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EP-4114919-B1 - AUTOMATED METHOD FOR PREPARING KERATINOCYTES

EP4114919B1EP 4114919 B1EP4114919 B1EP 4114919B1EP-4114919-B1

Inventors

  • MORIZUR, Lise
  • LESUEUR, Léa
  • BALDESCHI, CHRISTINE

Dates

Publication Date
20260506
Application Date
20210302

Claims (11)

  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 in the step d) of the method, the cells are treated a first time with trypsin or trypsin-EDTA during 2-3 minutes at 37°C to eliminate contaminant cells like fibroblasts or aged keratinocytes, and a second time with trypsin or trypsin-EDTA during 5-10 minutes at 37°C to detach the keratinocyte progenitors and/or the K5/K14 positive cells exhibiting a keratinocyte-like phenotype, 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 2 .
  3. The automated method for preparing keratinocytes according to to claim 1 or 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 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.
  9. The automated method of any one of claims 1 to 8, wherein the remaining adherent cells can be banked or expanded over at least one passage.
  10. The automated method of claim 9, 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 2 ; and (iii) culturing the keratinocytes until the keratinocytes are 50 to 100% confluent, wherein the passages does not comprise a centrifugation step.
  11. The automated method of any one of claims 1 to 10, 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 .

Description

FIELD OF THE INVENTION 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. BACKGROUND OF THE INVENTION The skin consists of self-renewing layers organized into functional units of differentiating cells with their origin in a single basal stratum of proliferating keratinocytes. The dead and dying cells that comprise the stratum corneum are continually shed during desquamation and replaced by cells derived from epidermal stem cells found in the stratum germinativum. Loss of epidermal function leads to loss of thermal regulation, reduced microbial defences, risks of desiccation, inhibited wound repair, and cosmetic concerns. In the absence of sufficient autologous donor for skin grafts, coverage of wounds with cultured human keratinocytes represents a promising option for treatment. Furthermore, in vitro and in vivo models for human skin may represent tremendous tools for studying the lineage of epidermis cells or for testing cosmetic and pharmaceutical compounds for therapeutic or toxicological effects. For example the need for in vitro models is strengthened by the fact that there is an incentive to provide an alternative to the use of animals for testing compounds and formulations. In addition, a number of diseases affect the function of keratinocytes, either cell autonomously or through alteration of their ability to form the pluristratified epidermal tissue. In vitro and in vivo models for human skin may represent ways to reveal molecular mechanisms of diseases and, as a consequence, identify pharmacological or biological compounds endowed with therapeutic potentials. Thus, there is a need for methods for obtaining populations of human keratinocytes that may then be useful for skin therapy or for obtaining in vitro and in vivo models for human skin. Embryonic stem cells and somatic cells that are genetically reprogrammed in order to replicate all characteristics of embryonic stem cells (such as, for example, those called "iPS" cells, for "induced pluripotent stem" cells) are pluripotent stem cells with an extensive proliferative capacity and accordingly offer a great potential use in research and medicine. Several attempts have therefore been described in the prior art for obtaining human keratinocytes from pluripotent stem cells. To date, several groups have reported procedures to differentiate human ES/iPS cells into epidermal keratinocytes. For example, US2009075374 describes a method of generating p63-positive cells, comprising the step of culturing embryoid bodies (EBs) in a medium comprising a retinoid and a bone morphogenetic protein for about two days to about nine days. WO2016061071 describes a method for providing engraftable keratinocyte stem cells by differentiation of pluripotent stem cells comprising (a) forming aggregates of the pluripotent stem cells in a suspension culture in the presence of a defined basal medium; (b) culturing the aggregates in a suspension culture in the presence of an initiation culture medium comprising retinoic acid and BMP4 to effect the formation of initiated aggregates; (c) culturing the initiated aggregates in a keratinocyte progenitor culture medium comprising cholera toxin and a TGFβR1 kinase inhibitor to effect the formation of a cell population comprising keratinocyte progenitors; and (d) culturing the keratinocyte progenitors in a keratinocyte stem cell maturation medium to effect the formation of a cell population comprising engraftable keratinocyte stem cells. WO2009156398 describes a method for obtaining a population of human keratinocytes derived from human pluripotent stem cells comprising a step of co-culturing human pluripotent stem cells with a layer of feeder fibroblasts in the presence of a keratinocyte culture medium supplemented with BMP-4 and ascorbic acid. The keratinocyte culture medium is further supplemented with one or more agents selected from the group consisting of glutamine, epidermal growth factor (EGF), sodium pyruvate, adenine, insulin, hydrocortisone, choleric toxin and triodothyronin. The keratinocytes obtained from the pluripotent stem cells co-express the keratinocyte markers keratin 5 (K5) and keratin 14 (K14). However, the flow cytometry analysis of the expression of K5 and K14 in keratinocytes shows the presence of two types of keratinocytes. It is also possible to cite the following documents disclosing protocols for the preparation of a population of human keratinocytes derived from human pluripotent stem cells or methods associated with the isolation and cultivation of human primary keratinocytes, inter alia their enzymatic separation from other cells: WO2016039687, WO2016209166, WO2017091547, Differentiation of Human Induced Pluri