3D Skin Equivalents Research Articles

Beyond branching: multiknot structured polymer for gene delivery.

Polymer-based transfection vectors are increasingly becoming the preferred alternative to viral vectors thanks to their safety and ease of production, but low transfection potency has limited their application. Many polycationic vectors show high efficiency in vitro, but their excessive charge density makes them toxic for in vivo applications. Herein, we demonstrate the synthesis of new and unique disulfide-reducible polymeric gene nanocarriers that exhibit significantly enhanced transfection potency and low cytotoxicity, particularly in skin cells, surpassing the efficiency of the well-known transfection reagents polyethylenimine (PEI) and Lipofectamine2000. The unique three-dimensional (3D) "multiknot" vectors were synthesized from in situ deactivation enhanced atom transfer radical (co)polymerization (DE-ATRP) of multivinyl monomers (MVMs). The high transfection levels and low toxicity of this multiknot structured polymer in vitro, combined with its ability to mediate collagen VII expression in 3D skin equivalents made from cells of recessive dystrophic epidermolysis bullosa patients, demonstrates its use as a platform nanotechnology which should be investigated further for dermatological disease therapies. Our findings suggest that the marked improvements stem from the dense multiknot architecture and degradable property, which facilitate both the binding and releasing process of the plasmid DNA.

Fibroblasts Derived from Human Pluripotent Stem Cells Activate Angiogenic Responses In Vitro and In Vivo

Human embryonic and induced pluripotent stem cells (hESC/hiPSC) are promising cell sources for the derivation of large numbers of specific cell types for tissue engineering and cell therapy applications. We have describe a directed differentiation protocol that generates fibroblasts from both hESC and hiPSC (EDK/iPDK) that support the repair and regeneration of epithelial tissue in engineered, 3D skin equivalents. In the current study, we analyzed the secretory profiles of EDK and iPDK cells to investigate the production of factors that activate and promote angiogenesis. Analysis of in vitro secretion profiles from EDK and iPDK cells demonstrated the elevated secretion of pro-angiogenic soluble mediators, including VEGF, HGF, IL-8, PDGF-AA, and Ang-1, that stimulated endothelial cell sprouting in a 3D model of angiogenesis in vitro. Phenotypic analysis of EDK and iPDK cells during the course of differentiation from hESCs and iPSCs revealed that both cell types progressively acquired pericyte lineage markers NG2, PDGFRβ, CD105, and CD73 and demonstrated transient induction of pericyte progenitor markers CD31, CD34, and Flk1/VEGFR2. Furthermore, when co-cultured with endothelial cells in 3D fibrin-based constructs, EDK and iPDK cells promoted self-assembly of vascular networks and vascular basement membrane deposition. Finally, transplantation of EDK cells into mice with hindlimb ischemia significantly reduced tissue necrosis and improved blood perfusion, demonstrating the potential of these cells to stimulate angiogenic responses in vivo. These findings demonstrate that stable populations of pericyte-like angiogenic cells can be generated with high efficiency from hESC and hiPSC using a directed differentiation approach. This provides new cell sources and opportunities for vascular tissue engineering and for the development of novel strategies in regenerative medicine.

Generation of 3D Skin Equivalents Fully Reconstituted from Human Induced Pluripotent Stem Cells (iPSCs)

Recent generation of patient-specific induced pluripotent stem cells (PS-iPSCs) provides significant advantages for cell- and gene-based therapy. Establishment of iPSC-based therapy for skin diseases requires efficient methodology for differentiating iPSCs into both keratinocytes and fibroblasts, the major cellular components of the skin, as well as the reconstruction of skin structures using these iPSC-derived skin components. We previously reported generation of keratinocytes from human iPSCs for use in the treatment of recessive dystrophic epidermolysis bullosa (RDEB) caused by mutations in the COL7A1 gene. Here, we developed a protocol for differentiating iPSCs into dermal fibroblasts, which also produce type VII collagen and therefore also have the potential to treat RDEB. Moreover, we generated in vitro 3D skin equivalents composed exclusively human iPSC-derived keratinocytes and fibroblasts for disease models and regenerative therapies for skin diseases, first demonstrating that iPSCs can provide the basis for modeling a human organ derived entirely from two different types of iPSC-derived cells.

