Tissue Biology Research Unit

Engineering autologous dermo-epidermal skin composites in vitro Identifying and maintaining the epidermal stem cell compartment Accelerating vascularization in skin composites

Engineering autologous dermo-epidermal skin composites in vitro

Extensive skin loss may result from burns, soft tissue trauma, various skin diseases, and from removal of giant naevi. Taken together, these conditions still represent a significant clinical problem. Although most of these patients survive today, and although various skin transplantation techniques finally allow restoration of skin integrity, the functional and cosmetic results are far from ideal. This is mainly due to disfiguring and often disabling hypertrophic scarring and keloid formation which is a consistent and lifelong sequel after large scale skin transplantation, particularly in children. Thus, an autologous dermo-epidermal skin substitute (consisting of cultured human keratinocytes, endothelial cells, myoepithelial cells and fibroblasts) which is timely available, easily transplantable and that yields functional and cosmetic long-term results with minimal or even no scarring would be an optimal solution to repair large skin defects.

Our goal is to develop autologous dermo-epidermal skin composites (full thickness skin analogues) that can be used clinically to cover skin defects of any origin in one single surgical intervention. To achieve that, these composites have to fulfill the following criteria:

1. the dermo-epidermal graft serves as a skin analogue both     morphologically and functionally
2. it contains an intact stem cell compartment
3. it is rapidly and sufficiently vascularized after grafting) it is rapidly and sufficiently vascularized after grafting
4. it has adequate mechanical properties for transplantation, ind) it has adequate mechanical properties for transplantation, in particular is has developed a robust dermo-epidermal junction

We are currently testing different types of biodegradable matrices which serve as dermal templates with regard to being populated by endothelial cells and fibroblasts. Additionally, we are creating genetically engineered allogeneic fibroblasts overexpressing and releasing biologically active molecules, such as certain members of the Wnt, Notch and Hedgehog families, which are thought to be implicated in the maintenance of the stem cell compartment. This gene transfer approach is deliberately designed to bring about the transient expression of the “therapeutic” gene. The transient character of this strategy results from the fact that the genetically manipulated fibroblasts are allogenic and hence will be eliminated by the immune system within 3-4 weeks. Some of the cultured dermo-epidermal composites have already been transplanted onto immuno-incompetent rats to study take, engraftment, as well as early and late functional and cosmetic results.
As soon as a cultured skin substitute performs successfully in the animal model, a clinical pilot study will be envisioned.

top

Identifying and maintaining the epidermal stem cell compartment

Epidermal regeneration obtained with autologous cultured keratinocytes can be life saving for patients suffering from massive full-thickness burns. However, the widespread use of cultured epidermal autografts has been hampered by inconsistent clinical results reported by several leading burn centers world wide. An important cause for poor results is depletion of epidermal stem cells during the culture period.

Therefore, our ultimate goal is to transplant composite skin grafts containing a functional stem cell compartment. To achieve this we are currently focusing on:

1) the reliable identification of epidermal stem cells
2) the enrichment and/or maintenance of an appropriate number of
    vital epidermal stem cells throughout the culture period and beyond.

The identification of the so called side population (SP) has already been accomplished in our laboratory using a dye exclusion technique based on the function of membrane pumps of the ABC transporter family (also known as BCRP or MXR). These pumps were shown to be specifically expressed in stem cells of various organs. We have been able to highly enrich the keratinocyte SP by FACS sorting. In addition, we have developed a bioassay to determine the “stemness” of the isolated SP.

Despite these critical achievements we still have to answer the question whether the typical characteristics of epidermal stem cells can be maintained during a culture period of about 3 weeks. It is becoming increasingly clear that the properties required to ensure sustained stem cell function are vested in neighboring differentiated cells. Through specific signals (e.g. induced by Wnt growth factors), these cells control the behavior of stem cells. Thus, location rather than specialized patterns of gene expression appear to characterize stem cells. These specific locations are generally referred to as ‘niches’. A niche is considered to be a subset of cells and extracellular substrates that can house stem cells and control both self-renewal and progeny production in vivo.

Starting off from a highly enriched keratinocyte SP fraction and utilizing appropriate extracellular scaffolds and combinations of growth factors, we are aiming at creating a dermo-epidermal environment that will allow the maintenance of stem cells in vitro.

top

Accelerating vascularization in skin composites

To date, engineered tissue substitutes do not contain a vascular plexus that would warrant graft survival and rapid integration into defected organs. Therefore, the engineering of stable blood vessels within the mesenchymal (stromal) component of a given tissue substitute represents a fundamental challenge.

Our goal is to develop epithelio-mesenchymal tissue substitutes, such as dermo-epidermal skin grafts, that have been pre-vascularized in vitro. We hypothesize that such a pre-vascularized graft would allow for a rapid connection to the patient’s vascular system after transplantation. As a consequence, the transplant (consisting of a significant cell mass) would be sufficiently supplied with nutrients and oxygen and also be better protected against infection.

The rationale that underlies this approach is based on the observation that full and split thickness skin grafts are rapidly vascularized and survive after transplantation. This is possible because the vascular plexus present in these skin grafts readily connects to the patient’s circulatory system and hence ascertains perfusion.

The physiological pattern of the vascular network of a tissue or organ is determined by the proliferation, branching, remodeling, and pruning of its different segments. The functionality and stability of blood vessels are determined by the ratio of endothelial cells to mural cells (pericytes, smooth muscle cells).

We have developed a procedure to isolate microvascular endothelial cells from human skin. In addition, we have created a biodegradable scaffold and culture conditions that optimally support the formation of three dimensional, lumen forming capillaries. We are currently testing the properties of pre-vascularized dermal substitutes in animal experiments.

top