WO2005059119A2

WO2005059119A2 - Differentiated cells

Info

Publication number: WO2005059119A2
Application number: PCT/GB2004/005232
Authority: WO
Inventors: Colin Albert Buchanan Jahoda; Amanda Jane Reynolds; Claire Jenna Whitehouse; Nicholas Hole
Original assignee: University of Durham
Current assignee: University of Durham
Priority date: 2003-12-15
Filing date: 2004-12-14
Publication date: 2005-06-30
Anticipated expiration: 2006-06-15
Also published as: GB0328943D0; WO2005059119A3

Abstract

The adult hair follicle dermal papilla and dermal sheath cell are developmentally active cell populations with a proven role in adult hair follicle cycling activity and unique inductive powers. In stein cell biology, the hair follicle epithelium has recently been the subject of a great deal of investigation, but up to now the follicle dermis has been largely overlooked as a source of stein cells. Following the sporadic appearance of muscle, lipid and bone-type cells in discretely isolated follicle dermal papilla and sheath cell primary cultures, we demonstrated that cultured papilla and sheath cell lines were capable of being directed to lipid and bone differentiation. Subsequently, for the First time, we produced clonal dermal papilla and sheath lines that had extended proliferative capabilities. Dye exclusion has been reported to be an identifying feature of stein cells, therefore clonal papilla and sheath lines with differing capacity to exclude rhodamine (123) were cultured in medium known to induce adipocyte and osteocyte differentiation. Both dermal sheath and dermal papilla-derived clones showed the capacity to make lipid and to produce calcified material, however different clones had varied behaviour and there was no obvious correlation between their stein cell capabilities, and dye exclusion or selected gene expression markers. As a highly accessible source, capable of being discretely isolated, the follicle has important potential as a stein cell source for tissue engineering and cell therapy purposes. It will also be interesting to compare follicle dermal stein cell properties with the broader stein cell capabilities discovered in skin dermis, and investigate whether the follicle is a key dermal stein cell niche. Finally, the discovery of stein cells in the dermis may have implications for certain pathologies in which abnormal differentiation occurs in the skin.

Description

Differentiated Cells

The invention relates to isolated clonally derived stem cells with different differentiation potential.

Recent evidence suggests that many adult tissues possess a "reservoir" of stem cells, and when well characterised, can demonstrate an apparently high degree of plasticity. (1-5). To investigate adult stem cell activities, model systems are required which should be capable of being isolated discretely in tissue form, and further, if adult stem cells are to be used therapeutically, ease of access is going to be a crucial prerequisite. Researchers are therefore investigating adult stem cells from a variety of sources including adipose tissue (6,7), the synovial membrane (8,9), perivascular cells (10) and tooth dental pulp cells (11,12). In terms of accessibility, skin ranks highly and one group has already shown that stem cells from the skin dermis can differentiate in culture and give rise to muscle and neurons, and therefore have a broad level of potency (13). However the phenotype and origin of this stem cell population is unclear since the initial dermal population was heterogeneous.

In the context of stem cell biology, hair follicle epithelial cells have been the subject of intense study in relation to skin renewal (14, 15) and tumour biology (16). Melanocyte stem cell activity in the follicle has also been the subject of investigation (17). Unique to the hair follicle, dynamic epithelial-mesenchymal cross-talk persists from embryonic development into adulthood, as seen during the course of normal follicle growth and cycling, (18). Crucially, for research purposes, hair follicles contain discrete populations of interacting cells that are clustered in defined sites and that can be isolated, cultured and then experimentally manipulated. This work has shown that the two main dermal cell populations in the adult follicle, the dermal papilla (DP) (19,20) and to a lesser extent the dermal sheath (DS) (21) have rare inductive powers, and we now have greater understanding of the molecular events governing this behaviour (22). Thus the hair follicle is emerging as a major developmental model, encompassing paradigms of epithelial-mesenchymal interactions and epithelial stem cell behaviour, and highly accessible in the adult body. However, up to now, the follicle dermis has been largely overlooked as a source of stem cells.

Previously, we suggested that the follicle dermis acts as important stem cell repository for repair of the dermis after skin wounding (23), and more recently we demonstrated that there is haematopoietic stem cell activity in the follicle dermis (24). Based on these data, and because of unusual behaviour observed in some of our hair follicle dermal cell cultures, we have investigated the capacity of hair follicle dermal papilla and dermal sheath cells from rat vibrissa follicles to undergo adipogenic and osteogenic differentiation in primary culture using supplemented media. We then carried out similar investigations using clonally derived cell lines from the early DP and DS cell primary outgrowths. Since links have been made between stem cell activity and certain properties including the ability to exclude dyes (25,26) and proliferative activity (27), and since other stem cell-containing populations (such as bone marrow) express alpha smooth muscle actin (28) we investigated whether there were any correlations between these phenomena and the capacity of the clonal lines to assume other mesenchymal phenotypes. We also examined the expression of a limited number of markers by the clones.

