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US-12618047-B2 - Automated method for preparing keratinocytes

US12618047B2US 12618047 B2US12618047 B2US 12618047B2US-12618047-B2

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.

Inventors

  • Lise MORIZUR
  • Léa LESUEUR
  • Christine BALDESCHI

Assignees

  • CENTRE D'ETUDE DES CELLULES SOUCHES (CECS)
  • UNIVERSITE EVRY VAL D'ESSONNE

Dates

Publication Date
20260505
Application Date
20210302
Priority Date
20200302

Claims (12)

  1. 1 . An automated method for preparing keratinocytes derived from human pluripotent stem cells (hPSC), the method comprising: (a) forming and culturing aggregates or clumps of said pluripotent stem cells on a cell culture surface coated with a protein matrix that supports 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 so as 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 a population of the cells obtained in the culturing c) so as to remove non-conform cells and obtain a homogeneous population of keratinocytes, wherein the cells are treated a first time with trypsin-EDTA during a time in a range from 2 to 3 minutes at 37° C. so as to eliminate contaminant cells, the contaminant cells comprising fibroblasts and aged keratinocytes, and a second time with trypsin-EDTA a time in a range from 5 to 10 minutes at 37° C. so as to detach the keratinocyte progenitors, or K5/K14 positive cells exhibiting a keratinocyte-like phenotype, or a combination thereof; and (e) culturing the detached cells resulting in the treating d) corresponding to the keratinocyte progenitors, or the keratinocytes, or a combination thereof, in the presence of a culture medium, wherein the keratinocytes obtained after the culturing (e) comprise more than 99% of K5+/K14+ keratinocytes, and wherein at least one process selected from the (a)-(e) in the automated method is performed by an apparatus for large-scale automated production of cells, without direct intervention from an operator.
  2. 2 . The automated method for preparing keratinocytes according to claim 1 , wherein a day before of the culturing b), the pluripotent stem cells are seeded at a cell density in a range from 1000 to 10,000 cells/cm 2 .
  3. 3 . The automated method for preparing keratinocytes according to claim 1 , wherein the human pluripotent stem cell medium is a medium that supports hPSC self-renewal.
  4. 4 . The automated method for preparing keratinocytes according to claim 1 , wherein the culturing (b) is performed for a time period in a range from 5 to 8 days.
  5. 5 . The automated method for preparing keratinocytes according to claim 1 , wherein the culturing (c) is performed 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. 6 . The automated method for preparing keratinocytes according to claim 1 , the method comprising: (a) the forming and culturing aggregates or clumps of said pluripotent stem cells on a cell culture surface coated with a protein matrix that supports cell attachment and growth in the presence of a defined human pluripotent stem cell medium; (b) the 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 so as to generate keratinocyte progenitors for a time period in a range from 5 to 8 days; (c) the 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 in a range from 8 to 25 days; and (d) the treating the population of cells obtained in the culturing c) so as to remove the non-conform cells and obtain a homogeneous population of keratinocytes.
  7. 7 . The automated method according to claim 1 , wherein remaining adherent cells are configured to be banked or expanded over at least one passage.
  8. 8 . The automated method according to claim 7 , wherein the at least one passage comprises: (i) dissociating differentiated cells or keratinocytes in a first vessel so as to obtain a cell suspension; (ii) transferring the dissociated keratinocytes to new culture vessels at a cell seeding density in a range from 10,000 to 100,000 cells/cm 2 ; and (iii) culturing the keratinocytes until the keratinocytes are from 50 to 100% confluent, wherein the at least one passage does not comprise centrifugation.
  9. 9 . The automated method according to claim 1 , wherein the method is carried out with an apparatus for large-scale automated production of cells, the apparatus comprising: a) a robotic vessel handler that handles culture vessels; b) a cell seeder that inoculates cells into a culture; c) a medium exchanger that changes or adds medium to a culture; and d) a programmable control; wherein the apparatus is adapted to the differentiation of hPSCs toward keratinocytes and their amplification.
  10. 10 . The automated method for preparing keratinocytes according to claim 1 , wherein the keratinocyte expresses cytokeratin 5 and cytokeratin 14.
  11. 11 . The automated method for preparing keratinocytes according to claim 2 , wherein the cell density is in a range from 2000 and 8000 cells/cm 2 .
  12. 12 . The automated method for preparing keratinocytes according to claim 2 , wherein the cell density is in a range from 2000 and 4000 cells/cm 2 .

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. Although the differentiation of hPSCs into keratinocytes became more efficient during the last years, it still remains a long and laborious process requiring meticulous manipulations from hPSCs thawing to hPSC-keratinocytes banking. Many cell culture parameters, such as seeding homogeneity, the time spent by the cells out of the incubator or the method used to isolate clumps, could impact on the