Notch-induced skin inflammation. (A, B) Immunohistochemical staining of back skin sections from 40HT-treated wild type (WT) and K14NICDER transgenic littermate mice with antibodies to CD4 (A) and CD8 (B). Arrows indicate- T cells in epidermis and dermis. (C) Macroscopic phenotype of 10-week old K14NICDER transgenic mice treated with 40HT or 40HT and Dexamethasone (40HT/DEX) for 21 days. (D-F) Back skin sections of K14NICDER transgenic mice treated with acetone, 40HT or combination of 40HT and Dexamethasone (40HT/DEX) for 21 days. Sections were stained with H & E (D), or labeled with antibodies to CD4 (E), a6 integrin subunit (red) and Iaminin5 (green, F). Nuclei were counterstained with haematoxylin (A, B, E) or DAPI (blue, F). Transgenic and wild type mice were injected with Dexamethasone or saline for 24 days and treated with 40HT or acetone for 21 days (A-F). Scale bars: 10 μηι (F) or 50 pm (A, B, D, E).

Figure 3. Epidermal Notch activity induces accumulation of stromal cells in the upper dermis. (A-H) 4 μηι (A, B, D, F-H) or 150 μπι (C, E) thick back skin sections of 40HT-treated wild type (WT) and K14NICDER transgenic littermate mice were analysed. (A) Alkaline phosphatase activity (blue). Locations of dermal papilla (DP) and arrector pili muscle (AM) are indicated with arrows. Arrows in right hand panel show stromal cell accumulation. (B-D, F-H) Immunolabelling with antibodies to CRABP1 (B, brown), Nestin (C, red; D, green; F, green), laminin 5 (D, red), c-kit (F, red), desmin (G, red), and SM22a (H, red). Arrowheads: arrector pili muscle (G, H); arrows (G, H): stromal cells at epidermal/dermal junction. Asterisk: c-kit positive mast cell. (E) Brightfield image showing dermal melanocytes in K14NICDER transgenic skin. Sections were counterstained with fast red (A), haematoxylin (B), or DAPI (C, D, F-H, blue). Mice were 40HT-treated for 14 days (A-H). Scale bars: 50 m. Figure 4. Kinetics of appearance of Notch induced stromal cells. (A, B, D) Back skin sections of 40HT-treated K14NICDER transgenic mice stained with antibodies to Nestin (A, green or red; B, green), CRABP1 (A, B, red), Sm22a (A, green) and p75 (D, green). (A) Arrows: double-labeled cells; arrowheads: single-labeled cells. (B) Arrow: CRABP1 positive cell. (C) % CRABP1 (C), Nestin (N), Sm22a (S) single-positive cells and CRABPI/Nestin and CRABP1/S 22a double-positive cells at the epidermal- dermal boundary. (E) Skin-derived neurospheres 7 days after seeding, viewed by phase contrast (top) or (bottom) anti-Nestin labelling (red) and counterstained with DAPI (blue). Insert: labelling with secondary antibody alone. (F) % sphere formation by cells from wild type (WT) and K14NICDER transgenic (TG) littermate mice treated with 40HT for 10 or 14 days. (G) Cytospin preparation of dermal cells isolated from 40HT treated K14NICDER skin stained with antibodies to Nestin (green) and CRABP1 (red). Sections and cells were counterstained with DAPI (blue, A, B, D, E, G). Mice were 40HT-treated for 2 days (B), 5 days (B), 7 days (B), 10 days (A, left panel; G) or 14 days (A, middle and right panels; D). Scale bars: 50 pm (D) or 25 μιτι (A, B, G).

Figure 5. Nestin-positive dermal cells are not of epidermal origin. (A, B) Back skin sections of 40HT-treated wild type (WT) and K14NICDER transgenic littermate mice stained with antibodies to Slug 1 (A, brown), E-cadherin (B, green). (C, D) 150 μιη back skin sections (C) and cytospin preparations (D) of cells isolated from 40HTtreated K14CreER/CAG-CAT-EGFP mice and K14CreER/CAG-CATEGFP/ K14NICDER mice stained with antibodies to GFP (green) and Nestin (red). DAPI nuclear counterstain (blue). Mice were 40HT-treated for 14 days (A-D). Scale bars: 20 pm (D) or 50 pm (A- C).

Figure 6. Notch induces Jag1 in the epidermis and dermis. (A, C-E) sections of back skin from wild type (A, C), K14NICDER (A, D), and Kl 4-AN3cateninER ^catER; E) transgenic mice. (A) Back skin sections stained with antibodies to Jaggedl (red) and DAPI counterstained (blue). (B) Western blot of protein lysates from skin of wild-type (WT) and K14NICDER transgenic (K14NICDER) mice probed with anti-Jaggedl . Each lane contains protein from a different mouse. Arrow indicates position of Jaggedl protein. Lower molecular mass bands are nonspecific and serve as loading control. Molecular mass markers (kD) are indicated. (C-E) RNA in situ hybridization using a radiolabeled antisense probe to Jaggedl . Corresponding brightfield (BF) and darkfield (DF) panels show the same field. Red lines mark the epidermal/dermal boundary. Right hand panels in (D) are higher magnification views of boxed region in left hand panels. Mice were 40HT- treated for 21 days (A, B), 10 days (C, D) or 7 days (E). Scale bars: 50 pm.

Figure 7. Jag1 is required for Notch induced skin phenotype. (A-F) H & E stained sections of back skin of wild type (A, WT), K14NICDER (B), K14CreER/Jagi"^x (C), and K14CreER/ ag7"^ox/ffo7K14NICDER (D-F, triple) transgenic mice treated with 40HT for 10 days. In triple transgenics areas of normal (F) and thickened (E) epidermis were found. Panels E and F are higher magnifications views of boxed regions in (D). (G, H) Back skin sections of triple transgenic mice in areas of normal thickness stained with antibodies to Ki67 (G, brown) and K10 (H, green). Sections were counterstained with haematoxylin (G) or DAPI (H). Scale bars: 25 pm (E-H) or 100 pm (A-D).

Figure 8. Relationship between Jagged 1 expression and Notch-induced stromal cells. (A-C, F) K14CreER/Jag?^{ffox flox}/K14NICDER (triple) skin with either increased (Thick; A; B left panels) or normal thickness (Thin; B right panels; C, F right panel) epidermis and K14CreER/Jagi^{flox flox} littermate mice (D) were 40HT-treated for 10 days. (F, G) Wild type (F, right panel) and K14NICDER littermate mice (F, middle panel, G) were 40HT-treated for 14 days. (A, C, D, F, E) Skin sections were stained with antibodies to Nestin (A, C green; D red), Jaggedl (A, C, red), SM22a (D, green), phosphorylated p65 (serine 276; E, green; F, brown) and CRABP1 (E, red) and DAPI (A, C, D, E, blue) or haematoxylin (F) counterstain. Both panels in (A) and (E) show the same field. Arrow in (A) mark Jaggedl staining. Inserts in F show higher magnification views. (B) RNA in situ hybridization using a radiolabeled antisense probe to Jaggedl . Corresponding brightfield (BF, top) and darkfield (DF, bottom) panels show the same field. In (B), black arrows mark interfollicular epidermis and white arrow marks hair follicle infundibulum. Scale bars: 50 pm. (G) Quantitative polymerase chain reaction of cDNA from 40HT treated wild type (WT), K14NICDER (NICD) and K14CreER/Jag7"^{0 fl0} K14NICDER epidermis. Results are expressed relative to WT = 1 for each TAQman probe. (H) Schematic summary of results. Epidermal Notch activation results in increased expression of Jaggedl in epidermis and dermis, which contributes to the epidermal changes and accumulation of cells in the dermis.