Our findings showed that both papilla and sheath cells could be directed to undergo adipogenic and osteogenic differentiation. Unexpectedly, however, the clonal lines revealed variation in their capacity to undergo phenotypic change and no clear correlation with any of the stem cell markers. This technology may be applied to human dermal sheath cells and human dermal papilla cells.

According to an aspect of the invention there is provided a clonally derived dermal sheath stem cell wherein said cell has the potential to differentiate into at least one cell type.

According to a further aspect of the invention there is provided a clonally derived dermal papilla stem cell wherein said cell has the potential to differentiate into at least one cell type. In a preferred embodiment of the invention said stem cell is derived from cultures of early dermal sheath primary outgrowths.

In an alternative preferred embodiment of the invention said stem cell is derived from cultures of early dermal papilla primary outgrowths.

In a further preferred embodiment of the invention said differentiated cell is an adipocyte or an adipocyte-like cell, or a cell derived from an adipocyte.

In an alternative preferred embodiment of the invention said differentiated cell is an osteogenic cell, or an osteogenic-like cell, or a cell derived from an osteocyte.

According to a further aspect of the invention there is provided a cell culture of clonally derived dermal sheath stem cells.

According to a further aspect of the invention there is provided a cell culture of clonally derived dermal papilla stem cells.

According to a further aspect of the invention there is provided a method to clonally derive dermal sheath stem cells comprising the steps of: i) providing a preparation comprising at least one dermal sheath cell and cell culture media; and ii) cloning from said preparation a clonally derived dermal sheath cell.

According to a further aspect of the invention there is provided a method to clonally derive dermal papilla stem cells comprising the steps of: i) providing a preparation comprising at least one dermal papilla cell and cell culture media; and ii) cloning from said preparation a clonally derived dermal papilla cell. According to a further aspect of the invention there is provided a method for the differentiation of a clonally derived dermal sheath cell into at least one differentiated cell- type comprising the steps of: i) providing a clonally derived dermal sheath cell according to the invention; and ii) providing conditions which initiate and/or promote the differentiation of said stem cell into at least one differentiated cell- type.

According to a further aspect of the invention there is provided a method for the differentiation of a clonally derived dermal papilla cell into at least one differentiated cell-type comprising the steps of: i) providing a clonally derived dermal papilla cell according to the invention; and ii) providing conditions which initiate and/or promote the differentiation of said stem cell into at least one differentiated cell- type.

In a preferred method of the invention said differentiated cell is an adipocyte cell or an adipocyte-like cell, or a cell derived from an adipocyte.

In an alternative preferred method of the invention said cell is an osteocyte cell, or an osteocyte-like cell or a cell derived from an osteocyte.

According to a further aspect of the invention there is provided a therapeutic cell composition comprising a clonally derived dermal sheath stem cell.

According to a further aspect of the invention there is provided a therapeutic cell composition comprising a clonally derived dermal papilla stem cell. According to a yet further aspect of the invention there is provided a therapeutic cell composition comprising a differentiated cell obtained by the method according to the invention.

In a preferred embodiment of the invention said cell is an adipocyte.

In an alternative preferred method of the invention said cell is an osteocyte.

According to a further aspect of the invention there is provided a method of treatment of a condition comprising administering to an animal in need of treatment a clonally derived dermal sheath cell according to the invention.

According to a further aspect of the invention there is provided a method of treatment of a condition comprising administering to an animal in need of said treatment a clonally derived dermal papilla cell according to the invention.

According to a further aspect of the invention there is provided a method of treatment of a condition comprising administering to an animal in need of treatment a differentiated cell according to the invention.

In a preferred method of the invention said differentiated cell is an adipocyte.

In an alternative preferred method of the invention said differentiated cell is an osteocyte.

An embodiment of the invention will now be described by example only and with reference to the following figures: Figure 1: Spontaneous and directed differentiation of primary dermal sheath and papilla cell cultures. (A-C): Combined rat dermal sheath and papilla cultures: A: Spontaneous myotube formation (arrowed) B and C: Spontaneous appearance of adipocytes in early cell outgrowths. (D-F) Mouse primary cultures. Adipocytes in dermal sheath (D) and dermal papilla (E) cultures are confirmed by oil red O staining (F). Directed differentiation of rat dermal sheath cell cells is shown by Von Kossa staining (G-K). From day 1(G) when there was no staining, calcified deposits appeared in regions of cell aggregation by days 6 (H) and 8 (I) and increased in size until large clumps of calcified material were present after two or three weeks of treatement (J). Control cultures grown over the same period show no staining (K).

Figure 2: Initial characteristion of selected follicular dermal clonal cell lines. A: rhodamine 123 efflux (left panels), asma expression (centre panels) and morphology (right panels) of selected follicular dermal clonal cell lines (bar = 200μM). B: RT-PCR analysis of expression of selected genes in follicular dermal clonal cell cultures.

