AUTOMATED METHOD FOR PREPARING KERATINOCYTES

Abstract

The present invention relates to ex vivo methods for obtaining populations of human keratinocytes derived from human pluripotent stem cells (hPSCs). More particularly, the present invention relates to an automated method that combines in a sequential manner automated differentiation and amplification of a population of hPSC-derived keratinocytes.

Claims

1. An automated method for preparing keratinocytes derived from human pluripotent stem cells (hPSC) 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 retinoic acid and BMP4 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 BMP4; (d) treating the population of cells obtained in step c) to remove the non-conform cells and obtain an homogeneous population of keratinocytes, wherein at least one step of the automated method is performed by an apparatus for large-scale automated production of cells, without direct intervention from an operator.

2. The automated method for preparing keratinocytes according to claim 1, wherein the day before of the step b), the pluripotent stem cells are seeded at a cell density between 1000 and 10,000, preferably between 2000 and 8000, or preferably between 2000 and 4000, more preferably at 2000 or 4000 cells/cm.sup.2.

3. The automated method for preparing keratinocytes according to any one of claims 1 to 2, wherein the human stem cell medium is a medium suitable to support hPSC self-renewal.

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

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

6. The automated method for preparing keratinocytes according to any one of claims 1 to 5, 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.

7. The automated method for preparing keratinocytes according to any one of claims 1 to 6, 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 retinoic acid and BMP4 to generate 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 BMP4 for a time period of 8 to 25 days; (d) treating the population of cells obtained in step c) to remove the non-conform cells and obtain an homogeneous population of keratinocytes.

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

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

10. The automated method for preparing keratinocytes according to any one of claims 1 to 9, wherein the method for preparing keratinocytes further comprises (e) culturing the detached cells of step d) corresponding to the keratinocyte progenitors and/or 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.

11. The automated method of any one of claims 1 to 10, wherein the remaining adherent cells can be banked or expanded over at least one passage.

12. The automated method of claim 11, wherein the passages comprises (i) dissociating the differentiated cells or keratinocytes in a first vessel to obtain a cell suspension; (ii) transferring the dissociated keratinocytes to new culture vessels at a cell seeding density between 10,000 and 100,000, preferably between 20,000 and 50,000, more preferably between 30,000 and 40,000 cells/cm.sup.2; and (iii) culturing the keratinocytes until the keratinocytes are 50 to 100% confluent, wherein the passages does not comprise a centrifugation step.

13. The automated method of any one of claims 1 to 12, wherein the method is carried out with an apparatus for large-scale automated production of cells comprising: a) robotic means for handling culture vessels; b) means for inoculating cells into a culture; c) means for changing or adding medium to a culture; and d) programmable control means; wherein the apparatus is adapted to the differentiation of hPSCs toward keratinocytes and their amplification .

14. The automated method for preparing keratinocytes according to any one of claims 1 to 13, wherein the keratinocyte express cytokeratin 5 and cytokeratin 14.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0184] FIG. 1: Automated differentiation and amplification of a pure population of hPSC-derived keratinocytes. (A) Schematic representation of the differentiation strategy to generate keratinocytes from hPSCs. (B) Representative macroscopic observation of the hESC RC9 line after keratinocyte derivation and amplification (KER-RC9). (C) Doubling time of KER-RC9 during sequential passages. (D) Representative immunofluorescence analysis for keratinocyte markers keratin 5, keratin 14, p40, Itgβ4 and Itgα6 of KER-RC9 at P1. Scale: 10 .Math.m.

[0185] FIG. 2: Automated process to differentiate hPSCs into keratinocytes using the CompacT SelecT system. (A) Overview of the automated CompacT SelecT cell culture system: (1) T-flask carousel incubator, (2) Media peristaltic pumps, (3) Cedex automated cell counter, (4) 6-axis robotic arm, (5) Flask decappers, (6) Pipette head and (7) IncuCyte live cell analysis system. (B) Automated process to generate large banks of keratinocytes from hPSCs.

EXAMPLES

1. Preparation of Keratinocytes From hiPSC

Manual hiPSC Culture

[0186] Human iPSC 1432 line was used and cultured in feeder free conditions using iPS Brew® medium (Miltenyi) and vitronectin coating (Life Technologies). Cells were plated and grown until they reached 80 percent of confluence.

Differentiation of hiPSC Cells in Keratinocytes

[0187] hiPSC 1432 cells were incubated with EDTA at 37° to obtain clumps that were then seeded in a defined human stem cell medium for 1 day and then 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 .Math.M of trans retinoic acid (Sigma) were added on day 1 and day 3 or 4. Cells were grown in the same medium until clones of epithelial cells were isolated.

[0188] During all the differentiation process the medium was changed every ⅔ days.

[0189] 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 discarding trypsin, cells are secondary treated with trypsin during 5 to 10 minutes at 37° C. The harvested cells are either banked or amplified on collagen I coated dishes with CnT-07.HC medium

Macroscopic Analysis of hiPSC 1432 Line Dring the Dfferentiation Pocess

[0190] The morphology of the cells is monitored throughout the differentiation process and during the amplification phase. From day 3, cells present a differentiated cell morphology with large cells organized in colonies distinct from that of pluripotent stem cells. At day 8, 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 (not shown).

[0191] The methods according to the present invention allow the preparation of iPSC-derived keratinocytes that provide an homogeneous and pure population of keratinocytes.

2. Preparation of Keratinocytes From hESC

Manual hESC Clture

[0192] Human embryonic stem cell RC-9 line was used and cultured in feeder free conditions using StemPro medium (Gibco - Thermo Fisher Scientific) supplemented with stabilized FGF-2 on L7 matrix (Lonza). Cells were plated and grown until they reached 80 percent of confluence.

Differentiation of hPSC Cels in Kratinocytes

[0193] hES RC9 cells were incubated with EDTA at 37 ° to obtain clumps that were then seeded in a defined human stem cell medium for 1 day and then 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 .Math.M of trans retinoic acid (Sigma) were added on day 1 and day 3 or 4. Cells were grown in the same medium until clones of epithelial cells were isolated. During all the differentiation process the medium was changed every ⅔ days. The morphology of the cells is monitored throughout the differentiation process and during the amplification phase. From day 3, cells present a differentiated cell morphology with large cells organized in colonies distinct from that of pluripotent stem cells. At day 8, 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.

[0194] 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 eliminate contaminant cells. After discarding trypsin, cells are secondary treated with trypsin during 5 to 10 minutes at 37° C. The harvested cells are either banked or amplified on collagen I coated dishes with CnT-07.HC medium.

Immunostaining

[0195] Cells were fixed in 4% PFA for 10 min at room temperature (RT) and rinsed 3 times with PBS. After 1 hour in blocking solution (PBS containing 1 % BSA) at RT, cells were incubated overnight à 4° C. with primary antibodies at appropriate dilutions. After 3 washes in PBS, Alexa Fluor-conjugated secondary antibodies (Invitrogen) were added at 1:1000 for 45 min at RT.

[0196] Representative immunofluorescence analysis for keratinocyte markers keratin 5, keratin 14, p40, Itgβ4 and Itgα6 of KER-RC9 are shown at passage 1 (see FIG. 1D).

[0197] As shown in FIG. 1, the methods according to the present invention allow the preparation of hPSC-derived keratinocytes that provide an homogeneous and pure population of keratinocytes.