METHODS FOR PREPARING KERATINOCYTES

Abstract

The present invention relates to ex vivo methods for obtaining populations of human keratinocytes derived from human pluripotent stem cells.

Claims

1. A method for preparing keratinocytes derived from human pluripotent stem cells comprising or consisting in the steps of: (a) forming and culturing aggregates or clumps of said pluripotent stem cells on a cell culture surface coated with a protein matrix to support cell attachment and growth in the presence of a defined human pluripotent stem cell medium; (b) culturing the adherent aggregates or clumps of said pluripotent stem cells on a cell culture surface coated with a protein matrix in the presence of a defined keratinocyte culture medium comprising retinoïc acid and BMP-4 to generate keratinocyte progenitors; (c) culturing the keratinocyte progenitors on a cell culture surface coated with a defined protein matrix coating in the presence of a defined keratinocyte culture medium devoid of retinoic acid and BMP-4; (d) treating the population of cells obtained in step c) to remove the non-adherent cells and non-conform cells and obtain an homogeneous population of keratinocytes.

2. The method for preparing keratinocytes according to claim 1, wherein the human stem cell medium is a medium suitable to support hESC self-renewal supplemented with stabilized FGF2.

3. The method for preparing keratinocytes according to any one of claims 1 to 2, wherein the culturing in step (b) comprises two pulses of effective amounts of BMP-4 and retinoïc acid introduced in the culture medium at Day 1 and Day 3 or Day 4.

4. The method for preparing keratinocytes according to any one of claims 1 to 3, wherein the culturing in step (b) is accomplished for a time period of 5 to 8 days.

5. The method for preparing keratinocytes according to any one of claims 1 to 4, wherein the culturing in step (c) is accomplished for a time period of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 days.

6. The method for preparing keratinocytes according to any one of claims 1 to 5, comprising or consisting in the steps of: (a) forming and culturing aggregates or clumps of said pluripotent stem cells on a cell culture surface coated with a protein matrix to support cell attachment and growth in the presence of a defined human pluripotent stem cell medium; (b) culturing the adherent aggregates or clumps of said pluripotent stem cells on a cell culture surface coated with a protein matrix in the presence of a defined keratinocyte culture medium comprising retinoïc acid and BMP-4 to form keratinocyte progenitors for a time period of 5 to 8 days; (c) culturing the keratinocyte progenitors on a cell culture surface coated with a defined protein matrix coating in the presence of a defined keratinocyte culture medium devoid of retinoic acid and BMP-4 for a time period of 8 to 25 days; (d) treating the population of cells obtained in step c) to remove the non-adherent cells and non-conform cells and to obtain an homogeneous population of keratinocytes.

7. The method for preparing keratinocytes according to any one of claims 1 to 6, wherein the step d) of the method is a two-step dissociation procedure comprising or consisting in washing and treating the cells enzymatically.

8. The method for preparing keratinocytes according to any one of claims 1 to 6, wherein the cells are treated a first time with trypsin for a period of time sufficient to eliminate contaminant cells like fibroblasts and aged keratinocytes, and a second time with trypsin to detach the keratinocyte progenitors, or the K5/K14 positive cells exhibiting a keratinocyte-like phenotype.

9. The method for preparing keratinocytes according to any one of claims 1 to 8, wherein the method for preparing keratinocytes further comprises (e) culturing the detached cells of step d) corresponding to the keratinocyte progenitors and/or the keratinocytes in the presence of a culture medium, and wherein the keratinocytes obtained after said culturing step comprise more than 95, 96, 97, 98, 99% of K5+/K14+ keratinocytes.

10. The method for preparing keratinocytes according to any one of claims 1 to 9, wherein the keratinocyte express cytokeratin 5, cytokeratin 14 and cytokeratin 19.

11. An isolated pure homogenous population of keratinocytes derived from human pluripotent stem cells optionally obtainable by the method according to any one of claims 1 to 10, wherein the keratinocytes express Keratin 19 at 99.9%.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0125] FIG. 1:

[0126] (A) Schematic representation of the first protocol design for differentiation of hESCs into keratinocytes according to a method adapted from the prior art WO2009156398 (here, the cells are cultivated on a coating matrix, whereas in WO2009156398, the cells are cultivated on feeder cells). Keratinocyte differentiation was induced with BMP-4 (0.5 nmol/L) and L-ascorbic acid (AA) (0.03 mmol/L) throughout the kinetic. At Day 0, hESCs are manually passaged on clinical grade matrix in clinical grade GREEN medium supplement with BMP-4 and AA. Cells are maintained in culture until D40. At D40, the keratinocytes are isolated using trypsin treatment and amplified until banking at passage 1 (p1).