Figure 9: Hair reconstitution experiments were performed using wild type adult mouse keratinocytes combined with either wild type adult dermal cells (left mouse) or K14NICDER transgenic dermal cells (right mouse). In this assay, only K14NICDER transgenic adult dermal cells supported new hair follicle development.

Figure 10 shows the results of activating the NICDER transgene in skin tissue

Example 1 : Adult epidermal Notch activity induces dermal accumulation of T cells and neural crest derivatives through upregulation of Jagged 1

Summary

Notch signalling regulates epidermal differentiation and tumour formation via non cell autonomous mechanisms that are incompletely understood. Epidermal Notch activation via a 4-hydroxy-tamoxifen inducible transgene causes epidermal thickening, focal detachment from the underlying dermis and hair clumping. In addition, there is dermal accumulation of T lymphocytes and stromal cells, some of which localise to the blisters at the epidermal-dermal boundary. The T cell infiltrate was responsible for hair clumping but not for other Notch phenotypes. Notch-induced stromal cells were heterogeneous, expressing markers of neural crest, melanocytes, smooth muscle and peripheral nerve. Although Slugl expression was expanded in the epidermis the stromal cells did not arise through epithelial-mesenchymal transition. Epidermal Notch activation resulted in upregulation of Jagged 1 in both epidermis and dermis. When Notch was activated in the absence of epidermal Jagged 1 , Jagged 1 was not upregulated in the dermis, and epidermal thickening, blister formation, accumulation of T cells and stromal cells were inhibited. Gene expression profiling revealed that epidermal Notch activation resulted in upregulation of several growth factors and cytokines, including TNFa, whose expression was dependent on epidermal Jagged 1. Accordingly the inventors conclude that Jagged 1 is a key mediator of non-cell autonomous Notch signalling in skin.

Introduction

The skin is a bi-compartmental organ. The outer layer is maintained by stem cells and comprises a stratified epithelium, the interfollicular epidermis, with associated hair follicles, sebaceous glands and sweat glands. The sub-epidermal compartment comprises dermal fibroblasts, peripheral nerves, blood vessels, muscle and fat. Subpopulations of dermal cells, located in the dermal papilla and dermal sheath, regulate epidermal stem cell properties, most notably by controlling the hair growth cycle. A number of key developmental signaling pathways, including Notch, BMP, Wnt and FGF, mediate epidermal-dermal communication (Estrach et al., 2006).

Notch signalling is activated when ligand binding initiates cleavage of the Notch receptor, which releases the Notch intracellular domain (NICD) from the plasma membrane. The NICD translocates to the nucleus and interacts with its binding partners RBP-J and Mastermindl to activate transcription of downstream targets, including members of the Hes and Hey families of transcriptional repressors. Notch signalling is important in local cell-to-cell communication, as both ligands and receptors are tethered to the cell membrane. Nevertheless, recent studies demonstrate that the cleaved domain of Jagged 1 can be secreted and modulate Notch signaling over a longer range.

The importance of the Notch pathway in skin is well established. High expression of the Notch ligand Delta-like 1 (DIM ) is a marker of human epidermal stem cells and plays a dual role in promoting stem cell cohesion and stimulating differentiation of neighbouring epidermal cells in culture. Consistent with this, Notch acts as an epidermal tumour suppressor. Disrupting Notch signaling in embryonic and neonatal mouse epidermis though genetic ablation of RBP-JK, Hes1 , or Notc and Notch2 leads to failure to maintain the hair follicles, abnormal keratinocyte differentiation, barrier disruption, and neonatal lethality. Dill regulates differentiation of the interfollicular epidermis, while Jagged 1 , a β- catenin target gene, is required for hair follicle maintenance (Estrach et al., 2006). Conversely, forced Notch activation in developing interfollicular epidermis leads to loss of hemidesmosomes and promotion of keratinocyte differentiation.

While recent studies have tended to focus on the epidermal consequences of modulating Notch, there are also clear indications that Notch signalling controls dermal function. Epidermal deletion of Notch leads to increased epidermal production of thymic stromal lymphopoietin (TSLP), which triggers a B lymphoproliferative disorder with massive accumulation of B cells in the dermis and other body sites. Notch signalling in melanoblasts is required for their survival and when signalling is blocked the hair follicles become depigmented. In addition, the tumour suppressive function of Notch is not exclusively cell autonomous. The inventors previously generated transgenic mice in which 4-hydroxy-Tamoxifen (40HT) inducible expression of NICD is under the control of the keratin 14 promoter (K14NICDER transgenics; Estrach et al., 2006). In addition to hair clumping and thickening of the interfollicular epidermis, NICD activation causes changes in the cellularity of the dermis. The inventors have now explored those changes and present evidence that they result from signalling between the epidermis and dermis, mediated by Jagged 1.

Methods and Materials

Mice

All experimental procedures were subject to CRUK ethical review and performed under the terms of a UK government Home Office licence. K14NICDER (also known as K14N^ICDA0PER), K14CreER, Jag ° ° K14AN cateninER and CAG-CAT-EGFP mice have all been described previously (Estrach et al., 2006; Jensen et al., 2009; Kawamoto et al., 2000; Lo Celso et al., 2004). To activate the NICDER and CreER transgenes, 7-week-old mice were treated topically with 2 mg 4-hydroxy-tamoxifen (40HT, Sigma) dissolved in acetone (stock concentration of 10 mg/ml), as previously described (Estrach et al., 2006).

Some mice were injected with 4mg/kg Dexamethasone (or the equivalent volume of saline, as a control) into the abdominal subcutaneous space, starting three days prior to the initial 40HT treatment. Thereafter, mice received daily injections of Dexamethasone or saline, and 40HT or acetone was applied topically 3 times per week for 21 days.

Assay for epidermal barrier integrity

The Toluidine Blue dye exclusion assay was performed as previously described (Byrne and Hardman 2005). 2 cm² pieces of back skin were fixed overnight in 4% paraformaldehyde in PBS and then dehydrated and rehydrated through a graded methanol series. Skin was attached, dermal side down, to a Petri dish containing petroleum jelly (Vaseline), leaving only the epidermis exposed. 1% Toluidine Blue solution was added for 2 minutes, and then the epidermis was destained in PBS for 5 - 10 minutes. Samples were photographed, then cryo-embedded in OCT (Sakura). 25pm frozen sections were cut, air dried, and then imaged using a Leica MZ9.5 dissecting microscope.

Histology and immunostaining

For whole mount labeling, frozen sections, and paraffin-embedded sections, tissue was collected and processed as previously described (Braun et al., 2003; Estrach et al., 2006). Tissues were immunolabelled using the following antibodies (dilutions in brackets): K14 (1 :1000, Covance), K10 (1 :1000, Covance), Ki67 (1 :400, NeoMarkers), K17 (1 :1000, gift of P. Coulombe; McGowan and Coulombe, 2000), CD4 (1 :100, BD Biosciences), CD8 (1 :100, BD Biosciences), Hes1 (1 :1000, gift of N. Brown), a6 integrin subunit (1 :100, BD Biosciences), Laminin 5 (rabbit, 1 :500, gift of P. Marinkovitch), Nestin (rabbit, 1 :1000, gift of R. McKay; mouse monoclonal 1 :100, Cell Signaling Technologies), CRABP1 (1 :400, Sigma), Desmin (1 :100, Abeam), Sm22a (1 :200, Abeam), Jagged 1 (1 :100, Santa Cruz Biotechnology), Slugl , (1 :1000, Abeam), E-cadherin, (ECCD-2, 1 :600, Calbiochem), p75 (1:500, Abeam), phosphorylated p65 (1 :100, Abeam) and GFP (1 :1000, Abeam).

