Impact of microstructure on cell behavior and tissue mechanics in collagen and dermal decellularized extra-cellular matrices.

Skin models are used for many applications such as research and development or grafting. Unfortunately, most lack a proper microenvironment producing poor mechanical properties and inaccurate extra-cellular matrix composition and organization. In this report we focused on mechanical properties, extra-cellular matrix organization and cell interactions in human skin samples reconstructed with pure collagen or dermal decellularized extra-cellular matrices (S-dECM) and compared them to native human skin. We found that Full-thickness S-dECM samples presented stiffness two times higher than collagen gel and similar to ex vivo human skin, and proved for the first time that keratinocytes also impact dermal mechanical properties. This was correlated with larger fibers in S-dECM matrices compared to collagen samples and with a differential expression of F-actin, vinculin and tenascin C between S-dECM and collagen samples. This is clear proof of the microenvironment's impact on cell behaviors and mechanical properties. STATEMENT OF SIGNIFICANCE: In vitro skin models have been used for a long time for clinical applications or in vitro knowledge and evaluation studies. However, most lack a proper microenvironment producing a poor combination of mechanical properties and appropriate biological outcomes, partly due to inaccurate extra-cellular matrix (ECM) composition and organization. This can lead to limited predictivity and weakness of skin substitutes after grafting. This study shows, for the first time, the importance of a complex and rich microenvironment on cell behaviors, matrix macro- and micro-organization and mechanical properties. The increased composition and organization complexity of dermal skin decellularized extra-cellular matrix populated with differentiated cells produces in vitro skin models closer to native human skin physiology.

Elastin-like Polypeptide-Based Bioink: A Promising Alternative for 3D Bioprinting.

Three-dimensional (3D) bioprinting offers a great alternative to traditional techniques in tissue reconstruction, based on seeding cells manually into a scaffold, to better reproduce organs' complexity. When a suitable bioink is engineered with appropriate physicochemical properties, such a process can advantageously provide a spatial control of the patterning that improves tissue reconstruction. The design of an adequate bioink must fulfill a long list of criteria including biocompatibility, printability, and stability. In this context, we have developed a bioink containing a precisely controlled recombinant biopolymer, namely, elastin-like polypeptide (ELP). This material was further chemoselectively modified with cross-linkable moieties to provide a 3D network through photopolymerization. ELP chains were additionally either functionalized with a peptide sequence Gly-Arg-Gly-Asp-Ser (GRGDS) or combined with collagen I to enable cell adhesion. Our ELP-based bioinks were found to be printable, while providing excellent mechanical properties such as stiffness and elasticity in their cross-linked form. Besides, they were demonstrated to be biocompatible, showing viability and adhesion of dermal normal human fibroblasts (NHF). Expressions of specific extracellular matrix (ECM) protein markers as pro-collagen I, elastin, fibrillin, and fibronectin were revealed within the 3D network containing cells after only 18 days of culture, showing the great potential of ELP-based bioinks for tissue engineering.

A perfusable vascularized full-thickness skin model for potential topical and systemic applications.

Vascularization of reconstructed tissues is one of the remaining hurdles to be considered to improve both the functionality and viability of skin grafts and the relevance ofin vitroapplications. Our study, therefore, sought to develop a perfusable vascularized full-thickness skin equivalent that comprises a more complex blood vasculature compared to existing models. We combined molding, auto-assembly and microfluidics techniques in order to create a vascularized skin equivalent representing (a) a differentiated epidermis with a physiological organization and correctly expressing K14, K10, Involucrin, TGM1 and Filaggrin, (b) three perfusable vascular channels with angiogenic sprouts stained with VE-Caderin and Collagen IV, (c) an adjacent microvascular network created via vasculogenesis and connected to the sprouting macrovessels. Histological analysis and immunostaining of CD31, Collagen IV, Perlecan and Laminin proved the integrity of vascular constructs. In order to validate the vascularized skin potential of topical and systemic applications, caffeine and minoxidil, two compounds with different chemical properties, were topically applied to measure skin permeability and benzo[a]pyrene pollutant was systemically applied to evaluate systemic delivery. Our results demonstrated that perfusion of skin reconstructs and the presence of a complex vascular plexus resulted in a more predictive and reliable model to assess respectively topical and systemic applications. This model is therefore aimed at furthering drug discovery and improving clinical translation in dermatology.

Utilization of patterned bioprinting for heterogeneous and physiologically representative reconstructed epidermal skin models.

Organotypic skin tissue models have decades of use for basic research applications, the treatment of burns, and for efficacy/safety evaluation studies. The complex and heterogeneous nature of native human skin however creates difficulties for the construction of physiologically comparable organotypic models. Within the present study, we utilized bioprinting technology for the controlled deposition of separate keratinocyte subpopulations to create a reconstructed epidermis with two distinct halves in a single insert, each comprised of a different keratinocyte sub-population, in order to better model heterogonous skin and reduce inter-sample variability. As an initial proof-of-concept, we created a patterned epidermal skin model using GPF positive and negative keratinocyte subpopulations, both printed into 2 halves of a reconstructed skin insert, demonstrating the feasibility of this approach. We then demonstrated the physiological relevance of this bioprinting technique by generating a heterogeneous model comprised of dual keratinocyte population with either normal or low filaggrin expression. The resultant model exhibited a well-organized epidermal structure with each half possessing the phenotypic characteristics of its constituent cells, indicative of a successful and stable tissue reconstruction. This patterned skin model aims to mimic the edge of lesions as seen in atopic dermatitis or ichthyosis vulgaris, while the use of two populations within a single insert allows for paired statistics in evaluation studies, likely increasing study statistical power and reducing the number of models required per study. This is the first report of human patterned epidermal model using a predefined bioprinted designs, and demonstrates the relevance of bioprinting to faithfully reproduce human skin microanatomy.

