The concept - redifferentiation of skin cells

In 2007 Professor Philippe Collas at University of Oslo and former CEO of Regenics AS, Runhild Gammelsæter reported in the scientific journal Cloning and Stem Cells that a cytoplasmic extract of fish eggs showed the ability to express transcription factors associated with pluripotency in already differentiated human epithelial cells in culture (COLLAS and GAMMELSÆTER, Novel Approaches to Epigenetic Reprogramming of Somatic Cells, CLONING AND STEM CELLS, Volume 9, Number 1, 2007). This finding had previously been shown for other somatic cells, but the observation that a marine egg cell extract of unfertilized fish eggs, could trigger elements of dedifferentiation, was new.

Skin Fibroblast

The broader concept means that a differentiated somatic cell, like skin cells, under certain conditions can be epigenetically reprogrammed to express pluripotency markers, in essence also by factors present in the cytoplasm of an unfertilized egg cell. Epigenetics means that environmental influence can alter our genes, explained e.g. by author Richard C. Francis (book insert).

 Such cytoplasmic factors are found across egg species as it is shown e.g. for frog eggs. The epigenetically reprogrammed cells may thereby acquire more or less pluripotency or stem cell like properties; i.e. phenotypic changes such as growth, maturation, and differentiation characteristics.

 

Regenics’ business concept is based on this research and we attempt to utilize potential regenerative  properties of unfertilized salmon roe for therapeutic products. Human skin cells are profoundly affected by exposure to salmon roe extract by e.g. increased secretion of collagen, increased migration, even faster reepithelialization of induced burn wounds. However, we have not demonstrated that these effects on human skin cells are attributable to the induction of pluripotency factors.

We have shown that re-epithelialisation occurs faster with samon roe extract than placebo, meaning that keratinocytes closes the wound and also other skin cells orchestrate or fascilitates the wound closure. For this fundamental process to happen, we know that the changes the skin cells undergo resembles incuction of pluripotency properties in the skin cells. 

Most reprogramming experiments have demonstrated that induced pluripotent cells (iPS; induced pluripotent stem cells), rely on upregulation of just a few transcription factors (maybe less than 4) to create pluripotency and cell self-renewal. The pioneering work in this area from a.o. Shinya Yamanaka, Japan, showed in 2006 that the introduction of four specific genes encoding transcription factors could convert adult cells into pluripotent stem cells. It is now known that several techniques can induce pluripotency in differentiated cells. Yamanakas was awarded the 2012 Nobel Prize along with Sir John Gurdon "for the discovery that mature cells can be reprogrammed to become pluripotent.". Thus, induced stem cell properties are created by induction of specific genes and mechanistically executed through epigenetic processes such as methylation, demethylation, acetylation, which means adding or removing very small molecules directly on the DNA molecule or on proteins bound to DNA.

In figure 1, some techniques for achieving dedifferentiation of skin fibroblasts are shown:

In A, the classic nuclear transfer technique is shown for induction of pluripotency, in B, cell fusion can achieve similar results, while Regenics uses the technique shown in C, where a cell extract (not a cell nucleus) can induce changes in the skin cells, as depicted in skin fibroblasts.

Regenics works with skin cells and has shown that the skin cells in fact can undergo changes when exposed to salmon roe extract which may be attributable to actual redifferentiation. The essence of cutaneous wound healing is to restore the epidermal barrier to the external environment during a process called re-epithelialization. We have shown that re-epithelialization occur in induced wounds i human skin (explants) faster with salmon roe, than with placebo (ETRS-WHS, 2015) or see right column insert . 

A key feature of re-epithelialization is movement or migration of the epithelial cells under stimulation of injury signals. To accomplish this, the skin cells undergo a process that resembles pluripotency transformation called Epithelial-Mesenchymal Transition (EMT), EMT is characterized by loss of epithelial adhesion and gain of mesenchymal features or partial pluripotency characteritics. This empower epithelial cells, simply explained to loose their connection and move along to close the wound. Stationary keratinocytes in human skin may be activated and mobilized in wound healing in a manner that is reminiscent of EMT. The causative agents are injury signals such as inflammatory cytokines. We have shown that salmon roe rapidly and potently alters the inflammatory profile of the key cells in skin, particularly macrophages, keratinocytes and fibroblasts. The skin cells also boost their collagen synthesis, they show intense migration and show a series of changes that correlate to processes that are needed; e.g. for wounds to heal and close the wound space.

Collas and Gammelsæter showed in their Cloning and Stem Cell communication , that at least 2 critical transcription factors were upregulated when cells were exposed to salmon roe, namely Nanog and Oct-4.

In the picture to the right, the upper row of images shows control and the next two rows fluorescence signal from upregulated Nanog (left) and Oct-4 (right) when the epithelial 293T cells are exposed to salmon roe extract. These two factors have recently been shown to be critical and interplay to create pluripotency. The Swedish research team led by Victor Olariu, reported in Nature (Nature, Scientific Reports 6, 25438 (2016), that Oct4, Nanog and Tet1 include positive feedback loops involving DNA-demethylation around the promoters of Oct4 and Tet1 which leads to enhanced self-renewal and pluripotency.

Regenics early invested in broad patenting the potential ability of salmon roe to alter skin cellular properties. Regenics now has 6 patents covering US, Europe and Asia and 12 patents pending on the technology.

For any practical use of extracts, devices or drugs to have a positive effects on skin, the salmon roe substances need to reach the cells under the barrier of the "stratum corneum" in the skin; the barrier that protects us from infection and mechanically shields off the cell layers of the skin where the fibroblast and other cells are embedded. The skin is also constantly changing, growing, regenerating, but also aging, looses elasticity, tone and becomes wrinkly. 

The development plan for creating a Medical Device based on salmon roe extract (Vernex®) therefore relies on clinical testing and efficacy results on human skin. Regenics has tested salmon roe extract on intact skin in healthy volunteers and  published the result in a peer reviewed journal where a double blinded trial approach was used. We observed positive effects on normal skin after 4-8 weeks of exposure, a reasonable time to be expected for an effect as described above to occur. (Lønne et al, Int J Cosmet Sci. 2013 Oct;35(5):515-22). In another approach we have exposed real wounds induced on human transplanted living skin to salmon roe extracts. We have observed increased and more rapid reepithelialization of skin cells and thus wound healing in such human transplanted skin. These findings were recently published  (Heldrup. et al, European Tissue Repair Society and Wound Healing Society, 2015,Wound Rep Reg (2015) 23 A1–A37)

A popular description of the technology and company aspirations can be read in GENialt, the publication from the Norwegian Bioteknologi Rådet, in its October 19th 2016, issue. 

 Further reading on Regenics concepts:

Cloning and Stem Cell  paper by Regenics AS researchers P. Collas and R. Gammelsæther describing cellular dedifferentiation by salmon roe extract in epithelial cells.

 

European Tissue Repair & Wound Healing abstract by Regenics AS researchers M. Heldrup et al. describing blinded assesment of improved and faster wound healing over placebo in 2 degree burns on human skin explants.

 

International Journal of Cosmetic Science by Regenics AS researchers Lønne at al. describing a double-blind , placebo controlled study of effects of salmon roe  extract apllied to normal skin over 4-8 weeks.  

A popular description of the technology and company aspirations can be read in GENialt, the publication from the Norwegian Bioteknologi Rådet, in its October 19th 2016, issue.

 

Regenics AS has developed a number of assays and techniques to study effects of various substances on skin cells both in vitroin vivo and in the clinic. Our focus is to develop products and techniques for therapeutic skin applications and to improve skin appearance.