Figure 3: Adipogenic differentiation in follicular dermal clonal cell lines. DP clones (A- H) were cultured in adipogenic medium (A, C, E, G) or control medium (B, D, F, H) for 7 days, and DS clones were cultured in adipogenic medium (I, K, M, O) and control medium (J, L, N, P) for 7 days (M-P) or 21 days (I-L). Cultures were then fixed and stained with oil red O. DP4 (A, B), DP5 (C, D), DP11 (E, F), DP12 (G, H), DS5 (M, N) and DS7 (O, P) showed extensive adipogenic differentiation in adipogenic medium but not in control medium, whereas DS2 (I, J) and DS4 (K, L) did not differentiate after 21 days of culture in adipogenic medium, (bar = 50μM)

Figure 4: Examples of osteogenic differentiation in follicular dermal clonal cell lines. Clonal cell lines were cultured in osteogenic medium or control medium for 28-40 days, then fixed and examined for bone formation by Von Kossa staining (A-D). DP4 was extensively calcified after 30 days in osteogenic medium (B), although the cells did not aggregate. No staining was seen in the control culture (A). DS2 (C) rapidly formed large calcified aggregates in osteogenic medium, while calcification of DS5 (D) occurred both in cell clumps and in the monolayer between aggregates. This was confirmed by staining with an osteopontin antibody, showing that osteopontin was highly expressed in the aggregated areas in DS2 (E), and more widely expressed throughout differentiated cultures of DS5 (F). (bar = 200μM).

MATERIALS AND METHODS

General rat DP and DS cell culture

Dermal papillae (DP) were dissected from adult rat vibrissa follicles using previously described methods (29). Briefly, the mystacial pad was cut open, the skin inverted, and the end bulb region of isolated sinus follicles removed. Fine forceps were then used to invert the collagen capsule of the end bulb and expose the papilla and epithelial matrix. The matrix component was then removed, and any epithelial tissue still present on the papilla was teased off. The papilla was then extracted using fine forceps and transferred to a culture vessel. Dissected papillae were cultured initially in 20% foetal bovine serum (Seralab) and Eagles minimal essential medium (E-MEM) with Glutamax-I, Earles salts and 25mM Hepes (Invitrogen) containing Gentamycin (50μg/ml). Cell cultures were initiated in 35mm dishes (Falcon) and were continued in these vessels after the first passage. On the second passage the cells were transferred to 25cm2 flasks (Falcon). After the first passage the concentration of foetal bovine serum in the medium was reduced to 10%. Dermal sheath (DS) tissue was isolated from vibrissae follicles as described by Reynolds (30). During the DP dissection above, when the follicle end bulb was inverted the DS collapsed but remained attached to the base of the DP. It was then teased from the papilla using fine forceps, and pieces of DS tissue from several follicles were initially cultured in exactly the same manner described for DP cells above.

Preparation of Dermal Papilla and Dermal Sheath Cell Clones

Dermal papilla and dermal sheath tissue was obtained by microdissection from the vibrissa follicles of 3 month old female Wistar rats as described above. Individual explants were cultured in 24 well plates (Nunc) in MEM + 10%FBS supplemented with antibiotics (Sigma) containing 20% DP or DS primary culture conditioned medium (MEM+CM). Primary cultures were incubated at 37°C/5% CO₂ for 5 days to allow cells to grow out from the explant while limiting the amount of cell division. Cells from individual explants were collected by incubation with 0.25% trypsin for 5 minutes at

37°C, and cloned at a density of one cell per well in 96 well plates by limiting dilution. Wells containing single cells were identified after 24 hours by phase contrast microscopy, and these were cultured in MEM+CM for 28 days, with a medium change every 7 days.

Twelve clones of each cell type were transferred to 35mm dishes (Nunc), and expanded for a further 16-35 days in MEM+CM at 37°C/5% CO₂, with a medium change every 3-4 days. At confluence, the clones were transplanted into 12.5cm² tissue culture flasks (Greiner) and allowed to grow for a further 10-14 days in MEM+10%FBS. At this stage, cells were prepared for rhodamine 123 dye efflux assays, alpha-smooth muscle actin staining, and RNA extraction. Clone stocks were then routinely maintained in 25cm² tissue culture flasks (Nunc) at 37°C/5% CO_2, in MEM+10%FBS, with a medium change every 3-4 days. Clone stocks were routinely passaged every 21-35 days.

Rhodamine 123 Dye Efflux Assay

To determine the rate of rhodamine 123 accumulation and efflux, cells (50,000 per 35mm plate) were incubated in MEM + 2%FBS containing 6.5μM rhodamine 123 (rhol23) for 3 hours at 37°C/5% CO₂ (31). Free rhol23 was removed by washing thoroughly with ice cold PBS, and the cells photographed to determine the level of accumulation of rhol23 in the mitochondria. Cells were then incubated for a further 2 hours in MEM + 10%FBS and photographed again to determine the rate of efflux. For samples where the dye efflux was not complete after 2 hours, incubation was continued for a further 22 hours.

