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WO2005059119A2 - Cellules differentiees - Google Patents

Cellules differentiees Download PDF

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Publication number
WO2005059119A2
WO2005059119A2 PCT/GB2004/005232 GB2004005232W WO2005059119A2 WO 2005059119 A2 WO2005059119 A2 WO 2005059119A2 GB 2004005232 W GB2004005232 W GB 2004005232W WO 2005059119 A2 WO2005059119 A2 WO 2005059119A2
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WO
WIPO (PCT)
Prior art keywords
cell
dermal
cells
sheath
papilla
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Ceased
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PCT/GB2004/005232
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WO2005059119A3 (fr
Inventor
Colin Albert Buchanan Jahoda
Amanda Jane Reynolds
Claire Jenna Whitehouse
Nicholas Hole
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University of Durham
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University of Durham
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Publication of WO2005059119A3 publication Critical patent/WO2005059119A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0666—Mesenchymal stem cells from hair follicles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0627—Hair cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells

Definitions

  • the invention relates to isolated clonally derived stem cells with different differentiation potential.
  • 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.
  • a clonally derived dermal sheath stem cell wherein said cell has the potential to differentiate into at least one cell type.
  • a clonally derived dermal papilla stem cell wherein said cell has the potential to differentiate into at least one cell type.
  • said stem cell is derived from cultures of early dermal sheath primary outgrowths.
  • said stem cell is derived from cultures of early dermal papilla primary outgrowths.
  • said differentiated cell is an adipocyte or an adipocyte-like cell, or a cell derived from an adipocyte.
  • said differentiated cell is an osteogenic cell, or an osteogenic-like cell, or a cell derived from an osteocyte.
  • a cell culture of clonally derived dermal sheath stem cells is provided.
  • a cell culture of clonally derived dermal papilla stem cells 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.
  • 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.
  • 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.
  • 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.
  • said differentiated cell is an adipocyte cell or an adipocyte-like cell, or a cell derived from an adipocyte.
  • said cell is an osteocyte cell, or an osteocyte-like cell or a cell derived from an osteocyte.
  • a therapeutic cell composition comprising a clonally derived dermal sheath stem cell.
  • a therapeutic cell composition comprising a clonally derived dermal papilla stem cell.
  • a therapeutic cell composition comprising a differentiated cell obtained by the method according to the invention.
  • said cell is an adipocyte.
  • said cell is an osteocyte.
  • 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.
  • 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.
  • 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.
  • a method of treatment of a condition comprising administering to an animal in need of treatment a differentiated cell according to the invention.
  • said differentiated cell is an adipocyte.
  • said differentiated cell is an osteocyte.
  • 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).
  • Figure 2 Initial characteristion of selected follicular dermal clonal cell lines.
  • B RT-PCR analysis of expression of selected genes in follicular dermal clonal cell cultures.
  • FIG. 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
  • DS7 O, P
  • FIG 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).
  • Dermal papillae 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 (50 ⁇ g/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).
  • DS Dermal sheath
  • 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% CO 2 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
  • 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.
  • 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% CO 2 , with a medium change every 3-4 days.
  • the clones were transplanted into 12.5cm 2 tissue culture flasks (Greiner) and allowed to grow for a further 10-14 days in MEM+10%FBS.
  • cells were prepared for rhodamine 123 dye efflux assays, alpha-smooth muscle actin staining, and RNA extraction.
  • Clone stocks were then routinely maintained in 25cm 2 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.
  • 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.
  • RNA was prepared from confluent 25cm 2 flasks. The cells were washed with sterile
  • 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 were performed using l ⁇ l first strand cDNA in a 50 ⁇ l 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 A
  • 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 ddH 2 O. The stained cells were then briefly rinsed with 60% isopropanol, washed thoroughly with ddH 2 O and photographed.
  • calcium formol 4% formaldehyde, 1% calcium chloride
  • 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.
  • 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).
  • efflux efficiency and stem cell capabilities 25.
  • 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.
  • Clones for further study were selected on the basis of their initial rhodamine 123 staining intensity and subsequent rate of efflux.
  • Follicular dermal cells express high levels of alpha smooth muscle actin in vitro, although staining intensity in mixed populations of cells is variable (33).
  • DS7 very high
  • DS2, DS4, DS5 very low
  • alpha smooth muscle actin in the DP clones was generally at an intermediate level (Fig 2a, middle panels).
  • 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.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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).
  • Id4 was previously found Id4 to be expressed in follicle end bulbs grown in long term culture (53), making it a candidate "survival factor" in mesenchyme.
  • Lefl forms part of the canonical wnt/Beta catenin pathway that has been shown to be essential for embryonic follicle development and adult growth (54).
  • Lefl forms part of the canonical wnt/Beta catenin pathway that has been shown to be essential for embryonic follicle development and adult growth (54).
  • Lefl forms part of the canonical wnt/Beta catenin pathway that has been shown to be essential for embryonic follicle development and adult growth
  • follicle dermal stem cell activity remains to be investigated as does the relationship between stem cell activity in the follicle and the rest of the skin.
  • haematopoietic activity from the follicle dermis (24)
  • stem cells derived from skin dermis have been shown to have multipotent stem cell capabilities, including the ability to make neurons (13).
  • 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.
  • stem cells within the hair follicle may have a clinical perspective.
  • 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.
  • 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/- ⁇ catenin 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).
  • the meso-angioblast a multipotent, self-renewing cell that originates from the dorsal aorta and differentiates into most mesodermal tissues. Development. 2002: 129: 2773-2783.
  • Newcomb P M Herman I M. Pericyte growth and contractile phenotype: modulation by endothelial-synthesized matrix and comparison with aortic smooth muscle. J Cell Physiol 1993: 155: 385-393.

