Methods for producing hair microfollicles and de novo papillae and their use for in vitro tests and in vivo implantations

Abstract

The present invention relates to a method for producing hair microfollicles comprising the steps of: a) providing de novo papillae, b) providing other cell populations selected from the group of fibroblasts, keratinocytes and melanocytes, and co-culturing the de novo papillae with at least one other cell population in non-adherent culture vessels. The present invention relates also to methods of producing de novo papillae usable in said method for producing hair microfollicles.

Claims

1. A de novo papilla consisting of a cell aggregate of mammalian dermal hair papilla fibroblasts (DPF), the cell aggregate exhibiting the shape and the size of a physiological dermal papilla (DP) of a hair follicle after isolation and being coated with an extracellular matrix protein, wherein the de novo papilla is produced by a method comprising the steps of: (a) providing at least one dermal papilla (DP) from at least one mammal hair follicle; (b) isolating dermal hair papilla fibroblasts (DPFs) from the DP by mechanically fixing said DP at the surface of a cell culture vessel, whereby the basal lamina is perforated to allow said DPFs migrating out; (c) expanding the isolated DPFs in monolayer culture without collagen coating, wherein said DPFs are passaged at least once; (d) condensing the expanded DPFs in non-adhesive culture vessels in a cell concentration per vessel surface of 1,000 to 100,000 DPFs/cm.sup.2 into cell aggregate and differentiating the cell aggregate into cell aggregates that exhibit the size and shape of the physiological DP in a non-adhesive culture vessel; and (e) coating the cell aggregates produced in (d) with the extracellular matrix protein, thereby producing the de novo papilla.

2. The de novo papilla of claim 1, wherein in step (d) of the method the expanded DPFs are condensed for at least 48 h.

3. The de novo papilla of claim 1, wherein in step (d) of the method the expanded DPFs are condensed for 2 to 21 or 3 to 15 days.

4. The de novo papilla of claim 1, wherein in step (d) of the method non-inductive DPFs are condensed.

5. The de novo papilla of claim 1, wherein the extracellular matrix protein is collagen IV, fibronectin and/or laminin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Figures

(2) FIG. 1 shows: A) Punch biopsy of a donor skin taken from a lifting surgery. The dashed line indicates the cutting line of the dermosubcutaneous border for further hair follicle isolation. B) The “amputated” and dissected hair follicle (highlighted box) is dissociated by a novel technique: The connected tissue sheath is pulled diametrically over the hair shaft resulting in a pure separation of the hair shaft with outer and inner root sheath keratinocytes and melanocytes on the on hand and Dermal papilla and connective tissue sheath on the other hand. C) Dissected connective tissue sheath with its fibroblasts plus adjacent dermal papilla, which can accurately, been cut off (highlighted box). D) A dissected dermal papilla taken by electron microscopy. E) Outgrowth of dermal papilla fibroblast from the slightly scratched and anchored dermal papilla, leaving their capsule structure almost intact. F) Cultured dermal papilla fibroblast of the 3.sup.rd passage in uncoated culture 75 cm.sup.2 flasks. G) Dermal papilla fibroblast forming dermal papilla-like condensates on ultra low attachment 75 cm.sup.2 flasks. H) Magnification of a dermal papilla fibroblast condensation ready to be processed to a Neopapilla with extracellular matrix components in 6well low attachment plates.

(3) FIG. 2 shows: A) Neopapilla (white arrow) with extracellular matrix and surrounded keratinocytes, which have been added to the ultra low attachment plate forming a multi-cellular condensate. B) First signs of the formation of follicular structures after 24 h in ultra low attachment culture. C) Neofollicle formation after 1 week. Clearly visible is the formation of a primitive hair shaft. D) Neofolllicle formation taken by DIC light microscopy illustrating the intact dermal papilla structure after 1 week of culture. E) Neofollicles inserted in a skin equivalent showing defined hair follicle like structures. The highlighted box shows a down-growing hair follicle. With the inverted microscope you see the proximal portion of the follicle/skin equivalent. F) Further culture of the Neofollicles within the skin equivalent demonstrating a clear anchorage of the hair follicle and continued growth.

(4) FIG. 3 shows: Neofollicle produced using KC and MC derived from sources different than mammalian hair follicle durich different stages of development and formation: A) Stage 1: Neopapilla (without the addition of exogenous extracellular matrix proteins) surrounded by Melanocytes and keratinocytes, which have been added to the ultra low attachment plate forming a early multi-cellular condensate. B) Stage 2: First signs of the formation of follicular structures after 24 h in ultra low attachment culture (Note the protuberance on top). The attached Cells adhere to the Neopapilla and adopt a flattened shape C) Stage 3: Beginning Neofollicle formation after 1 week with hair follicle-like sheath development. D) Stage 4: Within the Neofolllicle a clearly visible formation of a primitive hair shaft becomes visible.