Alpha-Smooth Muscle Actin Staining

Cells plated at low density (50,000 cells/35mm dish) were washed 2 x 5mins PBS and fixed in ice cold 100% methanol for 2 minutes. The fixed cells were then washed 3 x 5 minutes in PBS (and stored in PBS at 4°C if necessary). For immunohistochemical detection of alpha smooth muscle actin, non-specific antibody binding was blocked by incubation with 20% FBS in PBS for 1 hr at room temperature, followed by incubation with anti-alpha smooth muscle actin (Sigma) diluted 1/1000 in PBS containing 1%FBS for 1 hr at room temperature. The cells were then washed 3 x 5 minutes in PBS to remove unbound primary antibody, then incubated in rabbit anti-mouse IgG-FITC (Dako) diluted 1/100 in PBS for lhr at room temperature in the dark. Unbound secondary antibody was removed by washing for 3 x 5 minutes with PBS. The cells were then mounted in Mowiol and images recorded using a Spot RT digital camera(Diagnostic Instruments Inc) on a Zeiss axiovert 135 microscope with fixed exposure settings for all of the specimens.

Determination of clonal growth rates (minimum doubling time) Cells were plated into 3x6 well plates at 10⁴ cells/well and three wells were trypsinised and counted at 4, 6, 8, 14 and 24 days. The period of maximum log growth was determined from the mean cell counts at each time point, and the minimum population doubling time was determined using the following formula (32): Population doubling time = hours of growth Number of divisions

Where number of divisions = logNl - log NO Log2

(NO = initial cell count, Nl = subsequent cell count)

Preparation of cDNA from Dermal Cell Clones

Total RNA was prepared from confluent 25cm² flasks. The cells were washed with sterile

PBS, and RNA was prepared using (an Ambion totally RNA extraction kit) according to the manufacturer's protocol. The total RNA was treated with RNase free DNasel (Ambion) to remove genomic DNA contamination, and cDNA was prepared from 2Dg total RNA, using lOOng random primers (Promega) and 200U SuperScriptll reverse transcriptase (Gibco Invitrogen) at 44°C for 1 hour. No RT controls were prepared in parallel to ensure that no genomic DNA was present in the prepared cDNA samples.

PCR Reactions

PCR reactions were performed using lμl first strand cDNA in a 50μl reaction volume. Primers and amplification conditions used for PCR were as follows: GAPDH 5' ATG GCC TAC ATG GCC TCC AAG G and 5' AGG CCC CTC CTG TTG TTA TGG G, 35 cycles of 95°C 30s, 58°C 30s, 72°C 50s; FLK-1 5' AAC AGA ATT TCC TGG GAC AGC and 5' TGC CCA CAG TGG CTT CCA CC, 35 cycles of 95°C 30s, 56°C 30s, 72C 70s; ID4 5' 5' TAG GCG AGC TGC GAA CTC CAG G and 5' CCA ACA GGG CAC GTT TAG ACA C, 40 cycles of 95°C 30s, 54°C 30s, 72°C 60s; LEF-1 5' CTG TTT TTA TTA GCC GAT TAG TG and 5' GCT CAG CAC GTT AAC TCA AAC TGG, 35 cycles of 95°C 30s, 58°C 30s, 72°C 50s. Aliquots (lOμl) of each PCR reaction were then electrophoresed on 2.0% agarose/lxTAE gels containing O.Sμgml^"1 ethidium bromide, and photographed using the BioRad GelDoc 2000 system (BioRad) to determine the relative abundance of expression of the selected transcription factors in each of the clones.

Adipogenic Assay

Selected clones were plated in duplicate at 100,000 cells per 35 mm dish and allowed to adhere overnight in MEM + 10%FBS at 37°C/5% CO₂. One dish of each pair was then incubated in MEM + 10%FBS (control), while the other was incubated in adipogenic medium (15% rabbit serum, 0.45mM isobutyl-methylxanthine, 2.07uM insulin, lOOnM dexamethasone - modified from reference 6). The cultures were maintained for 7-23 days with a medium change every 3-4 days.

Lipid Detection

Cultured cells were stained with oil red-O to detect lipid production. Briefly, the cells were washed in PBS, then fixed in calcium formol (4% formaldehyde, 1% calcium chloride) for lhr at room temperature. The cells were then incubated for 15 minutes in 60% isopropanol, then stained for 15 minutes with a filtered solution of 3 parts saturated oil red O in isopropanol, 2 parts ddH₂O. The stained cells were then briefly rinsed with 60% isopropanol, washed thoroughly with ddH₂O and photographed.

Osteogenic Assay

Selected clones were plated in duplicate at 100,000 cells per 35 mm dish and allowed to adhere overnight in MEM + 10%FBS at 37°C/5% CO₂. One dish of each pair was then incubated in MEM + 10%FBS (control), while the other was incubated in osteogenic medium (MEM + 10%FBS containing 0.1 uM dexamethasone and lOmM 0- glycerophosphate, supplemented every 3 days with 50uM ascorbate-2-phosphate (6). The cultures were maintained for 25-40 days, with a medium change every 3-4 days. In preliminary experiments, primary dermal papilla and dermal sheath cultures were grown under exactly the same conditions, using variable seeding densities, and treated and control cultures tested for mineral deposition at intervals up to three weeks.