A pilot study of the photoprotective effect of almond phytochemicals in a 3D human skin equivalent

UV exposure causes oxidative stress, inflammation, erythema, and skin cancer. α-Tocopherol (AT) and polyphenols (AP) present in almonds may serve as photoprotectants. Our objectives were to assess the feasibility of using a 3D human skin equivalent (HSE) in photoprotectant research and to determine photoprotection of AT and AP against UVA radiation. AT or AP was applied to medium (25 and 5μmol/L, respectively) or topically (1mg/cm2 and 14μg/cm2), followed by UVA. Photodamage assessed 96h post UVA included HSE morphology, keratinocyte proliferation, apoptosis, and differentiation. UVA induced disorganization of basal layer, alteration of epidermal development, and fibroblast loss which were alleviated by all nutrient pretreatments. UVA significantly decreased keratinocyte proliferation compared to controls, and all pretreatments tended to negate the reduction though only the medium AT effect was statistically significant (p⩽0.05). UVA led to a significant 16-fold increase in apoptosis of fibroblasts compared to the control which was alleviated by topical AP pretreatment and completely negated by topical AT (p⩽0.05). In conclusion, we validated the feasibility of using HSE in evaluation of photoprotectants and found that AT and AP, applied to medium or topically, provided some degree of photoprotection against UVA.

Protective Effect of Tropical Highland Blackberry Juice (Rubus adenotrichos Schltdl.) Against UVB‐Mediated Damage in Human Epidermal Keratinocytes and in a Reconstituted Skin Equivalent Model

Solar ultraviolet (UV) radiation, particularly its UVB (280-320 nm) spectrum, is the primary environmental stimulus leading to skin carcinogenesis. Several botanical species with antioxidant properties have shown photochemopreventive effects against UVB damage. Costa Rica's tropical highland blackberry (Rubus adenotrichos) contains important levels of phenolic compounds, mainly ellagitannins and anthocyanins, with strong antioxidant properties. In this study, we examined the photochemopreventive effect of R. adenotrichos blackberry juice (BBJ) on UVB-mediated responses in human epidermal keratinocytes and in a three-dimensional (3D) reconstituted normal human skin equivalent (SE). Pretreatment (2 h) and posttreatment (24 h) of normal human epidermal keratinocytes (NHEKs) with BBJ reduced UVB (25 mJ cm(-2))-mediated (1) cyclobutane pyrimidine dimers (CPDs) and (2) 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation. Furthermore, treatment of NHEKs with BBJ increased UVB-mediated (1) poly(ADP-ribose) polymerase cleavage and (2) activation of caspases 3, 8 and 9. Thus, BBJ seems to alleviate UVB-induced effects by reducing DNA damage and increasing apoptosis of damaged cells. To establish the in vivo significance of these findings to human skin, immunohistochemistry studies were performed in a 3D SE model, where BBJ was also found to decrease CPDs formation. These data suggest that BBJ may be developed as an agent to ameliorate UV-induced skin damage.

APR-246/PRIMA-1 MET rescues epidermal differentiation in skin keratinocytes derived from EEC syndrome patients with p63 mutations

p53 and p63 share extensive sequence and structure homology. p53 is frequently mutated in cancer, whereas mutations in p63 cause developmental disorders manifested in ectodermal dysplasia, limb defects, and orofacial clefting. We have established primary adult skin keratinocytes from ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome patients with p63 mutations as an in vitro human model to study the disease mechanism in the skin of EEC patients. We show that these patient keratinocytes cultured either in submerged 2D cultures or in 3D skin equivalents have impaired epidermal differentiation and stratification. Treatment of these patient keratinocytes with the mutant p53-targeting compound APR-246/PRIMA-1(MET) (p53 reactivation and induction of massive apoptosis) that has been successfully tested in a phase I/II clinical trial in cancer patients partially but consistently rescued morphological features and gene expression during epidermal stratification in both 2D and 3D models. This rescue coincides with restoration of p63 target-gene expression. Our data show that EEC patient keratinocytes with p63 mutations can be used for characterization of the abnormal molecular circuitry in patient skin and may open possibilities for the design of novel pharmacological treatment strategies for patients with mutant p63-associated developmental abnormalities.