(5) FIG. 4 shows: Formation of functional neopapillae at two different time points; A) after 48 hours condensation; and B) after 7 days of condensation; at day 7, the aggregation of cells is much more dense and the formation of self derived extracellular matrix becomes visible.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

EXAMPLES

Example 1

Isolation and Culture of Human Follicular Dermal Papilla Fibroblasts (DPF), Connective Tissue Sheath Fibroblasts (CTSF), Keratinocytes (KC), and Melanocytes (MC)

(6) Single hair follicles were obtained after micro-dissection of human scalp samples from excess of lifting surgeries, received under required regulations. To isolate matrix KC and MC, CTSF and fibroblasts of the dermal papilla (DPF), the skin was cut at the dermosubcutaneous interface with a scalpel and hair follicles at anagen stage (growing phase) were pull out with forceps under a dissecting microscope.

(7) In contrast to previously described isolation techniques (e.g. Magerl et al., Methods Exp. Dermat. 11, 381-385, 2002), pure fractions of the desired cell populations, were obtained by longitudinally slicing the connective tissue of the upper part of the dissected hair follicle. By fixing the hair shaft on the one site, the CTS can be diametrically pulled over the hair matrix with forceps towards the lower proximal portion. With this technique DP cells were automatically uncovered avoiding damage and mixing cell types. Thus, the CTS and the dermal papilla as well as the hair matrix containing KC and MC cells were clearly separated and can easily been cut off by a scalpel.

(8) DP and CTS isolates were separately cultured into 6-well cell culture plates (2-4 in one well) by fixing them to the culture plate with a needle. To enable a fast outgrowth but keeping the capsule morphology and niche of the fibroblast at the same time, the thin membrane was slightly scratched to loosen extracellular matrix components (ECM). The cells were submerged with DMEM+ (Gibco/Invitrogen) plus 10% fetal calf serum (FCS) until growth of fibroblast was observed after 1-2 weeks based on donor variations. The cells were then moved to a 25 cm2 culture flask for another week and were further passaged to a 75 cm.sup.2 culture flask. After reaching sub-confluence passage 3 were split into three 75 cm.sup.2-flasks obtaining 1.5-2 million DPF or CTSF, respectively.

(9) The KC and amelanotic hair follicle MC were removed from the remaining hair shaft and the attached hair matrix by trypsinisation (0.05% trypsin and 0.53 mM EDTA) and were separated by differential trypsinisation and cultured with standard methods as described in Tobin et al., J. Invest., Dermatology 104(1), 86-88, 1995.

Example 2

Formation of Neopapillae

(10) The optimized count of 500,000 DPF were seeded into a 75 cm.sup.2 ultra-low attachment culture flask (Corning) containing DMEM+ and were allowed to form cell aggregates. After 48 hours keeping them unmoved these aggregates shape the size of a native human hair follicle dermal papilla. Condensates were then transferred to a 6-well ultra-low attachment plate and a mixture of Laminin (final concentration 11.5 μg/ml), Fibronectin (final concentration 10 μg/ml) and Collagen IV (final concentration 40 μg/ml) is added to the wells. After 24-48 hours in culture, intrinsic ECM secretion and the added proteins built a stable matrix envelop and Neopapillae have formed. To facilitate faster ECM accumulation and differentiation, growth factors i.e. Hepatocyte Growth Factor (30 ng/ml) and/or Connective Tissue Growth Factor (20 ng/ml) can alternatively been added to the medium. These Neopapillae are ready to be implanted into skin in vivo to develop hair inductive properties.

Example 3

Formation of Neofollicles

(11) 250,000 KC and MC (10:1) were added to the Neopapillae in ultra-low attachment culture flask (6 well, Corning) and DMEM+Medium was changed to Defined Keratinocyte Serum-Free Medium (Gibco). After 1 week the covered Neopapillae form hair follicle like structures. These Neofollicles could already been used for testing. To elaborate a multilayered Neofollicle, which even better mimics hair follicle structure and function can be obtained by coating the generated Neofollicle with a mixture of collagen IV (60 μg/ml) with CTSF (200,000 cells) in ultra low attachment plates with DMEM+10% FCS for further 2 days and change of medium every further 3rd day.