Bone Detection

Production of calcified deposits and changes in cell morphology were detected by Von Kossa staining, and phase contrast microscopy. Clonal lines in osteogenic medium were also tested for expression of the bone marker osteopontin at intervals up to 30 days. For immunohistochemical detection cells were fixed and pre-blocked as described above for alpha smooth muscle actin. They were then incubated with the osteopontin antibody [(MPIIIBlO(l), supernatant from the Developmental Studies Hybridoma Bank] for 1 hour at room temperature, washed 3 5 minutes in PBS to remove unbound primary antibody, then incubated in rabbit anti-mouse IgG-FITC (Dako) diluted 1/100 in PBS for lhr at room temperature in the dark. Unbound secondary antibody was removed by washing for 3 x 5 minutes with PBS. The cells were then mounted in Mowiol and images recorded using a Spot RT digital camera (Diagnostic Instruments Inc) on a Zeiss axiovert 135 microscope, or with a Zeiss confocal microscope.

Example 1

Spontaneous and directed differentiation of hair follicle dermal cell cultures

Under routine fibroblast culture conditions, we have periodically observed the appearance of unexpected cell types in primary follicle dermal cell cultures from rat, mice and sheep over several years. Muscle differentiation has been seen in the form of myotubes containing multiple nuclei in combined dermal papilla and dermal sheath cultures (Fig la), and in dermal sheath cultures alone, but never in dermal papilla cultures. Intriguingly, we have also observed beating cells reminiscent of cardiomyocytes (data not shown). Adipocytes have been observed in combined papilla and sheath cultures (Fig.lb,c) and in dermal sheath (Fig. Id) and dermal papilla (Figle) cultures alone, their presence confirmed by oil red O staining (Fig. If). Primary dermal papilla and sheath cell cultures have also been directed to adipocyte differentiation using the methods described above (data not shown). Nodules and unidentified deposits were observed in routine primary cultures for some time before Von Kossa staining finally revealed them to be calcified material. Subsequently, primary dermal papilla and dermal sheath cultures were directed to show osteocyte differentiation (Fig.lg-k).

Example 2

Behaviour and morphology of clonal cell lines

Starting from a single cell, we estimated that at least 16 population doublings were required to produce a confluent 35mm culture dish. Thus the initial establishment of clonal lines required isolated cells to have considerable proliferative capabilities and only 12-15% of cell lines survived to this stage. The majority of single cells seeded into 96 well plates either failed to divide at all, or showed very limited division. Of the 24 clones showing sufficient growth for transfer to 35mm dishes, three failed to proliferate further. The remaining 21 clones were transferred to 12.5cm flasks, and subsequently the cultures were maintained for at least 26 passages, longer than would normally be seen with primary rat cell lines. They have also been intermittently cryopreserved and regrown for extended passaging and up to present have shown no sign of becoming senescent. All of the cells were fibroblast-like in appearance, though some displayed a pronounced bipolar phenotype (DS7, DP12), while others were more stellate (DP4) (Fig2a, right panels). The cell size of the clones was also very variable, with DS2 being significantly smaller than most of the other lines. Differing behaviour was apparent from the very early stages of culture, with some of the clones forming tight colonies (DS2, DS5, DP5), while others were much more mobile. Several of the DS clones showed a distinct tendency to form clumps at confluence, while perhaps surprisingly, none of the DP clones were clump forming. Population doubling time for the clones was generally slow, but quite variable. A summary of these, and the results of all work on the 8 selected clonal lines is shown in Table 1. Example 3

Dye Efflux Assay

The mdr-1 substrate rhodaminel23 has been used to identify cells exhibiting high levels of drug efflux activity, and there appears to be a distinct correlation between efflux efficiency and stem cell capabilities (25). We therefore stained our 21 clonal cell lines and mixed populations of dermal papilla and dermal sheath cells with rhodamine 123 to determine their uptake and efflux profiles. Mixed populations of DP cells stain fairly highly and uniformly with rhodamine 123 at timeO of efflux, but this lack of variation was not reflected in the clonal cell lines, with staining levels varying widely. Mixed populations of DS cells showed much greater variation in staining intensity at timeO, with the clonal DS lines also showing a wide range of staining intensity (Fig 2a, left panels). After 2 hours of efflux, much of the staining was lost, although some of the DS clones retained high levels (for example DS5), and there was an increase in the level of retention in areas where the cells were tightly clumped (for example in DS4). After 24 hours, all staining was gone except in clumps, where a low level was still evident.

Clones for further study were selected on the basis of their initial rhodamine 123 staining intensity and subsequent rate of efflux.

