Julie Fradette

IFATS Collection: Using Human Adipose‐Derived Stem/Stromal Cells for the Production of New Skin Substitutes

The ability to harvest and culture stem cell populations from various human postnatal tissues is central to regenerative medicine applications, including tissue engineering. The discovery of multipotent mesenchymal stem cells within the stromal fraction of adipose tissue prompted their use for the healing and reconstruction of many tissues. Here, we examined the influence of adipose‐derived stem/stromal cells (ASCs) on skins regenerative processes, from a tissue engineering perspective. Using a self‐assembly approach, human skin substitutes were produced. They featured a stromal compartment containing human extracellular matrix endogenously produced from either dermal fibroblasts or adipose‐derived stem/stromal cells differentiated or not toward the adipogenic lineage. Human keratinocytes were seeded on each stroma and cultured at the air‐liquid interface to reconstruct a bilayered skin substitute. These new skin substitutes, containing an epidermis and a distinctive stroma devoid of synthetic biomaterial, displayed characteristics similar to human skin. The influence of the type of stromal compartment on epidermal morphogenesis was assessed by the evaluation of tissue histology, the expression of key protein markers of the epidermal differentiation program (keratin [K] 14, K10, transglutaminase), the expression of dermo‐epidermal junction components (laminins, collagen VII), and the presence of basement membrane and hemidesmosomes. Our findings suggest that adipose‐derived stem/stromal cells could usefully substitute dermal fibroblasts for skin reconstruction using the self‐assembly method. Finally, by exploiting the adipogenic potential of ASCs, we also produced a more complete trilayered skin substitute consisting of the epidermis, the dermis, and the adipocyte‐containing hypodermis, the skins deepest layer.

Evolution of three dimensional skin equivalent models reconstructed in vitro by tissue engineering

Since the culture of keratinocytes on feeder layers, research to produce skin equivalents has been motivated by the challenge of treating large burns and chronic wounds and by European regulations which both require proof of the innocuousness and the effectiveness of cosmetic products, and which forbid animal testing. The dynamism in fundamental research, dermocosmetology and the pharmaceutical industry has led to the evolution and complexification of reconstructed skin. The Collagen-GAG-Chitosan sponge, as well as the self-assembly model, allow dermal reconstruction in which the neosynthesized extracellular matrix contains all of the desired macromolecules. It is deposited forming an ultrastructurally organised architecture. The quality of the dermis obtained allows the development and regeneration of a pluristratified and differentiated epidermis firmly anchored by an organised dermal-epidermal junction. Evolution of reconstructed skin into models which are more and more similar to the physiological skin results in higher graft take rates in the treatment of burns and chronic wounds, and brings to research, to dermocosmetology and to the pharmaceutical industry, a wide range of products such as pigmented, endothelialized, immunocompetent, and now adipose reconstructed skins. The present review will mainly concentrate on the latest developments in skin engineering and will mostly concern the studies carried out by our groups.

Adipose-tissue engineering: Taking advantage of the properties of human adipose-derived stem/stromal cells

Adipose tissue is now recognized as an important source of postnatal mesenchymal stem cells for regenerative medicine applications. For example, adipose-tissue engineering is an emerging approach that enables the development of autologous substitutes that could be used as an alternative to fat transplantation methods currently yielding variable outcomes for the long-term repair of soft-tissue defects. Here, we describe the production of unique tissue-engineered adipose tissues devoid of exogenous biomaterials produced from human adipose-derived stem/stromal cells. Our strategy is based on the dual self-assembly of extracellular components secreted and organized by the adipose-derived stromal cells after ascorbic acid stimulation, as well as their concomitant differentiation into adipocytes after adipogenic induction. When compared to stromal cells isolated from resected fat, lipoaspirated fat-derived cells featured an increased adipogenic potential and the enhanced ability to recreate three-dimensional adipose substitutes in vitro. These substitutes were histologically similar to native adipose tissue. They featured lipid-filled adipocytes embedded into an extracellular matrix rich in fibronectin as well as collagens I and V. On a functional level, the reconstructed adipose tissues expressed adipocyte-related transcripts and secreted adipokines typical of adipose tissue, such as leptin. Finally, the successful in vitro production of human adipose substitutes featuring an increased surface area (>30cm2) is described, reinforcing the notion that customized autologous reconstructed adipose tissues could be produced in the future to repair a wide range of soft-tissue defects.