Example 4

Generation of Skin Equivalents with Neofollicles

(12) Juvenile Human Foreskin Fibroblasts were isolated using previously described methods (Toma et al., Stem Cells 23, 727-37, 2005) and cultured in DMEM+10% FCS. 250,000 dermal fibroblast (also DPF or CTSF were used, but foreskin fibroblasts were preferred for handling reasons) were mixed to a Fibrinogen solution (Sigma, 3 mg/ml) and 2.0% (v/v) Aprotinin (20 μg/ml) and 2.5% (v/v) Thrombin 1.25 U/ml were added. This chilled solution was filled in transwell culture chambers avoiding air bubbles. After adjusting the temperature for 2-3 min at room temperature, 30-40 Neofollicles were gently introduced to the solution and polymerization to a gel was done for 10 min at 37° C. The composed matrix was soaked with DMEM+ medium for 48 h.

(13) Dermal Keratinocytes were isolated from skin biopsies obtained from face lift surgeries or from foreskin (Barrandon & Green, Proc Natl. Acad. Sci. 82, 5390-4, 1985). Briefly, the underlying fatty tissue and dermis were cut off and the remaining epidermis was digested overnight in a Trypsin/EDTA solution (PAA) at 4° C. KC were harvested using a cell scraper and after passing through a 70 μm cell strainer (Becton Dickinson) they were seeded onto collagen I coated culture flasks. Defined Keratinocyte Serum-Free Medium (Gibco) was changed twice a week and KC were passaged or harvested at 60-80% confluence.

(14) KC (250,000) were added on top of the matrix and let them adhere for 24 hours. Excessive cells were taken by changing the Defined Keratinocyte Serum-Free Medium (Gibco) and after having reached confluence, the transwell chambers were lifted to the air-liquid interphase to enable KC differentiation.

(15) Also cell lines (i.e. HaCat for KC and HS27 for fibroblasts) were tested for generating a similar skin model as they are easier to culture and reduce donor variations. They were grown in DMEM+ (Gibco) supplemented with 5% fetal calf serum (FCS, Gibco).

Example 5

Formation of Neofollicles Comprising Cells not Derived from Mammalian Hair Follicles

(16) Neofollicles have been prepared as described in Example 3 using KC and MC obtained as outlined below.

(17) Cell Culture Human Foreskin Keratinocytes and Fibroblasts:

(18) Human Foreskin Fibroblasts and Keratinocytes were isolated from circumcisions using previously described methods (Toma et al., Stem Cells 23, 727-37, 2005, Barrandon and Green, Proc Natl Acad Sci. 1985, 82:5390-4.). Briefly the underlying fatty tissue and dermis were cut off and the remaining epidermis was digested overnight in a Dispase solution (4 mg/ml, Sigma) at 4° C. overnight. Keratinocytes were then harvested using a cell scraper and after passing through a 70 μm cell strainer (Becton Dickinson) are then seeded into culture flasks. All Keratinocytes were grown in Collagen I coated cell culture flasks and Defined Keratinocyte Serum-Free Medium (Gibco). Medium was changed twice a week and the Keratinocytes were passaged or harvested at 60-80% confluence. Fibroblasts were cultured in DMEM+10% FCS.

(19) Cell Culture Human Epidermal Melanocytes:

(20) Starting from a cryovial of Human Adult Epidermal Melanocytes (purchased from CellMade) cell culture procedures were done according to the manufacturers protocol. Briefly, 15 ml Melanocytes Growth Medium was added to a T75 culture flask. The cells were thawed quickly by placing the lower half of the vial in a 37° C. water bath for 1 minute. The cells were the resuspended in into the T-75 flask containing Melanocytes Growth medium. The T-75 flask was placed in a 37° C., 5% CO2 humidified incubator. The Melanocytes Growth Medium was changed every 2-3 days. The cells were subcultured when the culture reached 80% confluent.

(21) Resulting neofollicles are depicted in FIG. 3 were the different stages of development of Neofollicles produced using KC and MC derived from sources different than mammalian hair follicle are shown. In stage 1, Neopapilla (without the addition of exogenous extracellular matrix proteins) are surrounded by Melanocytes and keratinocytes, which have been added to the ultra low attachment plate forming a early multi-cellular condensate. A loose aggregate has been created. In stage 2, first signs of the formation of follicular structures in ultra low attachment culture can be seen. The attached cells adhere to the Neopapilla and adopt a flattened shape while a protuberance has been built on top of the condensate. After approximately 1 week, the Neofollicle begins to form comprising hair follicle-like sheaths development.

(22) Within the established Neofolllicle, a clearly visible primitive hair shaft becomes visible.