Example 4

Alpha-Smooth Muscle Actin Staining

Follicular dermal cells express high levels of alpha smooth muscle actin in vitro, although staining intensity in mixed populations of cells is variable (33). We found a high degree of variability in our clonal cell lines, with DS clones having either very high (DS7) or very low (DS2, DS4, DS5) levels of expression, while expression of alpha smooth muscle actin in the DP clones was generally at an intermediate level (Fig 2a, middle panels). Example 5

Gene Expression in Cultured DP and DS Cells

The relative levels of expression of several genes in our clonal cell lines were examined by RT-PCR. IGF-2 and ID4 expression was generally higher in the DP lines, while FLK1 expression was higher in the DS clones. Two clones expressed all of the genes we selected at fairly high levels (DS7, DP4), but the remaining clones showed very variable patterns and levels of expression of the selected genes (Fig 2b). When combined with the morphology, growth rate, asma staining and rhol23 efflux data, this shows that cultured DP and DS are a very mixed population of biochemically and morphologically distinct cells.

Example 6

Adipogenic Assay

As spontaneous appearance of adipocytes in primary cultures, particularly of DP cells, is relatively common, we expected that the DP clones in particular would readily undergo adipogenic differentiation in suitable culture conditions. Primary DP and DS cell populations both produced large quantities of oil red O positive, lipid rich cells within 7 days (DP) or 21 days (DS) of treatment. All of the DP clones showed evidence of adipogenesis after 24 hours in selective medium, with large lipid globules appearing in all cells. After 7 days of culture, oil red O staining showed extensive adipogenesis in all of the clonal DP cell lines. Of the DS lines tested, DS5 and DS7 behaved similarly to the DP lines, with extensive adipogenesis after 7 days in culture, but DS2 showed no change after 23 days in adipogenic medium. DS4 did not undergo extensive adipogenesis even after 23 days in culture, but there was some evidence of increased lipid production in the cells (Fig 3). Example 7

Osteogenic assay

Only three of the clones tested (DS2, DS5, DP4) produced calcified deposits (determined by Von Kossa staining) when cultured in osteogenic medium. DP4 was not stimulated to aggregate in these culture conditions, and small areas of calcification were produced throughout the culture (Fig. 4a, b). The level of aggregation of DS5 increased slightly in osteogenic medium, but the production of calcified deposits was not limited to the aggregated areas alone (Fig. 4d, f). DS2 showed the greatest change in morphology, with formation of tight clumps of cells which became heavily calcified (Fig. 4c, e). All experiments were repeated.

In this paper, we report that cells from the hair follicle dermal papilla and dermal sheath can differentiate either spontaneously or in directed fashion along other mesodermal lineages in vitro. This adds to our recent discovery that cells present within the hair follicle dermis support haematopoietic reconstitution in vivo, and to recent ideas that follicle cells may act as a source of stem cells for fibroblasts in skin wound healing (23,24).

It is interesting to note that when spontaneous differentiation in primary cultures occurred, usually only one cell type was produced. It has been suggested that lineage commitment of mesenchymal stem cells may be governed by activation/inhibition of ERK (MAP kinase) and although the stem cells may be multipotent, the microenvironment determines an either/or differentiation response into adipocytes or osteocytes (34). The broader stem cell capabilities of the follicular dermis remain to be investigated, but in this context it is relevant that we have seen sporadic evidence of diverse stem cell activities. The observation of skeletal muscle in our cultures is consistent with dermal cells having the ability to form muscle. (35). Moreover, the observations of beating cells in cultures is consistent with a number of recent studies that have shown that mesenchymal stem cells from bone marrow (36) and adipose tissue (37) can become myocardiocytes.

Our observations of directed differentiation in mixed populations demonstrate that the capability exists, but sheds no light on whether the population contains a broad mixture of cells each with limited potential (ie committed progenitor cells from a number of different lineages), or few cells with broader capabilities. Also while the vibrissa follicle dermal papilla and dermal sheath were discretely microdissected, they do have a resident blood supply. Therefore it is possible is that the primary cultures contained transient multipotent cells from circulating mesenchymal stem cells (38), or that the observations were attributable to cells associated with blood vessels that have mesenchymal stem cell capabilities, such as pericytes (10) or even meso-angioblasts (39). To analyse stem cell potential precisely, the exclusion of other differentiated cells and a clonal assay system are required since their potential can only be precisely analysed in single cell based experiments. (40, 41, 42). We therefore produced a series of dermal papilla and dermal sheath cell lines derived from single cells isolated from tissue explants cultured for 7 days. To our knowledge this is the first paper in which either of these cell populations have been grown clonally. In effect, the culture methodology we used 'pre-selected' for those cells having a high proliferative potential and extended survival.

It has been widely reported that the ability of cells to efflux the fluorescent dyes rhodamine 123 and Hoescht-33342 via ABC transporters (ATP binding cassette transporter superfamily members) correlates closely with their stem cell capabilities in hematopoietic stem cell populations (43,44,25), and regulation of stem cell biology by ABC transporters has emerged as an important new field of investigation (45). Stem cell populations have been identified in the pancreas on the basis of their efflux of rhol23 and Ho33342 (26check).