[0127] (B) Schematic representation of the clinical and feeder free protocol design for differentiation of hESCs RC-9 into keratinocytes. At Day 0, the RC-9 are mechanically seeded into new dishes coated with L7 matrix and cultured in hESC condition with Stem Pro hESC medium supplemented with 10 ng/mL of stabilized FGF-2. The induction of KER differentiation is initiated with two pulses of BMP-4 (0.273 nmol/L) and retinoic acid (1 μmol/L) at Day 1 and Day 5, until Day 6, in defined Keratinocyte Serum Free medium (dKSFM). At Day 7 until the end of the induction of differentiation process, cells are cultured in differentiation medium composed with dKSFM medium only. At the end of differentiation, cells are isolated and purified with differential trypsinizination.

[0128] FIG. 2:

[0129] (A) Representative fluorescence-activated cell sorting analysis of keratin 5 and keratin 14 in keratinocytes KER-RC9 and NHKs at passage 2 (p2) (B1-A and B2-A correspond to a relative fluorescence average in a specific channel depending secondary antibody fluorochromes). Those data illustrate that K5 present different expression profiles between KER-RC9 and NHKs. In NHKs, the population is homogenous with only one K5 and K14 peak whereas in KER-RC9, cells are homogenous for K14 marker but two distinct populations are present for K5 marker, suggesting the presence of 2 two types of KER-RC9 in the population (according to a method adapted from the prior art WO2009156398)

[0130] (B) Representative fluorescence-activated cell sorting analysis of keratin 5 and keratin 14 in keratinocytes KER-RC9 at p2 (B1-A and B2-A correspond to a relative fluorescence average in a specific channel depending secondary antibody fluorochromes). The FACS analysis show that the population is homogenous with only one K5 and K14 peak. KER-RC9 present a pure population expressing the two keratinocyte markers keratin 5 and keratin 14 at more than 98% (respectively 100% and 100%).

[0131] (C) Representative fluorescence-activated cell sorting analysis of K8/18, ΔNp63, K5 and K14 in cells during the differentiation (B1-A, B2-A and R1-A correspond to a relative fluorescence average in a specific channel depending on conjugated antibodies fluorochromes APC, FITC and PE/555). During the process, cells were analysed in order to determine the end of differentiation step. Cells highly express K8/K18 markers (epithelial markers, not expressed by adult keratinocytes but present in embryo skin) at the beginning of the differentiation. The K8/K18 signal is progressively lost and at D20 the majority of cells do not express those markers. Cells express ΔNp63 after 10 days of differentiation and it is stable until the end. It is followed up with keratin 5 and keratin 14. Those two markers appeared progressively during the differentiation and are stable after 15 days of differentiation. The increase in keratin 5 and 14 is concomitant with the decrease of keratin 8/18.

[0132] (D) Representative fluorescence-activated cell sorting analysis of Keratin19, (R1-A correspond to a relative fluorescence average in a specific channel depending on conjugated antibody fluorochrome APC). The FACS analysis show that the population is homogenous for KER-RC-9 (K-hESC) with the presence of a pure population expressing Keratin 19 at 99.9% whereas the primary KER (HPEK), only a small fraction of cells express Keratin 19 (28.2%) suggesting sternness of KER-RC9.

[0133] FIG. 3:

[0134] (A) Haematoxylin-eosin staining and immuno-histological analysis of in vitro epidermal reconstitutions of KER-RC9 at p2 on polycarbonate insert or on plasma based matrix containing human primary fibroblasts as equivalent dermis. In the two in vitro epidermis culture system, KER-RC9 are capable to reconstruct a pluristratified epidermis with a basal layer, a spinous layer, a granular layer and finally a corneal layer. Those reconstructed stratified epidermis express basal keratinocyte marker as keratin 5 and the suprabasal marker keratin 10.

[0135] (B) Haematoxylin-eosin staining and immunohistological analysis of in vivo of KER-RC9 at p2 seeded on plasma based matrix (without primary fibroblasts) grafted on the back of a nude mouse during 12 weeks. After 10-12 weeks, human epidermis is detected with a specific suprabasal human Involucrin marker. This antibody fail to detect mouse involucrin whereas the keratin 10 antibody detect all species (human and mouse). The epidermis is pluristratified with all four layer of human epidermis like in in vitro assay. To conclude, KER-RC9 are fully functional in vitro as well as in vivo.

EXAMPLES

Manual hESCs Culture

[0136] Clinical-grade hESC line RCe013-A (RC-9), was used and cultured in feeder free conditions using StemPro™ hESC Medium (Invitrogen) and stabilized FGF2 (Miltenyi) at 10 ng/ml and L7™ coating (Lonza). Cells were plated and grown until they reached 80 percent of confluence.