Example 5

Gene Expression Analysis of Different Human Dermal Papilla Derived Cell Samples Using Agilent Whole Human Genome Oligo Microarrays (One-Color)

(23) 1. SuperAmp™ RNA Amplification

(24) Two isolated native Dermal Papillae, 1×10.sup.3 momolayer-cultured Dermal Papilla Fibroblasts, re-condensed Dermal Papilla Fibroblasts after 48 hours and re-condensed Dermal Papilla Fibroblasts after 14 days were prepared as described above. The Four human cell samples were lysed using SuperAmp™ Lysis Buffer.

(25) TABLE-US-00001 TABLE 2 List of samples Sample no. Cell Sample ID 1 DP 1 2 MONO 2 3 KOND 1 3 4 KOND 2 4

(26) SuperAmp RNA amplification was performed according to Miltenyi Biotec's procedure. Briefly, the amplification is based on a global PCR protocol using mRNA-derived cDNA. mRNA was isolated via magnetic bead technology. Amplified cDNA samples were quantified using the ND-1000 Spectrophotometer (NanoDrop Technologies).

(27) TABLE-US-00002 TABLE 3 Summary of cDNA yields Cell Concentration Ratio Total amount sample (ng/μL) (260/280) Volume (μl) of cDNA (μg) 1 161.92 1.81 20 3.2 2 143.79 1.86 20 2.9 3 123.05 1.83 20 2.5 4 117.41 1.84 20 2.3

(28) The integrity of the cDNA was checked via the Agilent 2100 Bioanalyzer platform (Agilent Technologies). The results of the Bioanalyzer run have been analysed using a gel image and an electropherogram using the Agilent 2100 Bioanalyzer expert software. The average length of the highly amplified cDNA products ranged between 200-1,000 bp. 2. Hybridization of Agilent Whole Genome Oligo Microarrays

(29) 250 ng of each of the cDNAs were used as template for Cy3 labeling which was performed according to Miltenyi Biotec's protocol. The Cy3-labeled cDNAs were hybridized overnight (17 hours, 65° C.) to an Agilent Whole Human Genome Oligo Microarrays 4×44K (table 4) using Agilent's recommended hybridization chamber and oven.

(30) TABLE-US-00003 TABLE 4 Hybridisation schedule Experiment no. Cy3 Microarray No. 1 1 251485031842_1_1 2 2 251485031842_1_2 3 3 251485031842_1_3 4 4 251485031842_1_4

(31) Finally, the microarrays were washed once with 6×SSPE buffer containing 0.005% N-lauroylsarcosine for 1 min at room temperature followed by a second wash with pre-heated 0.06×SSPE buffer (37° C.) containing 0.005% N-lauroylsarcosine for 1 min. The last washing step was performed with acetonitrile for 30 sec. 3. Scanning Results

(32) Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent's Microarray Scanner System (Agilent Technologies). 4. Image and Data Analysis

(33) The Agilent Feature Extraction Software (FES) was used to read out and process the microarray image files. The software determines feature intensities (including background subtraction), rejects outliers and calculates statistical confidences. For determination of differential gene expression FES derived output data files were further analyzed using the Rosetta Resolver® gene expression data analysis system (Rosetta Biosoftware). This software offers—among other features—the possibility to compare two single intensity profiles in a ratio experiment. All samples were labeled with Cy3, here, the ratio experiments are designated as control versus (vs.) sample experiments (automated data output of the Resolver® system). Please note, that the ratios are always calculated by dividing sample signal intensity through control signal intensity. 5. Gene Lists Single-experiment Raw Data List Single-Experiment Normalized Data List Gene Ratio List/Pre-selected Candidate Gene List

(34) The output data of the Agilent Feature Extraction software includes gene lists with the complete raw data sets, referred to as single-experiment raw data list. Furthermore, the signal intensities from the single-experiment raw data lists are normalized by dividing the intensity values by their median. These normalized signal intensities are joined to a common table the single-experiment normalized data list. This list comprises in addition to the normalized intensity values the feature glsPosAndSignif which indicates with a value=1 that the signal intensity is positive and significant above the background and a value=0 that the signal intensity is not positive and significant above the background. The Resolver® Software allows the export of a gene list with all normalized sample/controllog 10 ratios and -fold changes, sequence descriptions, p-values, etc., referred to as gene ratio list (of all genes). For example: A “−10 fold change” in the gene ratio lists therefore indicates a 10-fold higher gene expression in the control compared to the sample. Putative candidate genes with a fold change >2 and p-value <0.01 are summarized in a pre-selected candidate gene list. An extract of such a list comprising some of the differentially expressed genes is shown in table 5.

