Methods for preparing human skin substitutes from human pluripotent stem cells

A technology of human pluripotent stem cells and substitutes, applied in the field of preparation of human skin substitutes, which can solve the problems of not obtaining human keratinocytes

Inactive Publication Date: 2011-09-14
INST NAT DE LA SANTE & DE LA RECHERCHE MEDICALE (INSERM)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, to date, no prior art methods have obtained human keratinocytes derived from human pluripotent stem cells, which have been shown to be effective when following techniques using adult basal keratinocytes from donors (see for example Green, 2008) Exhibits the ability to form a multilayered epidermis when processed (either in vitro or following xenografting in animals)

Method used

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  • Methods for preparing human skin substitutes from human pluripotent stem cells
  • Methods for preparing human skin substitutes from human pluripotent stem cells
  • Methods for preparing human skin substitutes from human pluripotent stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Example 1: Method for Preparation of Keratinocyte Populations and Human Skin Substitutes from hES

[0113] Materials and methods

[0114] Maintenance of hES cells

[0115] hESC (SA-01 and H9) at 37°C, 5% CO 2 Grow on a feeder layer of mouse fibroblasts supplemented with 20% (v / v) Knockout Serum Replacement (KSR, Invitrogen), 1 mM glutamine, 0.1 mM non-essential amino acids (Invitrogen), 4ng / ml recombinant human bFGF (PeProTech) and STO in DMEM / F12 (Sigma) of 0.1mM 2-mercaptoethanol (inactivated with 10mg / ml mitomycin C and treated at 30000 / cm 2 vaccination). For passaging, hESC colonies were dissected and passaged every 5 days.

[0116] Derivation of hES cells from keratinocytes.

[0117] For derivation, colonies were plated on mitomycin C-treated 3T3 fibroblasts in FAD medium consisting of 2 / 3DMEM, 1 / 3 HAM:F12 and 10% fetal calf serum (FCII , Hyclone), supplemented with 5μg / ml insulin, 0.5μg / ml hydrocortisone, 10 -10 M cholera toxin, 1.37 ng / ml T3, 24 μg / ml aden...

Embodiment 2

[0147] Example 2: Method for Preparation of Keratinocyte Populations and Human Skin Substitutes from iPS

[0148] The same differentiation protocol as described in Example 1 was performed with human induced pluripotent stem (iPS) cells. Briefly, iPS were seeded on mitomycin C-treated 3T3 fibroblasts in FAD medium consisting of 2 / 3 DMEM, 1 / 3 HAM:F12, and 10% fetal calf Serum (FCII, Hyclone) composition supplemented with 5 μg / ml insulin, 0.5 μg / ml hydrocortisone, 10 -10 M cholera toxin, 1.37 ng / ml triiodothyronine, 24 μg / ml adenine and 10 ng / ml recombinant human EGF. Induction of ectodermal differentiation was performed when 0.5 nM human recombinant BMP-4 (R&D Systems Europe, UK) and 0.3 mM ascorbic acid (Sigma) were added. Cells were grown in the same medium until colonies of epithelial cells were isolated. Cells were then plated on the same feeder layer in FAD medium without BMP4 and ascorbic acid. As a control, primary human keratinocytes (HK) were cultured on mitomycin C...

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Abstract

The present invention relates to an ex vivo 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 cells that support ectodermal differentiation in presence of an agent that stimulates epidermal induction and a agent that stimulates terminal differentiation of keratinocytes. A further object of the invention relates to a method for preparing a human skin substitute comprising a step of providing an organotypic culture of the substantially pure homogenous population of human keratinocytes derived from human pluripotent stem cells obtained according to the method of the invention.

Description

technical field [0001] The present invention relates to an in vitro method for obtaining a population of human keratinocytes derived from human pluripotent stem cells and a method for preparing a human skin substitute. Background technique [0002] The skin is composed of self-renewing layers forming functional units of differentiated cells originating from a single basal layer of proliferating keratinocytes. Dead and dying cells that make up the stratum corneum are continuously shed during desquamation and are replaced by cells derived from epidermal stem cells in the germinal layer. Loss of epidermal function leads to loss of thermoregulation, reduced microbial defenses, risk of dryness, inhibited wound repair, and cosmetic concerns. In the absence of sufficient autologous donors for skin grafts, covering wounds with cultured human keratinocytes represents a promising therapeutic option. [0003] Furthermore, in vitro and in vivo models of human skin can serve as excelle...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N5/071
CPCC12N2500/40C12N2501/01C12N5/0629C12N2501/155C12N2501/33C12N2501/11C12N2506/02C12N2506/45C12N2501/395C12N2502/13A61P17/02A61F2/10A61K35/36A61L27/38C12N5/0606
Inventor 欣德·盖农吉勒·勒迈特克里斯蒂娜·巴尔代斯基马克·佩先斯基
Owner INST NAT DE LA SANTE & DE LA RECHERCHE MEDICALE (INSERM)
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