To visualize alkaline phosphatase activity, frozen sections were fixed in 0.4% paraformaldehyde for 15 seconds, washed in phosphate buffered saline and then reacted with 4.5 μΙ 75 mg/ml Nitro Blue Toluidine (Roche) and 3.5 μΙ 5 mg/ml 5- Bromo- 4-chloro-3-indolyl phosphate substrate (Roche). Sections were post-fixed in 10% neutral buffered formalin, counterstained with Fast Red, and mounted using Permount (Fisher Scientific).

Electron microscopy

Adult mouse dorsal skin was fixed in 2.5% glutaraldehyde and 4% paraformaldehyde in Sorensen's buffer (pH 7.4), then embedded in araldite resin. 100 nm sections were cut on a Reichert ultracut S ultramicrotome and stained with 1.5% uranyl acetate and lead citrate. Specimens were analysed on a JEOL 1010 electron microscope equipped with a US1000 camera (Gatan) using Digital Micrograph software (Gatan).

RNA in situ hybridization

In situ hybridization was performed as previously described. DNA plasmids used to make 35S-labeled antisense and sense probes for Hes1 (gift of R. Kageyama), HeyL (gift of M. Gessler) and Jagged 1 (gift of J. Lewis), have been described previously. Slides were photographed using bright- and darkfield illumination on an Olympus Darkfield Microscope.

Dermal cell dissociation

The isolation of dermal-derived, sphere forming cells has previously been described (Wong et al., 2006). In brief, approximately 60 mm² of back skin from 40HT-treated wild type or K14NICDER mice was collected. The subcutaneous fat and muscle were removed by scraping with a sterile scalpel and then the remaining skin (epidermis and dermis) was diced into 2-5 mm² pieces. Skin pieces were digested in 1mg/ml crude Type I Collagenase (C-9891 , Sigma) in 1:1 DMEM/F12 culture medium (31331-028, Invitrogen) containing Fungizone (2 g/ml; 15290-018, Invitrogen) and penicillin and streptomycin (1x; 15140-148, Invitrogen) at 37°C for 1 hour, followed by mechanical dissociation. The resulting cell suspension was filtered using a 70 μιτι cell strainer to remove hair and any undigested tissue.

Sphere culture

Isolated dermal cells were counted using a ViCellXR cell counter (BD), and plated in quadruplicate into 48-well plates at a density of 5x10⁴ cells/ml in DMEM/F12 medium containing 2% B27 supplement (Invitrogen), Fungizone, penicillin and streptomycin. Seven days after plating, each well of the 48-well tissue culture dish was photographed using a Nikon TE1000 microscope with a motorized stage and a Plan Apo 4x objective. Using Nikon NIS-Elements automated imaging software, the same 5 areas of each well were photographed and the number of spheres was counted. The sphere forming capacity was compared for wild type and K14NICDER transgenic littermate mice treated with 40HT for 10 (WT n=6; K14NICDER n=6) or 14 days (WT n=6; K14NICDER n=6). Wild type sphere forming capacity was designated as 100%.

RNA isolation and microarray analysis

For microarray studies, 7-week old female mice (3 wild type and 3 transgenic) were treated with 40HT for 14 days. The back skin was collected, bisected, immersed directly in RNAIater (Qiagen) and stored overnight at 4°C. One piece of the bisected skin was subsequently heated at 60°C for 10 seconds and then scraped gently with a scalpel to separate epidermis from dermis. Total RNA was isolated from epidermis, dermis and whole skin using the RNeasy Mini Kit with on-column DNase digestion (Qiagen) and hybridized to Affymetrix Mouse 430_2 gene chips (Patterson Microarray Facility, Manchester, UK). The average MAS5-calculated signal intensity of replicate samples (n=3) of epidermis, dermis and whole skin from wild type (n=3) and transgenic mice (n=3) was determined. Fold change represents the average difference in transgenic versus wild type signal intensity. Microarray data sets have been deposited in the MIAME-VICE public repository

(http://bioinformatics.picr.man.ac.uk/vice/ExternalReview.vice?k=9Tm6w0aSIZSRBbEt hDvtWMer5ZI%3D). qPCR

Total RNA was reverse transcribed using a Superscript III first-strand synthesis kit (Invitrogen) and quantitative PCR was performed under standard conditions with an ABI 7500 fast real-time PCR machine. Samples were run in triplicate for each probe and quantification was based on ΔΔΟΤ calculations. Samples were normalized to β- actin and GAPDH as loading controls and calibrated to wild type levels. Pre-designed TAQman probes were purchased from Applied Biosystems and are listed in Supplemental Material.

Western blotting

Transgenic and wild type littermate control mice were treated with 40HT as described above. 0.5-1 cm² sections of treated back skin were collected and immediately snap- frozen and stored in liquid nitrogen. Frozen tissues were homogenised in RIPA buffer (150 mM NaCI, 50 mM Tris-HCI, pH 7.5, 1% Nonidet P-40, 0.25% Sodium deoxycholate, and complete mini ethylenediaminetetraacetic acid-free protease Roche cocktail inhibitor tablets) using a Polytron tissue homogeniser. Lysates were subjected to electrophoresis on a 4-12% gradient polyacrylamide gel (Invitrogen), transferred to PVDF membrane by electro-blotting, blocked with 3% cold water fish skin gelatin (Sigma)/0.2%Tween-20/PBS and hybridized with goat polyclonal antibodies to Jagged 1 (C-terminal, c-20 1 :100; Santa Cruz Biotechnologies). Blots were rinsed in 0.2%Tween-20/PBS, incubated with an HRP-conjugated anti-goat secondary antibody (Sigma) and visualized by reaction with ECL Western Blotting Substrate (Pierce).

Microscopy and image processing

Stained tissues were imaged using a Zeiss 510 confocal microscope, a Leica Tandem SP5 confocal microscope or a Nikon 90i brightfield microscope. Quantitation of the results is described in Supplemental Material. Images were adjusted for brightness using Adobe Photoshop CS3 software. Results

Epidermal Notch activation leads to changes in skin architecture

The inventors activated the Notch signaling pathway in basal keratinocytes by applying 4- hydroxy-tamoxifen (40HT) to the skin of 7-week-old K14NICDER transgenic mice and their wild type littermates for 21 days (Estrach et al., 2006). K14NICDER transgenic mice had a thickened epidermis (Figure 1A; Estrach et al., 2006). In addition, the dermal cells immediately adjacent to the interfollicular epidermis were more numerous and had an elongated morphology compared to the surrounding dermal cells (Figure 1A).

One readout of Notch activity is upregulation of Hes and Hey genes. In wild type back skin, Hes1 was only detected in rare suprabasal epidermal cells (Figure 1 B). In K14NICDER transgenic skin treated with 40HT for 15 days, Hes1 protein was detected in basal and suprabasal epidermal cells, and in occasional underlying dermal cells (Figure 1 B). Upregulation of Hes1 mRNA in epidermis and dermis was confirmed by in situ hybridisation (Figure 1 C). In addition, HeyL mRNA was strongly and selectively upregulated in the dermis on epidermal Notch activation (Supplemental Figure 1 ). Thus epidermal Notch activation leads to upregulation of the Notch signalling pathway in both epidermis and dermis.

Notch activation resulted in an increase in the number of keratin 14 positive epidermal layers, with keratin 14 protein being detected throughout the viable suprabasal layers (Figure 1 B). This was accompanied by patchy loss of keratin 10 (Figure 1D). The remaining keratin 10 positive cells were located mainly in the interfollicular epidermis adjacent to hair follicles (Figure D; Supplemental Figure 2A). Reduced keratin 10 expression correlated with epidermal hyperproliferation: keratin 17 (McGowan and Coulombe, 2000) was expressed in the interfollicular epidermis and most basal layer cells were Ki67 positive (Figure 1 E, F; Supplemental Figure 2B).