Senescence-associated SIN3B promotes inflammation and pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDAC) is strikingly resistant to conventional therapeutic approaches. We previously demonstrated that the histone deacetylase-associated protein SIN3B is essential for oncogene-induced senescence in cultured cells. Here, using a mouse model of pancreatic cancer, we have demonstrated that SIN3B is required for activated KRAS-induced senescence in vivo. Surprisingly, impaired senescence as the result of genetic inactivation of Sin3B was associated with delayed PDAC progression and correlated with an impaired inflammatory response. In murine and human pancreatic cells and tissues, levels of SIN3B correlated with KRAS-induced production of IL-1α. Furthermore, evaluation of human pancreatic tissue and cancer cells revealed that Sin3B was decreased in control and PDAC samples, compared with samples from patients with pancreatic inflammation. These results indicate that senescence-associated inflammation positively correlates with PDAC progression and suggest that SIN3B has potential as a therapeutic target for inhibiting inflammation-driven tumorigenesis.

Regulation and function of FoxO transcription factors in normal and cancer stem cells: what have we learned?

Forkhead FoxO transcription factors exert critical biological functions in response to genotoxic stress. In mammals four FoxOs proteins are known. FoxOs induce cell cycle arrest, repair damaged DNA, or initiate apoptosis by modulating genes that control these processes. In particular, FoxO proteins are critical regulators of oxidative stress by modulating the expression of several anti-oxidant enzyme genes. This function of FoxO is essential for the regulation of stem and progenitor cell pool in the hematopoietic system and possibly other stem cells. Overall functions of FoxOs are consistent with their role as tumor suppressors as has been shown in animal models. As such, FoxOs are suppressed in various cancer cells. However, recent reports strongly suggest that FoxOs are critical for the maintenance of leukemic stem cells. The diverse functions of FoxOs are orchestrated by tight regulations of expression and activity of its family members. Here we discuss the recent progress in understanding the function of FoxOs specifically in normal and cancer stem cells and what is known about the regulation of these proteins in various cell types and tissues including in the physiological setting of primary cells in vivo. These studies underscore the importance of regulation of FoxO proteins and whether these factors play distinct or redundant functions. Understanding how FoxOs are modulated is critical for devising novel therapies based on targeted restoration/or inhibition of FoxO function in cancer and in other diseased cells in which FoxOs have a key function.

Early alteration of the self-renewal/differentiation threshold in trophoblast stem cells derived from mouse embryos after nuclear transfer.

Development after nuclear transfer (NT) is subjected to defects originating from both the epiblast and the trophoblast parts of the conceptus and is always accompanied by placentomegaly at term. Here we have investigated the origin of the reprogramming errors affecting the trophoblast lineage in mouse NT embryos. We show that trophoblast stem (TS) cells can be derived from NT embryos (ntTS cells) and used as an experimental in vitro model of trophoblast proliferation and differentiation. Strikingly, TS derivation is more efficient from NT embryos than from controls and ntTS cells exhibit a growth advantage over control TS cells under self-renewal conditions. While epiblast-produced growth factors Fgf4 and Activin exert a fine-tuned control on the balance between self-renewal and differentiation of control TS cells, ntTS cells exhibit a reduced dependency upon their micro-environment. Since the supply of growth factors is known do decrease at the onset of placental formation in vivo we propose that TS cells in NT embryos continue to self-renew during a longer period of time than in fertilized embryo. The resulting increased pool of progenitors could contribute to the enlarged extra-embryonic region observed in the early trophoblast of in vivo grown mouse NT blastocysts that results in placentomegaly.

Trophoblast stem cell derivation, cross-species comparison and use of nuclear transfer: new tools to study trophoblast growth and differentiation.

The trophoblast is a supportive tissue in mammals that plays key roles in embryonic patterning, foetal growth and nutrition. It shows an extensive growth up to the formation of the placenta. This growth is believed to be fed by trophoblast stem cells able to self-renew and to give rise to the differentiated derivatives present in the placenta. In this review, we summarize recent data on the molecular regulation of the trophoblast in vivo and in vitro. Most data have been obtained in the mouse, however, whenever relevant, we compare this model to other mammals. In ungulates, the growth of the trophoblast displays some striking features that make these species interesting alternative models for the study of trophoblast development. After the transfer of somatic nuclei into oocytes, studies in the mouse and the cow have both underlined that the trophoblast may be a direct target of reprogramming defects and that its growth seems specifically affected. We propose that the study of TS cells derived from nuclear transfer embryos may help to unravel some of the epigenetic abnormalities which occur therein.