Generation of keratinocytes from normal and recessive dystrophic epidermolysis bullosa-induced pluripotent stem cells

Embryonic stem cells (ESCs) have an unlimited proliferative capacity and extensive differentiation capability. They are an alternative source for regenerative therapies with a potential role in the treatment of several human diseases. The clinical use of ESCs, however, has significant ethical and biological obstacles related to their derivation from embryos and potential for immunological rejection, respectively. These disadvantages can be circumvented by the alternative use of induced pluripotent stem cells (iPSCs), which are generated from an individual's (autologous) somatic cells by exogenous expression of defined transcription factors and have biological characteristics similar to ESCs. In recent years, patient-specific iPSCs have been generated to study disease mechanisms and develop iPSC-based therapies. The development of iPSC-based therapies for skin diseases requires successful differentiation of iPSCs into cellular components of the skin, including epidermal keratinocytes. Here, we succeeded in generating iPSCs not only from normal human fibroblasts but also from fibroblasts isolated from the skin of two patients with recessive dystrophic epidermolysis bullosa. Moreover, we differentiated both of these iPSCs into keratinocytes with high efficiency, and generated 3D skin equivalents using iPSC-derived keratinocytes, suggesting that they were fully functional. Our studies indicate that autologous iPSCs have the potential to provide a source of cells for regenerative therapies for specific skin diseases.

Fibroblasts derived from human embryonic stem cells direct development and repair of 3D human skin equivalents

IntroductionPluripotent, human stem cells hold tremendous promise as a source of progenitor and terminally differentiated cells for application in future regenerative therapies. However, such therapies will be dependent upon the development of novel approaches that can best assess tissue outcomes of pluripotent stem cell-derived cells and will be essential to better predict their safety and stability following in vivo transplantation.MethodsIn this study we used engineered, human skin equivalents (HSEs) as a platform to characterize fibroblasts that have been derived from human embryonic stem (hES) cell. We characterized the phenotype and the secretion profile of two distinct hES-derived cell lines with properties of mesenchymal cells (EDK and H9-MSC) and compared their biological potential upon induction of differentiation to bone and fat and following their incorporation into the stromal compartment of engineered, HSEs.ResultsWhile both EDK and H9-MSC cell lines exhibited similar morphology and mesenchymal cell marker expression, they demonstrated distinct functional properties when incorporated into the stromal compartment of HSEs. EDK cells displayed characteristics of dermal fibroblasts that could support epithelial tissue development and enable re-epithelialization of wounds generated using a 3D tissue model of cutaneous wound healing, which was linked to elevated production of hepatocyte growth factor (HGF). Lentiviral shRNA-mediated knockdown of HGF resulted in a dramatic decrease of HGF secretion from EDK cells that led to a marked reduction in their ability to promote keratinocyte proliferation and re-epithelialization of cutaneous wounds. In contrast, H9-MSCs demonstrated features of mesenchymal stem cells (MSC) but not those of dermal fibroblasts, as they underwent multilineage differentiation in monolayer culture, but were unable to support epithelial tissue development and repair and produced significantly lower levels of HGF.ConclusionsOur findings demonstrate that hES-derived cells could be directed to specified and alternative mesenchymal cell fates whose function could be distinguished in engineered HSEs. Characterization of hES-derived mesenchymal cells in 3D, engineered HSEs demonstrates the utility of this tissue platform to predict the functional properties of hES-derived fibroblasts before their therapeutic transplantation.

The basement membrane microenvironment directs the normalization and survival of bioengineered human skin equivalents

Epithelial–mesenchymal interactions promote the morphogenesis and homeostasis of human skin. However, the role of the basement membrane (BM) during this process is not well-understood. To directly study how BM proteins influence epidermal differentiation, survival and growth, we developed novel 3D human skin equivalents (HSEs). These tissues were generated by growing keratinocytes at an air–liquid interface on polycarbonate membranes coated with individual matrix proteins (Type I Collagen, Type IV Collagen or fibronectin) that were placed on contracted Type I Collagen gels populated with dermal fibroblasts. We found that only keratinocytes grown on membranes coated with the BM protein Type IV Collagen showed optimal tissue architecture that was similar to control tissues grown on de-epidermalized dermis (AlloDerm) that contained intact BM. In contrast, tissues grown on proteins not found in BM, such as fibronectin and Type I Collagen, demonstrated aberrant tissue architecture that was linked to a significant elevation in apoptosis and lower levels of proliferation of basal keratinocytes. While all tissues demonstrated a normalized, linear pattern of deposition of laminin 5, tissues grown on Type IV Collagen showed elevated expression of α6 integrin, Type IV Collagen and Type VII Collagen, suggesting induction of BM organization. Keratinocyte differentiation (Keratin 1 and filaggrin) was not dependent on the presence of BM proteins. Thus, Type IV Collagen acts as a critical microenvironmental factor in the BM that is needed to sustain keratinocyte growth and survival and to optimize epithelial architecture.