Differentiation of hES Cells in Keratinocytes

[0137] Clumps of hESC were seeded in the defined Keratinocyte SFM (dKSFM-Gibco-Thermo Fisher Scientific) which is a serum-free medium optimized for the isolation and expansion of human keratinocytes without the need for bovine pituitary extract (BPE) supplementation or the use of fibroblast feeder layers. The induction of ectodermal differentiation was done when 0.273 nM of human recombinant BMP-4 (Peprotech) and 1 μM of trans retinoic acid (Sigma) were added. Cells were grown in the same medium until clones of epithelial cells were isolated.

[0138] During all the differentiation process the medium was changed every 2/3 days.

[0139] At the end of differentiation, cells are isolated and purified with differential trypsinizination. Briefly, cells are first treated with trypsin for 2-3 minutes in 37° C. to eliminated contaminant cells like fibroblasts. After discard trypsin, cells are secondary treated with new trypsin during 5 to 10 minutes at 37° C. The harvested cells are then amplified on collagen I coated dishes with CnT-07.HC medium or on L7 matrix dishes with dKSFM medium until cell banking at the end of passage 1.

[0140] Microscopy analysis of hESC RC9 line during keratinocyte differentiation process. The RC-9 colonies growth and differentiate until D6. At Day 10, the differentiating colonies are still growing and present 2 distinct morphologies: the centre part is composed of small cells and the outer part (like a ring around the centre part), is composed of migrating bigger cells. After D15, the outer part is bigger: cells escape from the centre. After D20, the majority of cells are out of the centre of colonies and the centre of colonies is composed with cells bigger than at D6.

[0141] Representative fluorescence-activated cell sorting analysis of K8/18, ΔNp63, K5, K14 and optionally K19 in cells during the differentiation (B1-A, B2-A and R1-A correspond to a relative fluorescence average in a specific channel depending on conjugated antibodies fluorochromes APC, FITC and PE/555). During the process, cells were analysed in order to determine the end of differentiation step. Cells highly express K8/K18 markers (epithelial markers, not expressed by adult keratinocytes) at the beginning of the differentiation. The K8/K18 signal is progressively lost and at D20 the majority of cells do not express those markers. Cells express ΔNp63 after 10 days of differentiation and it is stable until the end. It is followed up with keratin 5, keratin 14 and optionally keratin 19. Those two markers appeared progressively during the differentiation and are stable after 15 days of differentiation. The increase in keratin 5 and 14 is concomitant with the decrease of keratin 8/18.

[0142] As shown in FIG. 2C, the methods according to the present invention allow the preparation of iPSC- or ES-derived keratinocytes that provide an homogeneous and pure population of keratinocytes.

FACS Analyses

[0143] Cells were detached from culture plates using Trypsin-EDTA (Invitrogen) and fixed and permeabilized using BD CytoFix/CytoPerm kit (BD Biosciences) according to the manufacturer instruction. Conjugated antibodies diluted at 1:50 were incubated 15 to 30 min at room temperature in PBS containing 0.1% FCS. Control samples were done using specific isotype. Stained cells were analyzed on a MAC5 Quant flow cytometer (Miltenyi) using FlowJo software.

Immunocytochemistry

[0144] Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature before permeabilized and blocking in PBS supplement with 0.4% Triton X-100 and 5% BSA (Sigma). Primary antibodies were incubated overnight at 4° C. in blocking buffer. Rabbit anti-K14 is from Covance and, mouse anti-K5 is from Abcam. Cells were stained with the species specific fluorophore-conjugated secondary antibody (Invitrogen) for 1 hour at room temperature and nucleus were dye using DAPI. Immunofluorescence images were acquired on a Zeiss Z2 microscope using Axiovision imaging software.

Organotypic Cultures

[0145] Human epidermis substitutes are reconstructed according a modified version of described protocol by Poumay, Y et al. 2004. Culture of the keratinocytes derived from human pluripotent stem cells is performed on polycarbonate culture inserts. These cells may be maintained for 5 to 7 days in CnT07.HC medium (amplification phase) and 14 days in 3D Prime CnT medium (CELLnTEC) for stratification. The cells were exposed to the air-liquid interface by removing the culture medium in the insert for 14 days for stratification.

[0146] For in vivo testings, human epidermis substitutes are generated with keratinocytes derived from human pluripotent stem cells previously seeded on a plasma based matrix (fibrin) for 7 to 10 days in CnT07.HC medium to obtain a monolayer of keratinocytes (KER). For grafting, nude mice are aseptically cleansed. Full-thickness wounds are then created on the dorsum of mice and finally applied with the human skin substitute of the invention is performed under sterile conditions. Fatty gauzes are put on the graft area. The graft and the fatty gauzes are put inside the mouse skin borders to avoid suturing. The piece of mouse skin is devitalized by liquid nitrogen and PBS1X bath cycles and resutured on the graft to provide the graft with a “biological” dressing. 10-12 weeks may be then sufficient to obtain a human skin on said animal. Implants were harvested between 4-12 weeks after grafting, and the tissue specimens fixed in 10% buffered formalin for paraffin embedding.