We investigated the ability of mixed populations and cloned lines of dermal papilla and dermal sheath cells to efflux rhodamine 123, and found that their dye-efflux capabilities varied widely. A report by Uchida et al (25) suggested that rhodamine 123 efflux could be used to identify functional subsets of hematopoietic stem cells - only those cells which were able to efficiently efflux rhol23 were able to repopulate the blood system of lethally irradiated mice. We therefore selected clones showing low, intermediate and high dye- exclusion capabilities to test for stem cell activity. Uchida et al further reported that those cells which could most efficiently efflux rhol23 tended to have the lowest growth rates but the longest survival in vitro. We determined the population doubling time for each of our selected clones and found that those DP clones with the greatest ability to efflux rhodamine 123 tended to divide more slowly, whereas there was no apparent correlation between rhodamine efflux and doubling time for the DS clones.

A common feature of adipose derived cells (6), bone marrow stromal cells (28) and osteoblasts (46) is that they harbour alpha smooth muscle actin expressing cells, and one group has looked at contractability and smooth muscle actin expression in human mesenchymal stem cells (47). Cultured follicle DP and DS cells express particularly high levels of alpha smooth muscle actin although the level of expression in individual cells can vary considerably (33). As expected, all of the clones expressed asma, although the level of expression was very variable. Cultured pericytes (which can also differentiate into mesodermal derivatives) express high levels of smooth muscle actin on regular tissue culture substrates (48). In this study the two clones showing the highest potential to form bone and fat cells both expressed low levels of alpha smooth muscle actin, making it unlikely that the clones were pericyte-derived.

We further tested four markers with relevant biological roles to investigate whether clones with differentiation (or indeed non-differentiation) capabilities had particular expression profiles. Since blood cell precursors and meso-angioblasts that differentiate into multiple mesodermal derivatives (39) express Flkl, to investigate the possibility that our cloned lines were blood cell derived, we examined their expression of Flkl. Although Flkl is highly expressed in follicle end bulbs, we found that Flkl expression in the clones was highly variable and did not correlate with any pattern of differentiation. IGF-II, a promoter of mesoderm formation in the developing embryo (49) that was recently shown to be upregulated by sonic hedgehog in pluripotent mesenchymal stem cells (50) was also expressed variably in the clones, and its expression did not correlate with their differentiation potential. The same was true of Id4, which has been identified as a negative regulator of haematopoiesis in vitro (51) and has also been found to be to be downregulated in senescent human fibroblast cells (52). We previously found Id4 to be expressed in follicle end bulbs grown in long term culture (53), making it a candidate "survival factor" in mesenchyme. The only marker to be expressed in all clones (albeit to a variable extent) was Lefl, which forms part of the canonical wnt/Beta catenin pathway that has been shown to be essential for embryonic follicle development and adult growth (54). Overall, however, none of the gene expression markers could be related positively or negatively to stem-cell behaviour of clones, although the observations are preliminary with small sample sizes.

Although we restricted our experimental investigation to whether follicle-derived cells could be directed to become adipocytes and osteocytes, both these lineages are significant in the context of other work. Previously a group investigating leptin expression showed that human follicle dermal papilla cells expressed leptin, but did not convert to adipocytes when treated with adipocyte differentiation medium (55). In our work, all the vibrissae follicle DP clones tested turned into adipocytes. Previous reports have shown that follicle dermal cells express osteogenic markers including alkaline phosphatase (56), and osteopontin (57). In the latter report, expression of osteopontin was correlated with papilla activity in the hair cycle. Our preliminary finding showing a correlation between the expression of osteopontin by specific clones, and their capacity to make bone, implies that the expression of bone-related markers by papilla cells might be significant in areas of biology over and above the hair follicle cycle.

The limits of follicle dermal stem cell activity remain to be investigated as does the relationship between stem cell activity in the follicle and the rest of the skin. We have previously reported haematopoietic activity from the follicle dermis (24), and interestingly, stem cells derived from skin dermis have been shown to have multipotent stem cell capabilities, including the ability to make neurons (13). However, the phenotype and origin of this stem cell population is unclear as the initial dermal population was heterogeneous. Indeed it is interesting that all of the cells came from haired skin. In other reports, dermal cultures from interfollicular skin could not be induced to differentiate into adipocytic, chondrocytic or osteocytic lineages (1), suggesting that there is a relationship between the presence of hair follicles in skin and dermal stem cell activities. Therefore from a biological and therapeutic perspective it will be important to establish precisely how much of the reported skin stem cell activity comes from hair follicles and whether stem cell populations in follicles and interfollicular skin overlap completely or differ quantitatively and qualitatively.

Finally, the finding of stem cells within the hair follicle may have a clinical perspective. For example, in conditions such as osteoma cutis in which skin undergoes ossification it has been suggested that the mechanism by which bone deposition occurs involves resident mesenchymal stem cells differentiating into osteoblasts.(58, 59). This may represent an in vivo manifestation of what we have observed in vitro and makes it all the more important to know whether stem cells are uniformly distributed in skin dermis, or have particular niches such as the hair follicle.