Expression of α-Smooth Muscle Actin Determines the Fate of Mesenchymal Stromal Cells

Summary Pro-fibrotic microenvironments of scars and tumors characterized by increased stiffness stimulate mesenchymal stromal cells (MSCs) to express α-smooth muscle actin (α-SMA). We investigated whether incorporation of α-SMA into contractile stress fibers regulates human MSC fate. Sorted α-SMA-positive MSCs exhibited high contractile activity, low clonogenicity, and differentiation potential limited to osteogenesis. Knockdown of α-SMA was sufficient to restore clonogenicity and adipogenesis in MSCs. Conversely, α-SMA overexpression induced YAP translocation to the nucleus and reduced the high clonogenicity and adipogenic potential of α-SMA-negative MSCs. Inhibition of YAP rescued the decreased adipogenic differentiation potential induced by α-SMA, establishing a mechanistic link between matrix stiffness, α-SMA, YAP, and MSC differentiation. Consistent with in vitro findings, nuclear localization of YAP was positively correlated in α-SMA expressing stromal cells of adiposarcoma and osteosarcoma. We propose that α-SMA mediated contraction plays a critical role in mechanically regulating MSC fate by controlling YAP/TAZ activation.

Regeneration of Skin and Cornea by Tissue Engineering

Progress in tissue engineering has led to the development of technologies allowing the reconstruction of autologous tissues from the patients own cells. Thus, tissue-engineered epithelial substitutes produced from cultured skin epithelial cells undergo long-term regeneration after grafting, indicating that functional stem cells were preserved during culture and following grafting. However, these cultured epithelial sheets reconstruct only the upper layer of the skin and lack the mechanical properties associated to the connective tissue of the dermis. We have designed a reconstructed skin entirely made from human cutaneous cells comprising both the dermis and the epidermis, as well as a well-organized basement membrane by a method named the self-assembly approach. In this chapter, protocols to generate reconstructed skin and corneal epithelium suitable for grafting are described in details. The methods include extraction and culture of human skin keratinocytes, human skin fibroblasts as well as rabbit and human corneal epithelial cells, and a complete description of the skin reconstructed by the self-assembly approach and of corneal epithelium reconstructed over a fibrin gel.

Vibrissa hair bulge houses two populations of skin epithelial stem cells distinct by their keratin profile

Defining the properties of postnatal stem cells is of interest given their relevance for tissue homeostasis and therapeutic applications, such as skin tissue engineering for burn patients. In hair follicles, the bulge region of the outer root sheath houses stem cells. We show that explants from the prominent bulge area, but not the bulb, in rodent vibrissa follicles can produce epidermis in a skin model of tissue engineering. Using morphological criteria and keratin expression, we typified epithelial stem cells of vibrissa bulge. Two types of slow‐cycling cells (Bb, Bs1) featuring a high colony‐forming capacity occur in the bulge. Bb cells are located in the outermost basal layer, express K5, K15, K17, and K19, and feature a loosely organized keratin network. Bs1 cells localize to the suprabasal layers proximal to Bb cells and express K5/K17, corre lating with a network of densely bundled filaments. These prominent bundles are missing in K17‐null mice, which lack vibrissa. Atypically, both the Bb and Bs1 keratinocytes lack K14 expression. These findings show heterogeneity within the hair follicle stem cell reposi tory, establish that a subset of slow‐cycling cells are suprabasal in location, and point to a special role for K5/K17 filaments in a newly defined subset of stem cells. Our results are discussed in the context of long‐term survival of engineered tissues after grafting that requires the presence of stem cells.—Larouche, D., Tong, X., Fradette, J., Coulombe, P. A., Germain, L. Vibrissa hair bulge houses two populations of skin epithelial stem cells distinct by their keratin profile. FASEB J. 22, 1404–1415 (2008)

Cell sheet technology for tissue engineering: the self-assembly approach using adipose-derived stromal cells.

In the past years, adipose tissue has spurred a wide interest, not only as a source of adult multipotent stem cells but also as a highly eligible tissue for reconstructive surgery procedures. Tissue engineering is one field of regenerative medicine progressing at great strides in part due to its important use of adipose-derived stem/stromal cells (ASCs). The development of diversified technologies combining ASCs with various biomaterials has lead to the reconstruction of numerous types of tissue-engineered substitutes such as bone, cartilage, and adipose tissues from rodent, porcine, or human ASCs. We have recently achieved the reconstruction of connective and adipose tissues composed entirely of cultured human ASCs and their secreted endogenous extracellular matrix components by a methodology known as the self-assembly approach of tissue engineering. The latter is based on the stimulation of ASCs to secrete and assemble matrix components in culture, leading to the production of cell sheets that can be manipulated and further assembled into thicker multilayer tissues. In this chapter, protocols to generate both reconstructed connective and adipocyte-containing tissues using the self-assembly approach are described in detail. The methods include amplification and cell banking of human ASCs, as well as culture protocols for the production of individual stromal and adipose sheets, which are the building blocks for the reconstruction of multilayered human connective and adipose tissues, respectively.

