Cell therapy for genodermatosis
Gilles Lemaître : Research engineer (Evry University), bound to the Genodermatoses team
Benoite Champon : Technician (CECS)
Aims and background :
The convergence of stem cell research with medical application has been a source of exaltation for the scientific community. Pluripotent stem cells offer the hope for treatment of individuals suffering from cellular degeneration caused by either disease or injury. Human Embryonic Stem (hES) cells have already been successfully differentiated in vitro into various cell types, such as neurons, oligodendrocytes, cardiomyocytes, hematopoietic precursor cells and keratinocytes. More importantly, the therapeutic potential of these pluripotent stem cells has been effectively demonstrated in animal models. Diseases of the skin are a major public health problem. Much of these diseases can be treated by skin grafting. The epidermal substitute supply is a problem because the vast majority of treatments are using autologous keratinocytes. The embryonic stem cells can be a tool to address this lack. By their self-renewal capacity and the ability to differentiate into keratinocytes, we can imagine preparing a bank of allogenic keratinocytes derived from hES cells to manufacture a skin dressing. The first step in developing a protocol for keratinocytes derived from hESCs has been achieved in our group. We have now a functional skin substitute graft onto mice which has been obtained by designing a protocol that takes into account the long-term succession of biological steps that lead to epidermis formation during ontogenesis. The current challenge is the transition from bench to bedside.
Strategy, means and methods :
The protocol of differentiation of keratinocytes derived from hES cells follows the chronobiology of epidermal development. hES cells are seeded onto a feeder layer in a specific keratinocyte medium and treated by ascorbic acid and BMP4 during 40 days in culture. Keratinocytes are isolated and amplified to obtain a pure and stable population of keratinocytes derived hES cells.
In appropriate conditions, hES cells are able to form a functional human pluristratified epidermis on the back of immunodeficient mice.
Once placed on an artificial matrix, keratinocytes derived from hES cells were able to form a pluristratified epidermis replicating normal human epidermis in vitro. In order to verify the functional capacities of hES cells derived epidermis under physiological conditions; keratinocytes derived from hES cells were grafts on mice. Results showed that grafted mice exhibited a completely normal and functional human pluristratified epidermis consistent with a mature epidermis.
The next step to have a ready to use, temporary skin substitute would be to obtain a quality controlled product; produced in Good Manufacturing Protocol (GMP) conditions at all stages from derivation to safety deliver. In order to produce a skin substitute derived from hES cells, we must establish quality control stages during product development and take into account the safety of the product to be grafted.Results and future prospects :
Skin substitutes offer promise in the treatment of chronic and acute wounds. Diabetic foot and venous leg ulcers are frequent and costly complications of their underlying diseases and thus represent a critical issue for public health. Besides these classically described leg ulcers a chronic disease may also occur in patients with sickle cell disease. Several requirements are necessary for the treatments of ulcers during this chronic disease implicate (1) ease of use (2) an absence of side effects and (3) the ability to be reused repeatedly.
To produce an epidermis derived from hES cells which might be grafted on ulcers, industrial production in GMP conditions needs to be established. Since we decided to make our first clinical trial on leg ulcers in patients with sickle cell disease, we must present a complete product containing both a matrix (dermal equivalent) and epidermis. In the case of regenerative medicine of the epidermis we rely on three decades of autologous and allogenic skin grafting. For the scaling, we have adapted the basic research to industrial development research. 
hES derived keratinocyte cell expressing a specific keratin 14 protein (in green).
Publications :
Guenou H et al., Human embryonic stem-cell derivatives for full reconstruction of the pluristratified epidermis: a preclinical study. Lancet. 2009 Nov 21;374(9703):1745-1753.
Peschanski M et al., Epidermis grafting: from adult to embryonic stem cells. Regen Med. 2010 Mar;5(2):157-159.
Harfouche G et al., Fibroblast growth factor type 2 signaling is critical for DNA repair in human keratinocyte stem cells. Stem Cells. 2010 Sep;28(9):1639-1648.
Peschanski M et al., Epidermis grafting: from adult to embryonic stem cells. Regen Med. 2010 Mar;5(2):157-159.
Harfouche G et al., Fibroblast growth factor type 2 signaling is critical for DNA repair in human keratinocyte stem cells. Stem Cells. 2010 Sep;28(9):1639-1648.
Patent :
WO/2009/156398 (2009-12-30) GUENOU Hind (FR); LEMAITRE Gilles (FR); BALDESCHI Christine (FR); PESCHANSKI Marc (FR) Methods for preparing human skin substitutes from human pluripotent stem cells.