Since loss of Notch leads to perturbation of the epidermal barrier, the inventors investigated whether increased Notch activity also affected barrier function. After 21 days of 40HT treatment, the skin of K14NICDER transgenic mice was permeable to toluidine blue (Byrne and Hardman, 2005), whereas that of wild type littermates was not (Supplemental Figure 3), indicating that sustained Notch activation led to disruption of the epidermal barrier. Nevertheless, epidermal cells still underwent complete terminal differentiation, as evidenced by the presence of spinous, granular and cornified layers (Figure 1A).

In wild type skin, the hemidesmosome component α6β4 integrin and its extracellular matrix ligand laminin 5 co-localised at the basal surface of keratinocytes (Figure 1G). In contrast, co-localisation of these markers was disrupted in 40HT treated K14NICDER skin (Figure 1G; Supplemental Figure 2C). Small blisters formed through separation of the epidermis from the underlying dermis. As a result, the a6 integrin subunit and laminin 5 were still detected on the basal surface of epidermal keratinocytes, but in addition laminin 5 was detected on the exposed dermis underlying detached epidermal cells. The blisters contained nucleated cells, as detected by DAPI staining (Figure 1G).

It has previously been reported that Notch activation in embryonic epidermis leads to loss of hemidesmosomes. Consistent with this, transmission electron microscopy revealed a marked reduction in hemidesmosomes in 40HT treated K14NICDER transgenic compared to wild type skin (Figure 1 H).

Ectopic Notch activity induces a dermal inflammatory infiltrate

To investigate whether the increase in dermal cell density on epidermal Notch activation was due to an inflammatory infiltrate, the inventors stained back skin sections of 40HT-treated K14NICDER transgenic mice and wild type littermates with antibodies to the T lymphocyte markers CD3, CD4 and CD8 (Figure 2A and data not shown). In 40HT-treated K14NICDER back skin, there was a massive infiltrate of CD3 positive T cells (data not shown). These comprised CD4 positive cells, with few CD8 positive cells being present in epidermis or dermis (Figure 2A, B).

To determine the contribution of the T cell infiltrate to the phenotype of 40HTtreated K14NICDER transgenic skin, the inventors treated 7-week old mice with the antiinflammatory drug Dexamethasone for 24 days. In addition, mice received topically applied 40HT or acetone for 21 days. Wild type mice injected with Dexamethasone or saline and treated with 40HT or acetone, and acetone-treated transgenic mice injected with Dexamethasone or saline, were indistinguishable from untreated wild type control mice (data not shown). The number of CD4-positive cells was reduced to wild type levels in Dexamethasone treated K14NICDER mice (Figure 2E). Notch activation results in hair shaft clumping, giving mice a "tufted" appearance (Estrach et al., 2006; Figure 2C). K14NICDER mice that received both Dexamethasone and 40HT had reduced hair clumping compared to K14NICDER mice treated with 40HT alone (Figure 2C). However, epidermal thickening (Figure 2D) and blister formation (visualized by dissociation of laminin 5 and a6 integrin staining; Figure 2F) were unaffected by Dexamethasone treatment.

The inventors conclude that epidermal Notch activation induces a T lymphocyte infiltrate that is responsible for hair clumping, but not for other skin phenotypes.

Epidermal Notch activity results in accumulation of dermal cells that express neuronal, muscle, dermal papilla and neural crest markers

To examine the effect of epidermal Notch activity on the composition of the underlying dermal compartment the inventors stained sections with markers to the various cell populations resident within the dermis. Alkaline phosphatase is a marker of both dermal papilla and arrector pili muscle (Figure 3A). In 40HT treated K14NICDER mice, alkaline phosphatase was additionally detected in dermal cells at the epidermal-dermal junction (Figure 3A). The dermal papilla marker CRABP1 was also expressed by these cells (Figure 3B). Nestin is an intermediate filament protein that is highly expressed by the peripheral nerve cluster adjacent to the hair follicle bulge and also by neural crest stem cells, melanocyte precursors and dermal papilla cells. In K14NICDER mice treated with 40HT for 14 days, there was marked accumulation of Nestin-positive cells at the epidermal-dermal boundary (Figures 3C) and in the subepidermal blisters of 40HTtreated K14NICDER skin (Figure 3D). Cells in this region also expressed neurofilament protein, an intermediate filament protein highly expressed in neuronal cells, but were negative for two additional markers of skin peripheral neurons, Sox2 and NCAM (data not shown). In addition to the accumulation of Nestin positive cells, there was an increase in differentiated, melanin positive melanocytes (Figure 3E). The melanocytes expressed c-kit (Figure 3F), and tended to lie below the Nestin positive cells at the epidermal/dermal boundary (Figure 3F). When transgenic skin was treated with 40HT for 21 days the number of dermal c-kit positive cells increased approximately 3 fold (Supplemental Figure 4A). Subepidermal dermal cells in 40HT-treated K14NICDER skin expressed the smooth muscle markers desmin and SM22a, but did not express the vascular endothelial marker, CD31 (Figure 3G, H and data not shown).

In wild type adult back skin, Nestin, CRABP1 and SM22ct are individual markers of peripheral nerves, dermal papilla cells, and arrector pili muscle cells, respectively (Figure 3 B, C, H; WT panels). In back skin of 40HT-treated K14NICDER mice cells that were singly positive for each marker were also present (Figure 4A, arrowheads; Figure 4C). However, many cells co-expressed Nestin and CRABP1 or Sm22a and CRABP1 (Figure 4A, arrows; Figure 4C). Quantitation of double and single positive cells was performed both on skin sections (Figure 4C) and on cytospin preparations of disaggregated dermis (Supplemental Figure 4B).

The inventors detected a gradient in the intensity of staining for Nestin and SM22a, with those cells immediately adjacent to the epidermis being most strongly labeled (Figure 4A). In contrast, CRABP1 staining was equally strong, regardless of cell location (Figure 4A). The inventors conclude that the cells that accumulate at the epidermal-dermal boundary of 40HT treated K14NICDER skin are heterogeneous and distinct from the cell types resident in the dermis of wild type skin.

To investigate the timing of the appearance of Nestin, CRABP1 and S 22a positive dermal cells, the inventors examined the skin of K14NICDER mice that had been treated with 40HT for 0 to 21 days. Occasional CRABP1 and Nestin positive cells were detected after 2 days of 40HT treatment, with the majority of CRABP1 positive cells initially located in unblistered areas of the subepidermal dermis (Figure 4 B and data not shown). The number of CRABP1 positive cells increased progressively throughout the treatment period, whereas the number of Nestin positive cells was maximal by 14 days (Figure 4B, C). Nestin positive cells often co-expressed CRABP1 (Figure 4B, C). Sm22a positive cells were not detected until K14NICDER mice had been treated with 40HT for 7 days and increased in abundance thereafter (Figure 4B, C). Notch induced stromal cells tended to form stable intercellular adhesions that were not disrupted under conditions used to isolate single cell suspensions from the dermis (Figure 4G).

The origin of cells at the epidermal-dermal boundary following Notch activity

During development, peripheral neurons, smooth muscle cells, facial dermal papilla cells and melanocytes are all derived from the embryonic neural crest, and Nestin and CRABP1 positive cells are readily detected in embryonic and early post-natal dermis (Supplemental Figure 5A, D). Thus the markers expressed by Notch-induced dermal cells are consistent with a neural crest origin. In support of this conclusion, the neural crest marker p75 was also detected in cells at the epidermal/dermal junction in 40HT- treated K14NICDER transgenic mice (Figure 4D).

The dermal papilla contains multipotent stem cells (Skin-derived Precursors; SKPs) that have similarities to neural crest stem cells and can form Nestin-positive neurospheres in culture (Figure 4E; Wong et al., 2006). The inventors examined whether epidermal Notch activation affected the number of dermal-derived neurosphere forming cells (Figure 4E, F). The percentage of sphere-forming cells was lower in 40HT-treated K14NICDER transgenic than control back skin, regardless of length of treatment (Figure 4E, F and data not shown). Thus the appearance of dermal cells that expressed neural crest markers correlated with a reduction in the number of multipotent dermal stem cells.