Tissue engineering of skin and cornea : Development of new models for in vitro studies

Human beings are greatly preoccupied with the unavoidable nature of aging. While the biological processes of senescence and aging are the subjects of intense investigations, the molecular mechanisms linking aging with disease and death are yet to be elucidated. Tissue engineering offers new models to study the various processes associated with aging. Using keratin 19 as a stem cell marker, our studies have revealed that stem cells are preserved in human skin reconstructed by tissue engineering and that the number of epithelial stem cells varies according to the donors age. As with skin, human corneas can also be engineered in vitro. Among the epithelial cells used for reconstructing skin and corneas, significant age‐dependent variations in the expression of the transcription factor Sp1 were observed. Culturing skin epithelial cells with a feeder layer extended their life span in culture, likely by preventing Sp1 degradation in epithelial cells, therefore demonstrating the pivotal role played by this transcription factor in cell proliferation. Finally, using the human tissue‐engineered skin as a model, we linked Hsp27 activation with skin differentiation.

Adipose‐derived stromal cells for the reconstruction of a human vesical equivalent

Despite a wide panel of tissue‐engineering models available for vesical reconstruction, the lack of a differentiated urothelium remains their main common limitation. For the first time to our knowledge, an entirely human vesical equivalent, free of exogenous matrix, has been reconstructed using the self‐assembly method. Moreover, we tested the contribution of adipose‐derived stromal cells, an easily available source of mesenchymal cells featuring many potential advantages, by reconstructing three types of equivalent, named fibroblast vesical equivalent, adipose‐derived stromal cell vesical equivalent and hybrid vesical equivalent – the latter containing both adipose‐derived stromal cells and fibroblasts. The new substitutes have been compared and characterized for matrix composition and organization, functionality and mechanical behaviour. Although all three vesical equivalents displayed adequate collagen type I and III expression, only two of them, fibroblast vesical equivalent and hybrid vesical equivalent, sustained the development of a differentiated and functional urothelium. The presence of uroplakins Ib, II and III and the tight junction marker ZO‐1 was detected and correlated with impermeability. The mechanical resistance of these tissues was sufficient for use by surgeons. We present here in vitro tissue‐engineered vesical equivalents, built without the use of any exogenous matrix, able to sustain mechanical stress and to support the formation of a functional urothelium, i.e. able to display a barrier function similar to that of native tissue. Copyright

Harvesting the Potential of the Human Umbilical Cord: Isolation and Characterisation of Four Cell Types for Tissue Engineering Applications

The human umbilical cord (UC) has attracted interest as a source of cells for many research applications. UC solid tissues contain four cell types: epithelial, stromal, smooth muscle and endothelial cells. We have developed a unique protocol for the sequential extraction of all four cell types from a single UC, allowing tissue reconstruction using multiple cell types from the same source. By combining perfusion, immersion and explant techniques, all four cell types have been successfully expanded in monolayer cultures. We have also characterised epithelial and Wharton’s jelly cells (WJC) by immunolabelling of specific proteins. Epithelial cell yields averaged at 2.3 × 105 cells per centimetre UC, and the cells expressed an unusual combination of keratins typical of simple, mucous and stratified epithelia. Stromal cells in the Wharton’s jelly expressed desmin, α-smooth muscle actin, elastin, keratins (K12, K16, K18 and K19), vimentin and collagens. Expression patterns in cultured cells resembled those found in situ except for basement membrane components and type III collagen. These stromal cells featured a sustained proliferation rate up to passage 12 after thawing. The mesenchymal stem cell (MSC) character of the WJC was confirmed by their expression of typical MSC surface markers and by adipogenic and osteogenic differentiation assays. To emphasise and demonstrate their potential for regenerative medicine, UC cell types were successfully used to produce human tissue-engineered constructs. Both bilayered stromal/epithelial and vascular substitutes were produced, establishing the versatility and importance of these cells for research and therapeutic applications.