(35) TABLE-US-00004 TABLE 5 Level (Ratio) of defined genes measured by microarray analysis within fibroblasts (monolayered, condensed 48 hours, condensed 14 days) compared to native dermal papilla fibroblasts. Gene Name Cellular Function Ratio Mono Ratio 48 h Ratio 14 d Biological Process COMP Matrix −26.07 −100 −16 noncollagenous extracellular matrix glycoprotein, matrix integrity, cell adhesion MMP10 Matrix −43.08 −12.25 8.47 matrix metalloproteinase (MMP) family are involved in the of extracellular matrix (tissue) remodeling MGP Matrix −100 −8.25 −3.87 Extracellular matrix structural constituent, Multicellular organismal development, Cell differentiation CDK8 Cell Cycle 23.89 16.07 4.47 member of the cyclin-dependent protein kinase (CDK) family. Regulation of transcription, CDH3 Adhesion −100 −52.61 −34.49 Zinc ion binding, Regulation of transcription, PCDH85 Adhesion −100 −100 −10.12 Protocadherin beta 5, specify differential cell-cell connections L1CAM Adhesion 22.49 7.95 −1.65 Regulation of actin cytoskeleton PCDH20 Adhesion −28.97 −27.7 1.76 Protocadherin 20, establishment and function of specific cell-cell connections PECAM1 Adhesion −71.51 −100 −46.87 Cell to Cell Adhesion Signaling Jag2 Cytokine −100 −100 −21 Impact in: cell differentiation, Notch signaling pathway, Regulation of apoptosis, cell proliferation, Cell communication Multicellular organismal development, Cell cycle, Cell fate determination, Morphogenesis of embryonic epithelium, cell adhesion, cell migration TNFSF10 Cytokine −75 −100 −10.24 Apoptosis LEF1 Transcription Factor −100 −100 −8.5 LEF1 is a nuclear protein, Regulation of Wnt receptor signaling pathway SPRY1 Growth Factors −100 −100 −9.25 Sprouty regulation of tyrosine kinase signals CTGF Growth Factors 2 −20 4 Connective tissue growth factor, major connective tissue mitoattractant, Response to wounding, Proteinaceous extracellular matrix

(36) What can be deduced from table 5 is that with prolonged culture time, the expression level of genes involved in three dimensional arrangement and tissue formation is approaching the expression level observed in native dermal papilla fibroblasts.

(37) TABLE-US-00005 TABLE 1 DMEM +− Dulbecco's modified Eagle Medium-Composition (Gibco) COMPONENTS Molecular Concentration Molarity Amino Acids Glycine 75 37.5 0.500 L-Alanine 8.9 L-Arginine hydrochloride 84 L-Asparagine 13.2 L-Aspartic acid 13.3 L-Cystine 2HCl 63 L-Glutamic Acid 14.7 L-Histidine 42 hydrochloride-H2O L-Isoleucine 105 L-Leucine 105 L-Lysine hydrochloride 146 L-Methionine 30 L-Phenylalanine 66 L-Proline 11.5 L-Serine 52.5 L-Threonine 95 L-Tryptophan 16 L-Tyrosine disodium salt 104 L-Valine 94 Vitamins Ascorbic Acid phosphate 2.5 Choline chloride 4 D-Calcium pantothenate 477 4 0.00839 Folic Acid 441 4 0.00907 i-Inositol 7.2 Niacinamide 4 Pyridoxine hydrochloride 4 Riboflavin 0.4 Thiamine hydrochloride 4 Inorganic Salts Calcium Chloride (CaCl2) 111 200 1.80 Ferric Nitrate 0.1 (Fe(NO3)3″9H2O) Magnesium Sulfate 97.67 (MgSO4) Potassium Chloride (KCl) 400 Sodium Bicarbonate 3700 (NaHCO3) Sodium Chloride (NaCl) 6400 Sodium Phosphate dibasic 125 Proteins AIbuMAX ® II 400 Human Transferrin (Holo) 7.5 Insulin Recombinant 10 Full Chain Trace Elements Ammonium Metavanadate 0.0003 Cupric Sulfate 0.00125 Manganous Chloride 5 Sodium Selenite 0.005 Other Components D-Glucose (Dextrose) 4500 Ethanolamine 1.9 Glutathione (reduced) 307 1 0.00326 Phenol Red 15 Sodium Pyruvate 110 Penicillin | Streptomycin 100 u/ml | 100 μ/ml GlutaMAX ™