Nestin-positive cells at the epidermal/dermal boundary in K14NICDER mice are not of epidermal origin

During development, the neural crest arises from neuroectoderm and correlates with upregulation of the transcription factor Slugl . Since Slugl is a direct Notch target gene the inventors examined Slugl expression in transgenic and control skin. In wild type skin, Slugl was detected in some basal and suprabasal epidermal cells and scattered dermal cells (Figure 5A). In 40HT-treated K14NICDER back skin Slugl was detected in all layers of the epidermis, and was strongly expressed in dermal cells at the epidermal/dermal junction (Figure 5A).

In mammary epithelial cells, upregulation of Slugl results in downregulation of Ecadherin and induces an epithelial to mesenchymal transition. However, in both 40HT-treated K14NICDER and wild type mice E-cadherin was strongly expressed in basal and suprabasal epidermal layers (Figure 5B). To further investigate whether the dermal cells accumulating in response to Notch activation were derived from the epidermis, the inventors generated triple transgenic mice by crossing the following strains: K14NICDER, K14CreER and CAG-CAT-EGFP, which contains a flox-stop-flox GFP reporter (Estrach et al., 2006; Kawamoto et al., 2000). Since the keratin 14 promoter is specifically active in basal epidermal cells, Cre-induced expression of GFP will only occur in cells of epidermal origin. 7-week old K14CreER/CAG-CAT-EGFP (n=10) and K14Cre/EP CAG-CATEGFP/K14NICDER (n=1 1 ) littermates were treated with 40HT for 14 days to activate both the CreER and NICDER transgenes. In 40HT- treated K14CreER/CAGCAT-EGFP and K14CreER/CAG-CAT-EGFP/K14NICDER mice GFP expression was patchy, indicating that 40HT treatment led to incomplete recombination in the epidermis (Figure 5C). There was also a low level of GFP expression in mice that had not received 40HT, consistent with the previously reported leakiness of the CreER founder line (Jensen et al., 2009; data not shown). GFP positive cells were only detected within the epidermal compartment, never the dermis, in 40HT-treated in mice, whether or not the mice expressed the K14NICDER transgene (Figure 5C). This was confirmed by double labelling disaggregated epidermal and dermal cells for GFP and Nestin (Figure 5C, D).

The inventors conclude that cells expressing CRABP1 or Nestin at the epidermal/dermal boundary in 40HT-treated K14NICDER mice were not of epidermal origin and thus that Notch did not induce an epithelial to mesenchymal transition.

Epidermal Notch activation results in upregulation of Jagged 1 expression in epidermis and dermis

In developing skin Jagged 1 is detected both in the epidermis and dermis (Supplemental Figure 6), whereas in adult skin, Jagged 1 is primarily expressed in the bulb of anagen follicles (Estrach et al., 2006). In 40HT treated K14NICDER skin Jagged 1 was upregulated in the interfollicular epidermis (Figure 6A), consistent with a previous report that Jag1 is positively regulated by Notch signaling. Jaggedl protein was also detected in the upper dermis of 40HT treated K14NICDER skin (Figure 6A), correlating with dermal expression of Hes1 and HeyL (Figure 1 C; Supplemental Figure 1 ). The increase in Jagged 1 protein was confirmed by Western blotting of total skin lysates (Figure 6B). The Notch-induced increase in Jaggedl expression and accumulation of dermal cells expressing CRABP1 , Sm22a and other markers was also observed in transgenic mice treated with Dexamethasone (Supplemental Figure 6), indicating that they were independent of the Notch-induced inflammatory infiltrate.

The inventors performed in situ hybridization to determine whether the dermal accumulation of Jagged 1 (Figure 6A) was due to epidermal secretion (Aho, 2004) or Jagged 1 transcription in dermal cells (Figure 6C-E). Jag1 mRNA was detected at low levels in wild type telogen back skin (Figure 6C; Estrach et al., 2006). When β-catenin activity was induced in the epidermis by 40HT treatment of K14ANil-cateninER transgenic mice Jag1 was upregulated in the hair follicles and interfollicular epidermis, but in the not dermis (Figure 6E; Estrach et al., 2006). In contrast, in 40HT-treated K14NICDER back skin Jag"\ mRNA levels were increased in both epidermis and dermis (Figure 6D).

The inventors conclude that activation of Notch in the epidermal basal layer results in upregulation of Jagged 1 expression in both epidermis and dermis.

Accumulation of Nestin-positive cells at the epidermal/dermal junction is dependent on epidermal Jagged 1 expression

To examine the role of Jagged 1 in the Notch-induced skin phenotype, the inventors crossed K14NICDER, K14CreER and Jag ^{ox nm} strains of mice. In these mice 40HT treatment results in deletion of Jag1 in the same cells in which Notch is activated. K14NICDER/K14CreER/Jagf^ffox/to (triple) transgenic mice were compared with untreated K14NICDER/K14CreER/Jagi^{flox flox} mice and littermates that were wild type for Notch. 40HT treatment of K14CreER/Jagi^{flox flox} and K14NICDER mice induced minimal changes to the overall health of the mice (Estrach et al., 2006 and data not shown). However, all 40HT treated triple transgenic mice rapidly lost weight and developed a hunched appearance; the mice drooled and their belly hairs were matted with saliva (data not shown). Therefore, 7-week old mice triple and control mice were treated for a maximum of 10 days.

The inventors analysed the histology of K14 ICDER/K14CreER/Jag1^{flox f,ox} (triple), K14NICDER, K14CreER/Jagi^floxflox and wild type skin (Figure 7A-F and Table 1). The back skin of treated K14NICDER mice had the expected phenotypic characteristics, including thickened, hyperproliferative epidermis, blistering and ccumulation of dermal cells at the epidermal/dermal junction (Figure 7B). In 40HT reated K14CreER/Jagi^flox/flc>x mice the epidermis was also thicker than wild type controls, consistent with our previous finding that K5Cre/ Jag ι"^οχΠοχ mice have thickened dorsal epidermis (Figure 7C; Estrach et al., 2006). In addition, there was a marked dermal infiltrate of CD3 positive T-cells (Figure 7C and data not shown; Table 1 ; Estrach et al., 2006). Triple transgenic mice treated with 40HT for 10 days developed two distinct back skin phenotypes (Figure 7D-F). In some areas the skin displayed a similar phenotype to K14NICDER transgenic back skin (Figure 7E), whereas in other areas the epidermis was similar in thickness to wild type, but with a reduction in nucleated cells (Figure 7F; Table 1 ). In the areas of reduced epidermal thickness the number of Ki67 positive, proliferating cells was similar to wild type and was markedly reduced compared to K14NICDER transgenic and K14CreER/Jag1^flo^^flox mice (Figure 1 F; Figure 7G and data not shown). In addition, Keratin 10 was expressed in all suprabasal epidermal cells and a significant proportion of basal cells (Figure 7H). Blistering was reduced in thin epidermis of triple transgenic mice, but present in thick epidermis (Figure 7E, F and data not shown; Table 1 ).

