The skin as a biofactory for systemic secretion of erythropoietin: potential of genetically modified keratinocytes and fibroblasts

Abstract

In summary, while both transduced KC and FB are able to synthesize and secrete human EPO, KC show higher potential in serving as possible target cells for therapeutic substitution with EPO, probably because of improved transduction rates and increased secretory activity.

In vivo synthesis and secretion of erythropoietin by genetically modified primary human keratinocytes grafted onto immunocompromised mice

“…Primary human KC and fibroblasts were isolated from human foreskin as described by Scheidemann et al. (25), and cultivated in KC‐serum‐free medium (SFM) with supplements (Gibco‐Invitrogen, Karlsruhe, Germany) and Dulbecco’s modified Eagle’s medium (DMEM; Biochrom, Berlin, Germany), supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin and 2 m m of l ‐glutamine (all Gibco‐Invitrogen), respectively. Transduction was performed by incubation of first passage cells on consecutive days with either the control vector QGIM (containing the bicistronic vector cassette GFP/MDR) or the selectable EPO‐expressing retroviral vector QEIM (containing the vector cassette EPO/MDR; Fig.…”

“…Transduction was performed by incubation of first passage cells on consecutive days with either the control vector QGIM (containing the bicistronic vector cassette GFP/MDR) or the selectable EPO‐expressing retroviral vector QEIM (containing the vector cassette EPO/MDR; Fig. 1a) (25) at an MOI of 2 together with polybrene (5 μ g/ml; Sigma, Taufkirchen, Germany). For selection of transduced cells, different concentrations of colchicine (20 or 50 ng/ml; Sigma) were added to the culture media every other day.…”

“…Another promising cell type to be employed as biofactory is human keratinocytes (KC) (21–24). Previously, we have shown that KC transduced with a retroviral EPO‐vector can express and release high levels of human EPO in vitro , which significantly exceeds the amount of EPO secreted by transduced fibroblasts (25).…”

Sensory Systems of Epidermal Keratinocytes

“…Thus, the level of erythropoietin, i.e., the number of red blood cells in the blood, is infl uenced not only by respiration but also by the oxygen level at the surface of the skin. Another study confi rmed that keratinocytes synthesize erythropoietin [ 97 ] . Thus, epidermal keratinocytes might sense the environmental concentration of oxygen and be involved in appropriately modulating the number of red blood cells in the blood.…”

Vectors for gene therapy of skin diseases

“…Examples which have already been successfully tested in preclinical animal models are the secretion of erythropoi‐etin [43], blood coagulation factors [44], and atrial natriuretic protein [45]. It is advisable to check beforehand which cell population is the most suitable for the respective GV as transduction efficiency and intensity of expression in ker‐atinocytes or fibroblasts can vary [46, 47].…”