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Abstract

Les cellules des papilles dermiques et des gaines dermiques des follicules pileux adultes constituent des populations de cellules actives dans le développement ayant un rôle prouvé dans l'activité cyclique des follicules pileux adultes et ayant des pouvoirs inducteurs uniques. Dans la biologie des cellules souches, l'épithélium des follicules pileux a récemment fait l'objet d'une grande masse d'investigations mais, jusqu'à présent, le derme du follicule a été largement surestimé comme source de cellules souches. A la suite de l'apparition sporadique de cellules de muscles, de lipides et de type osseux dans des cultures primaires de cellules de papilles et de gaines dermiques de follicules isolées de façon discrète, nous avons démontré que les lignées de cellules de papilles et de gaines cultivées pouvaient être dirigées vers la différentiation entre lipides et os. Par conséquent, pour la première fois, nous avons produit des lignées de papilles et de gaines dermiques clonales qui possédaient des capacités de prolifération étendues. On a constaté que l'exclusion par colorant constituait une fonction d'identification des cellules souches, de telle sorte que des lignées de papilles et de gaines clonales avec une capacité différente d'exclure la rhodamine (123) ont été cultivées dans un milieu connu pour son pouvoir inducteur de la différentiation entre adipocytes et ostéocytes. Des clones dérivés à la fois de gaines dermiques et de papilles dermiques on montré une capacité à fabriquer des lipides et à produire de la matière calcifiée, cependant que différents clones présentaient un comportement varié et qu'il n'y avait pas de corrélation évidente entre leur capacité de cellules souches et les marqueurs d'exclusion par colorant ou d'expression de gènes sélectionnés. Comme source hautement accessible, pouvant être isolé de façon discrète, le follicule possède un potentiel important comme source de cellules souches à des fins de génie génétique des tissus et de thérapie cellulaire. Il sera également intéressant de comparer les propriétés des cellules souches dermiques des follicules avec les capacités des cellules souches plus larges découvertes dans le derme de la peau, et d'investiguer si le follicule constitue une niche de cellules souches dermiques clé. Enfin, la découverte de cellules souches dans le derme peut avoir des implications pour certaines pathologies dans lesquelles une différenciation anormale se produit dans la peau.
PCT/GB2004/005232 2003-12-15 2004-12-14 Cellules differentiees Ceased WO2005059119A2 (fr)

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GBGB0328943.6A GB0328943D0 (en) 2003-12-15 2003-12-15 Differentiated cells
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007037486A1 (fr) * 2005-09-30 2007-04-05 Phoenixbio Co., Ltd. Procédé de culture de cellules de la cuticule d'un follicule pileux
US9109204B2 (en) 2006-02-28 2015-08-18 The Trustees Of Columbia University In The City Of New York Methods for compact aggregation of dermal cells
CN113201481A (zh) * 2021-04-19 2021-08-03 清华大学深圳国际研究生院 皮肤微球及其制备方法和应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3573488B2 (ja) * 1994-04-11 2004-10-06 独立行政法人 科学技術振興機構 毛乳頭細胞の長期継代培養法
DE10224982A1 (de) * 2002-06-05 2003-12-24 Rolf Hoffmann Mesenchymale Stammzellen des Haarfollikels und deren Verwendung

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007037486A1 (fr) * 2005-09-30 2007-04-05 Phoenixbio Co., Ltd. Procédé de culture de cellules de la cuticule d'un follicule pileux
US8206980B2 (en) 2005-09-30 2012-06-26 Phoenixbio Co., Ltd. Method for cultivation of hair follicular dermal sheath cells
JP5227024B2 (ja) * 2005-09-30 2013-07-03 株式会社フェニックスバイオ 毛包真皮毛根鞘細胞の培養法
US9109204B2 (en) 2006-02-28 2015-08-18 The Trustees Of Columbia University In The City Of New York Methods for compact aggregation of dermal cells
US9550976B2 (en) 2006-02-28 2017-01-24 The Trustees Of Columbia University In The City Of New York Methods for compact aggregation of dermal cells
CN113201481A (zh) * 2021-04-19 2021-08-03 清华大学深圳国际研究生院 皮肤微球及其制备方法和应用
CN113201481B (zh) * 2021-04-19 2023-10-13 清华大学深圳国际研究生院 皮肤微球及其制备方法和应用

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WO2005059119A3 (fr) 2005-08-11

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