As reported previously, Jagged 1 protein was undetectable in the epidermis of 40HTtreated K14CreER/Jag i^flo '^?ox back skin (data not shown; Estrach et al., 2006). However, in 40HT treated triple transgenics, Jagged 1 deletion was incomplete. The areas of thick epidermis that retained the K14NICDER Notch activation phenotype expressed Jagged 1 protein and mRNA in both epidermis and adjacent dermis (Figure 8A, B), whereas Jagged 1 was absent in thin epidermis and underlying dermis (Figure 8B, C). The incomplete deletion of Jag1 is consistent with the partial expression of the CAG-CAT-eGFP reporter (Figure 5C) and indicates that 40HT treatment was not sufficient to efficiently activate two ER transgene fusion proteins simultaneously. Sections of 40HT treated K14NICDER and triple transgenic back skin were labeled with antibodies to Jagged 1 and Nestin, CRABP1 , or SM22a (Figure 8A, C and data not shown). In K14NICDER dermis, Nestin, CRABP1 and SM22a positive cells colabeled with Jagged 1 (data not shown). In triple transgenic sections, positively stained cells at the epidermal/dermal boundary were only detected in the thick epidermal regions that retained Jagged 1 in the epidermis and dermis (Figure 8A). Dermal cells did not accumulate below thin, Jagged 1 negative, regions in the triple transgenics, nor in the back skin of 40HT treated KMCreER/Jagl"⁰^ mice (Figure 8C, D). The inventors conclude that epidermal Jagged 1 is required for epidermal Notch induced dermal Jagged 1 expression and the accumulation of dermal cells that express neural crest markers.

Jagged 1 dependent upregulation of TNFa signalling To investigate how epidermal Notch activation via Jagged 1 led to changes in the underlying dermis, the inventors performed gene expression profiling of epidermis, dermis and whole skin from transgenic and wild type mice treated with 40HT for 14 days. The microarray data were analysed using GeneSpring GX10 and Ingenuity Pathway Analysis software programmes after average signal intensities had been determined by MAS5-calculation. The full gene lists are shown in Supplementary Tables 1-3 and can be viewed via the following link: http://bioinformatics.picr.man.ac.uk/vice/ExternalReview. vice?k=9Tm6w0aSIZSRBbEth DvtWMer5ZI%3D.

Within the epidermis, Notch activation resulted in changes in many genes that are associated with barrier formation and integrity, including several metalloproteinases, S100A8, Sprrl b and Filaggrin (Supplemental Table 4). Within the skin, markers of melanocytes (such as Tyrpl ) and regulators of neural crest cell specification (such as Edn1 ) and glial and neuronal differentiation (for example, Sox11 ) were strongly upregulated (Supplemental Table 4). These changes in gene expression are consistent with the epidermal and dermal phenotypes of 40HT treated K14NICDER skin. To gain insights into potential mechanisms by which epidermal Notch activation could induce Jagged 1 in the underlying dermis, the inventors examined the microarrays for secreted growth factors and cytokines that were upregulated in the epidermis. The list included three growth factors that were upregulated more than 10 fold: Neuregulin 1 , Inhibin β A, and Tumour necrosis factor a (TNFa). (Supplemental Table 4). The microarray results were validated by quantitative RT-PCR of epidermal mRNA using predesigned TAQman probes. All three factors were strongly upregulated in 40HTtreated K14NICDER epidermis (Figure 8G). Relative mRNA abundance was normalized to endogenous wild type levels (equals 1 ).

The upregulation of TNFa was of particular interest because it is linked to epidermal barrier disruption and skin inflammation and also to the Notch pathway. TNFa activates the NF-KB family of transcription factors that includes p65 (RelA). There is cross-talk between the Notch and NF-KB pathways in many tissue types and in cultured human keratinocytes exposure to Jagged 1 activates the NF-κΒ pathway. To confirm that the NF- Β pathway was activated in 40HT-treated K14NICDER skin, sections were stained with an antibody specific to active, serine-phosphorylated (residue 276) p65. Occasional phospho-p65 positive nuclei were detected in the epidermis of wild type control sections but were largely absent from the dermis (Figure 8F). In 40HT treated transgenic skin, phospho-p65 positive nuclei were detected in all epidermal layers and in the dermis (Figure 8F). The majority of phospho-p65 positive dermal cells expressed CRABP1 (Figure 8E).

To further examine the link between Jagged 1 and p65, the inventors used Ingenuity Pathway Analysis (IPA; Supplemental Figure 7). Three factors directly linking p65 activity and Jagged 1 expression in the epidermis and dermis of 40HT treated K14NICDER mice were identified: TNFa, interferon-y and transforming growth factor beta (TGFP). Interferon-γ was increased 2.5 fold and TGF3 3.0 fold in the epidermal microarrays. Q-PCR performed on epidermis treated with 40HT for 10 days revealed an increase in TGF but not interferon-γ mRNA (Figure 8G).

To examine whether upreguiation of TNFa and other secreted factors was dependent on epidermal Jagged 1 expression, the inventors performed Q-PCR of mRNA from 40HT treated K14NICDERyK14CreER/Jagi^{flox flox} (triple) transgenics (Figure 8G). Levels of Inhibin β A and IFNy did not decline on Jag1 deletion; indeed Inhibin β A levels were further elevated. However, levels of TNFa, TGFp, Edn1 and Nrg1 were all decreased on Jag1 deletion. Furthermore, in areas of Jag1 deletion in 40HT treated K14NICDER/K14CreER/Jagi^ffo '^tof mice phospho-p65 was not detected in the epidermis or underlying dermis (Figure 8F).

The inventors conclude that the mechanism by which epidermal Notch activation exerts its pleiotropic effects on the skin (Figure 8H) involves Jagged 1 dependent induction of a number of secreted factors and their downstream pathways, including NF- B.

Discussion

The inventors report that Notch activation in the basal layer of the epidermis not only results in thickening and blistering of the interfollicular epidermis, as reported previously (Estrach et al., 2006, but also causes remarkable changes in the dermis. These include a CD4 positive T-cell infiltrate and accumulation of cells that express neural crest markers. Our data uncover a previously unappreciated role for Notch signalling in epidermal-dermal interactions. Epidermal deletion of Notch leads to dermal accumulation of B cells through epidermal production of TSLP. Impaired presenilin function also leads to a skin inflammatory infiltrate of B and CD4 positive T cells. It is interesting that epidermal Notch activation also led to increased TSLP expression (Supplemental Table 4) and accumulation of CD4 positive T cells. This suggests that the inflammatory changes are a consequence of epidermal barrier dysfunction rather than direct effects of activating or inhibiting Notch activity (Supplemental Table 4).

The Notch induced accumulation of T cells and hair clumping were blocked by Dexamethasone treatment, but accumulation of stromal cells and epidermal thickening and blistering were not. There are interesting parallels between the hair phenotype of K14NICDER skin and a human skin condition known as tufted hair folliculitis, in which multiple hairs emerge from a single follicular opening. The human condition, like the mouse phenotype, is associated with inflammation. Tufted hair folliculitis can be successfully treated with antibiotics or anti-fungal agents.

The importance of reciprocal signalling between the epidermis and dermis is well established in the context of the hair follicle, since hair development and postnatal maintenance depend on interactions between the epidermis and specialized dermal cells that constitute the dermal papilla. Some of the genes that are specifically expressed in the dermal papilla and mediate dermal papilla signalling have been identified. Compared to our knowledge of dermal papilla signalling, little is known about the factors that mediate signaling between the interfollicular epidermis and the underlying dermis.

Notch activation in the epidermis led to accumulation of cells that expressed markers of smooth muscle, peripheral nerve, neural crest and melanocytes, but not of endothelial cells. Notch activity has been linked to Sm22a and Nestin expression in other cell types and is known to control melanocye survival. The markers expressed on Notch induced stromal cells are consistent with a neural crest origin, and the increase in p75 might indicate an expansion of neural crest stem cells. The inventors established by lineage tracing that the origin of the dermal cells was not the epidermis. Thus, although the proportion of cells expressing Slugl in the epidermis was increased by Notch activation, the cells did not undergo an epithelial-tomesenchymal transition. An alternative is that the dermal cells were derived from SKPs, which declined in number with time of Notch activation. Since the first stromal cells always appeared adjacent to the epidermis, SKP progeny would have to migrate from the dermal papilla to the epidermal-dermal boundary prior to differentiation. The appearance of Notch induced stromal cells was not correlated with increased proliferation in the dermis (Figure 1 ). The inventors therefore propose that Notch induced stromal cells arise by transdifferentiation of cells already present in the upper dermis, migration and subsequent differentiation of SKP progeny, or, given the observed cellular heterogeneity, by a combination of both processes.

