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EP-4114921-B1 - METHODS FOR PREPARING KERATINOCYTES

EP4114921B1EP 4114921 B1EP4114921 B1EP 4114921B1EP-4114921-B1

Inventors

  • BALDESCHI, CHRISTINE
  • DOMINGUES, SOPHIE
  • LEMAITRE, GILLES

Dates

Publication Date
20260506
Application Date
20210302

Claims (8)

  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 retinoic 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, 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 to detach the keratinocyte progenitors and/or the K5/K14 positive cells exhibiting a keratinocyte-like phenotype.
  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 claim 1 or 2, 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.
  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 retinoic 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 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.
  8. The method for preparing keratinocytes according to any one of claims 1 to 7, wherein the keratinocyte express cytokeratin 5, cytokeratin 14 and cytokeratin 19.

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. 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 (ESC) 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 retinoïd 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 BMP-4 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 Pluripotent Stem Cells into a Keratinocyte Lineage, I. Kogut et al., Methods in Molecular Biology, 2013; Differentiation of nonhuman primate pluripotent stem cells into functional keratinocytes, S. Domingue