Example 8

Further Clone Characterization

The mRNA from sub-confluent cultured clones was isolated and probed for a number of dermal or stem cell associated genes (Table 2 and 3). All clones expressed Versican, a cell surface proteoglycan normally restricted to the hair follicle dermal papilla (du Cros et al 1995). Similarly all clones expressed Lef-1 and Wnt5a of the canonical Wnt/-βcatenin pathway that has been shown to be essential for embryonic follicle development and adult growth (DasGupta and Fuchs 1999). The majority of clones also expressed Frizzled, a member of the Sonic hedgehog pathway and the anagen stage-specific gene Nexin (Jensen et al 2000). Moreover, consistent with the inherent stem cell properties of the clones expression of genes associated with adult and embryonic stem (ES) cells was observed (Table 2 and 3) . Expression of the hematopoteic stem cell marker CD 133, pluripotent ES cell markers FOXD3 and Nanog, and the embrynoic dermis transcription factor Dermo-1 was observed in a number of different clones.

To evaluate further the origin of the dermal cell clones immunohistochemisrty was performed on all clone cell cultures. This revealed the expression of other dermal associated proteins including fibronectin. Also evaluated was the expression of proteins not normally expressed in hair follicle dermal cells, such as the endothelial marker Von willebrands factor was also evaluated. These "non dermal" markers were absent from all clone lines. Immunohistochemistry also identified the expression of the laminin receptor in all clones.

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TABLE 1. Characterisation of follicular dermal papilla and dermal sheath clonal cell lines: summary of data.

Table 2 Gene expression within the dermal sheath derived clones (+ indicates expression - indicates an absents of expression)

Table 3 Gene expression within the dermal papilla derived clones(+ indicates expression - indicates an absents of expression)

Claims

1. An isolated clonally derived dermal sheath stem cell wherein said cell has the potential to differentiate into at least one cell type.

2 An isolated clonally derived dermal papilla stem cell wherein said cell has the potential to differentiate into at least one cell type.

3. A cell according to Claim 1 wherein said stem cell is derived from cultures of early dermal sheath primary outgrowths.

4. A cell according to Claim 2 wherein said stem cell is derived from cultures of early dermal papilla primary outgrowths.

5. A cell according to any of Claims 1-4 wherein said differentiated, cell is an adipocyte or an adipocyte-like cell, or a cell derived from an adipocyte.

6. A cell accoriding to any of Claims 1-4 wherein said differentiated cell is an osteogenic cell, or an osteogenic-like cell, or a cell derived from an osteocyte.

7. A cell culture of clonally derived dermal sheath stem cells.

8. A cell culture of clonally derived dermal papilla stem cells.

9. A method to clonally derive dermal sheath stem cells comprising the steps of: i) providing a preparation comprising at least one dermal sheath cell and cell culture media; and ii) cloning from said preparation a clonally derived dermal sheath cell.

10. A method to clonally derive dermal papilla stem cells comprising the steps of: i) providing a preparation comprising at least one dermal papilla cell and cell culture media; and ii) cloning from said preparation a clonally derived dermal papilla cell.

11. A method for the differentiation of a clonally dermal sheath cell into at least one differentiated cell-type comprising the steps of: i) providing a clonally derived dermal sheath cell according to the invention; and ii) providing conditions which initiate and/or promote the differentiation of said stem cell into at least one differentiated cell- type.

12. A method for the differentiation of a clonally dermal papilla cell into at least one differentiated cell-type comprising the steps of: i) providing a clonally derived dermal papilla cell according to the invention; and ii) providing conditions which initiate and/or promote the differentiation of said stem cell into at least one differentiated cell- type.

13. A method according to Claim 11 or 12 wherein said differentiated cell is an adipocyte cell or an adipocyte-like cell, or a cell derived from an adipocyte.

14. A method according to Claim 11 or 12 wherein said differentiated cell is an osteocyte cell, or an osteocyte-like cell or a cell derived from an osteocyte.

15. A therapeutic cell composition comprising a clonally derived dermal sheath stem cell.

16. A therapeutic cell composition comprising a clonally derived dermal papilla stem cell.

17. A therapeutic cell composition comprising a differentiated cell obtained by the method according to Claim 12.

18. A composition according to Claim 17 wherein said cell is an adipocyte.

19. A composition according to Claim 17 wherein said cell is an osteocyte.

20. A method of treatment of a condition comprising administering to an animal in need of treatment a clonally derived dermal sheath cell according to the invention.

21. A method of treatment of a condition comprising administering to an animal in need of said treatment a clonally derived dermal papilla cell according to the invention.

22. A method of treatment of a condition comprising administering to an animal in need of said treatment a clonally derived dermal papilla cell according to the invention.

23. A method of treatment of a condition comprising administering to an animal in need of treatment a differentiated cell according to the invention.

24. A method according to Claim 23 wherein said differentiated cell is an adipocyte.

25. A method according to Claim 23 wherein said differentiated cell is an osteocyte.