On activation of Notchl in the epidermis, Jagged 1 expression was upregulated in both epidermis and dermis (Figure 8H). This provides an explanation for the observed increase in Notch activity in the dermis and also for the accumulation of melanocytes, the differentiation of which is Notch dependent.

Gene expression profiling and quantitative RT-PCR identified several additional factors that were upregulated on epidermal Notch activation and are known to influence proliferation and differentiation of neural crest derivatives. These include Endothelin 1 and Neuregulin 1 , expression of both of which was partially dependent on epidermal Jagged 1 (Figure 8G). The inventors have thus identified several secreted factors that are likely to mediate the increase in neural crest derivatives in response to epidermal Notch activation. Gene expression profiling revealed one potential mechanism by which Jagged 1 is induced in the dermis. Epidermal Notch activation resulted in induction of TNFa in the epidermis and activation of NF-κΒ in both epidermis and dermis. TNFa induces Jagged 1 expression via NF- Β. Since Jagged 1 activates the NF-KB pathway the inventors envision a positive auto-regulatory loop involving TNFa and Jagged 1 expression in the skin. The upregulation of TNFa following Notch activation is also likely to contribute to the inflammation and barrier defects observed.

In conclusion, our results identify Jagged 1 as a key component of cell autonomous and non-cell autonomous Notch signalling in the skin. The mechanisms the inventors have identified may explain some of the previously reported changes in the stromal compartment of epithelial tumours in which Notch is activated.

Table 1. Effect of epidermal Jag1 deletion on Notch-induced skin phenotype.

NotchER: 40HT-treated K14NICDER skin. Jag1-/-: 40HT treated skin of K14CreER x _{Jagr i} fⁱoxfiox _mice NotchER, j_agi-/._: 40HT treated triple transgenics (K14CreER x Jag ^oMox x K14NICDER). -: wild type phenotype; +: detectable effect; ++: strong effect. IFE: interfollicular epidermis.

References

Braun, K.M. ef al (2003) Development 130, 5241-5255

Byrne, C, and Hardman, M. J. (2005) Methods Mol Biol. 289, 127-136

Estrach, S er a/ (2006) Development 133, 4427-4438

Jensen, K.B ef al (2009) Cell Stem Cell 4, 427-439

Kawamoto, S ef al (2000) FEBS Lett 470, 263-268

Lo Celso C ef al (2004) Development 131, 1787-99

McGowan, K.M., and Coulombe, P.A. (2000) J Invest Dermatol 1 14, 1 101-1 107

Wong, C.E ef al. (2006). J Cell Biol 175, 1005-1015.

Example 2: A protocol for isolating therapeutic neural cells

Cells were collected from back skin from K14NICDER mice treated with 40HT. Following dissection, the fat and muscle was removed from the skin by cutting or gently scraping the inside surface with a scalpel. The skin was then treated with dispase for 4 hours to overnight at 4°C. After dispase treatment the dermis was then digested with a mixture of Collagenase Type I (1 mg/ml), Collagenase Type II (0.5 mg/ml), Collagenase Type IV (0.5 mg/ml) and/or Hyaluronidase (0.2 mg/ml) in HamsFl2/DMEM 1 :1. Incubate at 37 °C Water bath in 15 ml Falcon for 30 minutes -1 hr monitoring the reaction. Shake every 5 minutes. Alternatively, digestion can be moved put in a bacterial shaker and mixed vigorously for 20-40 minutes at 37°C. Add HamsF12/DMEM 1 :1 media plus 10% serum, then filter using a 70 micron filter. Count cells using a hemocytometer or ViCell automated cell counter.

Neural cells are then isolated by fluorescence assisted cell sorting after labeling cells with antibodies to CD45, CD34, PDGFRa and p75 NTR. Only K14NICDER-induced dermal derived neural cells are positive for PDGFRalpha and p75 NTR and negative for CD45 and CD34, while contaminating dermal cells are positive for CD34 cells and PDGFRalpha. All blood cell lineages will be positive for CD45 and excluded from the sort.

Example 3: Hair follicle reconstitution assay

7-week-old wild type or K14NICDER transgenic mice were 40HT-treated for 14 days before mice were humanely sacrificed and treated skin removed. Subcutaneous muscle and fat was gently scraped from tissue using scalpel then skins were incubated in 2.5% Trypsin/EDTA overnight at 4°C. After overnight incubation, epidermal cells are scraped from skin tissue and removed. The remaining dermis is washed in FAD media with 10% fetal calf serum. Dermis is chopped into fine pieces and incubated in 1 mg/ml Collagenase Type 1 at 37°C for 30 minutes with constant stirring. When completely digested add FAD with serum and filter slurry using 70-micron cell strainer. Combine 5x106 dermal cells with 4-5x106 early passage (P1 ) adult wild type epidermal keratinocytes grown under standard keratinocyte culture conditions (Rhienwald and Green, 1975). Inject cell pellet into a silicon chamber grafted onto the back of an immunocompromised mouse (e.g. nude). Chambers were removed 14 days post surgery.

Example 4: Activation of the NICDER transgene in skin tissue.

Methods:

To activate the NICDER transgene, 7-week-old mice were treated topically with 2 mg 4-hydroxy-tamoxifen (40HT, Sigma) dissolved in acetone (stock concentration of 10 mg/ml). Mice were treated 3 times per week for 3 weeks (9 total treatments). At the end of treatment, mice were retained for 4 months before humane sacrifice. Tissues were processed and stained as previously described (Ambler and Watt, 2010 and Estrach et al., 2006).

Results:

Induced Notch activity in the epidermis causes thickening of the epidermis, inflammation and changes to the cellularity of the dermis (Figures 1-3). To determine if epidermal Notch activation induces transient or persistent changes to the epidermal and dermal compartments, 7-week old K14NICDER transgenic and control mice were treated with 40HT for 3 weeks followed by a 4-month chase period of no treatment (Figure 9). Analysis of sections from these mice shows no difference in epidermal thickness in wild type control and K14NICDER transgenic back skins. Additionally, dermal cell density was similar in transgenic and control animals as detected by distribution of cell nuclei (Figures 10 A and B). This suggests that inflammatory cell infiltration caused by forced Notch activity has resolved following the 4-month chase period.

As shown above, inducing epidermal Notch activity with 40HT for only 14 -21 days causes an increase in the number of dermal melanocytes (Figure 3E). Interestingly after 4-month period of no 40HT treatment a significant increase in the population of dermal melanocytes was detected in transgenic skin but not wild type controls (Figure 0B arrows). This suggests transient activation of Notch affects long-term changes to differentiated dermal melanoctyes.

Example 5: Isolation of differentiated cells by FACS dermal cell preparation: To prepare dermal cells for flow cytometry analysis humanely euthanise mice by C02 asphyxiation. Remove back skin and gently scrape or cut away subcutaneous fat and muscle. Next cut skin into 2cm² pieces and float each piece in 2mg/ml dispase in RP I media with the dermis side down overnight at 4°C. Following overnight incubation, remove epidermis from dermis with forceps. Mince remaining dermis tissue and incubate with 1 mg/ml Collagenase Type 1 and 1 mg/ml Collagenase Type 2 in RPMI media using an orbital mixer to agitate. Incubate in collagenase mixture for 2 hours, then strain cells using a 70 micron cells strainer. Count cells and label cells with cell surface marker antibodies pre-conjugated with fluorescent tags (0.5ul per 1 ,000,000 cells) for 20-30 minutes on ice in dark in an orbital mixer. Rinse with phosphate buffer saline three times for 5 minutes each. Analyse cells using a FACSCaliber, FACS LSRII or other equivalent flow cytometry analyzer.

Claims

CLAIMS:

1. One or more activators of Notch signalling for use in a method of preparing a population of differentiated cells for use in autologous cell replacement therapy, the method comprising contacting skin tissue of a patient in need of said therapy with one or more activators of Notch signalling, and subsequently isolating the required differentiated cells from the skin tissue for autologous cell replacement therapy.

2. One or more activators of Notch signalling according to claim 1 wherein the epidermal layer of the skin tissue is contacted with the one or more activators of Notch signalling, and the required differentiated cells are isolated from the dermal layer of the skin tissue.

3. One or more activators of Notch signalling according to claim 1 or 2 wherein the one or more activators of Notch signalling are applied to the skin tissue in situ.

4. One or more activators of Notch signalling according to claim 1 or 2 wherein the one or more activators of Notch signalling are applied to the skin tissue ex vivo.

5. One or more activators of Notch signalling according to any of the previous claims wherein the required differentiated cells are neural progenitor cells, melanocytes or dermal papilla cells.

6. One or more activators of Notch signalling according to claim 5 wherein the neural progenitor cells produce neurons and Schwann cells.

7. One or more activators of Notch signalling according to any of the previous claims wherein the activator is a ligand for Notch receptor proteins.

8. One or more activators of Notch signalling according to claim 7 wherein the ligand is Delta 1 , Jagged 2 and/or JAGGED 1 , or a fragment, variant or derivative of said ligands capable of activating Notch signalling.

9. One or more activators of Notch signalling according to claim 8 wherein the activator of Notch signalling is JAGGED 1 , or a fragment, variant or derivative of JAGGED 1 capable of activating Notch signalling.

10. One or more activators of Notch signalling according to claim 9 wherein the fragment of JAGGED 1 is Soluble JAGGED 1.

11. One or more activators of Notch signalling according to any of claims 1 to 6 wherein the activator of Notch signalling is NICD, or a fragment, variant or derivative of NICD capable of activating Notch signalling.

12. One or more activators of Notch signalling according to any of claims 1 to 6 wherein the activator of Notch signalling is ADA 10 or ADAM17, or a fragment, variant or derivative of ADAM10 or ADAM17 capable of activating Notch signalling.

13. One or more activators of Notch signalling according to any of the previous claims wherein the required differentiated cells are isolated using FACS.

14. A population of differentiated cells prepared according to the method described in any of claims 1 to 13 for use in a method of autologous cell replacement therapy.

15. Isolated differentiated autologous cells for use in a method of autologous cell replacement therapy, wherein the differentiated cells have been derived from skin tissue of a subject, in need of autologous cell replacement therapy, in which Notch signalling has been activated by one or more Notch signalling activators.

16. The isolated autologous cells according to claim 15 wherein the epidermal layer of the skin tissue is contacted with the one or more activators of Notch signalling, and the required differentiated cells are isolated from the dermal layer of the skin tissue.

17. The isolated autologous cells according to claim 15 or 16 wherein the one or more activators of Notch signalling are applied to the skin tissue in situ.

18. The isolated autologous cells according to claim 15 or 16 wherein the one or more activators of Notch signalling are applied to the skin tissue ex vivo.

19. The isolated autologous cells according to any of claims 15-18 wherein the required differentiated cells are neural progenitor cells, melanocytes or dermal papilla cells.

20. The isolated autologous cells according to claim 19 wherein the neural progenitor cells produce neurons and Schwann cells.

21 The isolated autologous cells according to any of claims 15-20 wherein the activator is a ligand for Notch receptor proteins.

22. The isolated autologous cells according to claim 21 wherein the ligand is Delta 1 , Jagged 2 and/or JAGGED 1 , or a fragment, variant or derivative of said ligands capable of activating Notch signalling.

23. The isolated autologous cells according to claim 21 wherein the activator of Notch signalling is JAGGED 1 , or a fragment, variant or derivative of JAGGED 1 capable of activating Notch signalling.

24. The isolated autologous cells according to claim 23 wherein the fragment of JAGGED 1 is Soluble JAGGED 1.

25. The isolated autologous cells according to any of claims 15 to 21 wherein the activator of Notch signalling is NICD, or a fragment, variant or derivative of NICD capable of activating Notch signalling.

26. The isolated autologous cells according to any of claims 15 to 21 wherein the activator of Notch signalling is ADA 10 or ADAM 7, or a fragment, variant or derivative of ADAM10 or ADAM17 capable of activating Notch signalling.

27. The isolated autologous cells according to any of claims 15-26 wherein the required differentiated cells are isolated using FACS.

28. One or more activators according to any of claims 1-13 or cells according to any of claims 15-27 wherein the differentiated cells are neural progenitor cells, and the autologous cell replacement therapy is for the treatment of a condition is characterised by degeneration, damage to, the loss of, or the disorder in nervous tissue.

29. One or more activators according to any of claims 1-13 or cells according to claims 15-27 wherein the condition is a spinal injury and the differentiated cells are neurons and/or Schwann cells.

30. One or more activators according to any of claims 1-13 or cells according to any of claims 15-27 wherein the differentiated cells are melanocytes and the autologous cell replacement therapy is for the treatment of a condition characterised by degeneration, damage to, the loss of, or the disorder in skin pigmentation.

31. One or more activators according to any of claims 1-13 or cells according to any of claims 15-27 wherein the disorder is vitiligo.

32. One or more activators according to any of claims 1-13 or cells according to any of claims 15-27 wherein where disorder is vitiligo associated with inflammation, the autologous cell replacement therapy comprises melanocytes in combination with one or more anti-inflammatory agents.

33. One or more activators according any of claims 1-13 or cells according to any of claims 15-27 wherein the differentiated cells are dermal papilla cells and the autologous cell replacement therapy is for the treatment of a condition characterised by degeneration, damage to, the loss of, or the disorder in hair follicle formation and/or growth.

34. One or more activators according to any of claims 1-13 or cells according to any of claims 5-27 wherein the disorder is alopecia or male pattern baldness.

35. One or more activators of Notch signalling for use in a method of prevention or treating a condition characterised by degeneration, damage to, the loss of, or the disorder in skin pigmentation said method comprising contacting skin tissue of a patient in need of therapy to one or more activators of Notch signalling.

36. One or more activators of Notch signalling for use in the method of claim 35 wherein the one or more activators of Notch signalling are defined in relation to any of claims 7 to 12.

37. An in vitro method of promoting the differentiation of dermal cells, the method comprising contacting a sample of skin tissue with one or more activators of Notch signalling, and subsequently isolating the differentiated cells from the skin tissue.

38. The method of claim 37 wherein the differentiated cells are neural progenitor cells, melanocytes or dermal papilla cells.

39. An in vitro method of preparing a substantially pure population of neural progenitor cells, melanocytes or dermal papilla cells, the method comprising contacting skin tissue to one or more activators of Notch signalling, and subsequently isolating the differentiated cells from skin tissue.

40. A composition formulated for administration to the skin comprising one or more activator of Notch signalling.

41. The composition of claim 40 wherein the formulation is for subcutaneous or topical administration.

42. The composition of claim 40 or 41 wherein the activator of Notch signalling is Delta 1 , Jagged 2, JAGGED 1 , Soluble JAGGED 1 , NICD, ADAM 10 and/or ADAM17, or a fragment, variant or derivative of said activators capable of activating Notch signalling.

43. A device comprising a composition according to claims 40 or 41 adapted for delivery of the composition to the epidermis or epidermal /dermal boundary of the skin.