[Show abstract][Hide abstract] ABSTRACT: 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/cm(2) and 14μg/cm(2)), 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.
Journal of photochemistry and photobiology. B, Biology 07/2013; 126C:17-25. · 3.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 01/2013; 8(12):e83755. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Current approaches to skin equivalents often only include the epidermis and dermis. Here, a full-thickness skin equivalent is described including epidermis, dermis, and hypodermis, that could serve as an in vitro model for studying skin biology or as a platform for consumer product testing. The construct is easy to handle and is maintained for >14 d while expressing physiological morphologies of the epidermis and dermis, seen by keratin 10, collagens I and IV expression. The skin equivalent produces glycerol and leptin, markers of adipose metabolism. This work serves as a foundation for understanding a few necessary factors needed to develop a stable, functional model of full-thickness skin.
[Show abstract][Hide abstract] ABSTRACT: The controlled differentiation of induced pluripotent stem cells (iPSC) towards clinically-relevant cell types has benefitted from epigenetic profiling of lineage-specific markers to confirm the phenotype of iPSC-derived cells. Mapping epigenetic marks throughout the genome has identified unique changes which occur in the DNA methylation profile of cells as they differentiate to specific cell types. Beyond characterizing the development of cells derived from pluripotent stem cells, the process of reprogramming cells to iPSC resets lineage-specific DNA methylation marks established during differentiation to specific somatic cell types. This property of reprogramming has potential utility in reverting aberrant epigenetic alterations in nuclear organization that are linked to disease progression. Since DNA methylation marks are reset following reprogramming, and contribute to restarting developmental programs, it is possible that DNA methylation marks associated with the disease state may also be erased in these cells. The subsequent differentiation of such cells could result in cell progeny that will function effectively as therapeutically-competent cell types for use in regenerative medicine. This suggests that through reprogramming it may be possible to directly modify the epigenetic memory of diseased cells and help to normalize their cellular phenotype, while also broadening our understanding of disease pathogenesis.
Molecular Aspects of Medicine 09/2012; · 10.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Platelet-derived growth factor receptor-beta (PDGFRβ) is required for the development of mesenchymal cell types, and plays a diverse role in the function of fibroblasts in tissue homeostasis and regeneration. In this study, we characterized the expression of PDGFRβ in fibroblasts derived from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), and showed that this expression is important for cellular functions such as migration, extracellular matrix production and assembly in 3D self-assembled tissues. To determine potential regulatory regions predictive of expression of PDGFRβ following differentiation from ESCs and iPSCs, we analyzed the DNA methylation status of a region of the PDGFRB promoter that contains multiple CpG sites, before and after differentiation. We demonstrated that this promoter region is extensively demethylated following differentiation, and represents a developmentally regulated, differentially methylated region linked to PDGFRβ expression. Understanding the epigenetic regulation of genes such as PDGFRB, and identifying sites of active DNA demethylation, is essential for future applications of iPSC-derived fibroblasts for regenerative medicine.
[Show abstract][Hide abstract] ABSTRACT: Reprogramming of somatic cells to induced pluripotent stem cells (iPSC) provides an important cell source to derive patient-specific cells for potential therapeutic applications. However, it is not yet clear whether reprogramming through pluripotency allows the production of differentiated cells with improved functional properties that may be beneficial in regenerative therapies. To address this, we compared the production and assembly of extracellular matrix (ECM) by iPSC-derived fibroblasts to that of the parental, dermal fibroblasts (BJ), from which these iPSC were initially reprogrammed, and to fibroblasts differentiated from human embryonic stem cells (hESC). iPSC- and hESC-derived fibroblasts demonstrated stable expression of surface markers characteristic of stromal fibroblasts during prolonged culture and showed an elevated growth potential when compared to the parental BJ fibroblasts. We found that in the presence of L: -ascorbic acid-2-phosphate, iPSC- and hESC-derived fibroblasts increased their expression of collagen genes, secretion of soluble collagen, and extracellular deposition of type I collagen to a significantly greater degree than that seen in the parental BJ fibroblasts. Under culture conditions that enabled the self-assembly of a 3D stromal tissue, iPSC- and hESC-derived fibroblasts generated a well organized, ECM that was enriched in type III collagen. By characterizing the functional properties of iPSC-derived fibroblasts compared to their parental fibroblasts, we demonstrate that these cells represent a promising, alternative source of fibroblasts to advance future regenerative therapies.
[Show abstract][Hide abstract] ABSTRACT: The microenvironment plays a significant role in human cancer progression. However, the role of the tumor microenvironment in the epigenetic control of genes critical to cancer progression remains unclear. As transient E-cadherin expression is central to many stages of neoplasia and is sensitive to regulation by the microenvironment, we have studied if microenvironmental control of E-cadherin expression is linked to transient epigenetic regulation of its promoter, contributing to the unstable and reversible expression of E-cadherin seen during tumor progression. We used 3D, bioengineered human tissue constructs that mimic the complexity of their in vivo counterparts, to show that the tumor microenvironment can direct the re-expression of E-cadherin through the reversal of methylation-mediated silencing of its promoter. This loss of DNA methylation results from the induction of homotypic cell-cell interactions as cells undergo tissue organization. E-cadherin re-expression is associated with multiple epigenetic changes including altered methylation of a small number of CpGs, specific histone modifications, and control of miR-148a expression. These epigenetic changes may drive the plasticity of E-cadherin-mediated adhesion in different tissue microenvironments during tumor cell invasion and metastasis. Thus, we suggest that epigenetic regulation is a mechanism through which tumor cell colonization of metastatic sites occurs as E-cadherin-expressing cells arise from E-cadherin-deficient cells.
Epigenetics: official journal of the DNA Methylation Society 01/2012; 7(1):34-46. · 4.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The repair of central nerves remains a major challenge in regenerative neurobiology. Regenerative guides possessing critical features such as cell adhesion, physical guiding and topical stimulation are needed. To generate such a guide, silk protein materials are prepared using electrospinning. The silk is selected for this study due to its biocompatibility and ability to be electrospun for the formation of aligned biofunctional nanofibers. The addition of Brain Derived Neurotrophic Factor (BDNF), Ciliary Neurotrophic Factor (CNTF) or both to the electrospun fibers enable enhanced function without impact to the structure or the surface morphology. Only a small fraction of the loaded growth factors is released over time allowing the fibers to continue to provide these factors to the cells for extended periods of time. The entrapped factors remain active and available to the cells as rat retinal ganglion cells (RGCs) exhibit longer axonal growth when in contact with the biofunctionalized fibers. Compare to non-functionalized fibers, the growth of neurites increased 2 fold on fibers containing BDNF, 2.5 fold with fibers containing CNTF and by almost 3-fold on fibers containing both factors. The results demonstrate the potential of aligned and functionalized electrospun silk fibers to promote nerve growth in the central nervous system, underlying the great potential of complex biomaterials in neuroregenerative strategies following axotomy and nerve crush traumas.
[Show abstract][Hide abstract] ABSTRACT: Human breast cancers are broadly classified based on their gene-expression profiles into luminal- and basal-type tumors. These two major tumor subtypes express markers corresponding to the major differentiation states of epithelial cells in the breast: luminal (EpCAM(+)) and basal/myoepithelial (CD10(+)). However, there are also rare types of breast cancers, such as metaplastic carcinomas, where tumor cells exhibit features of alternate cell types that no longer resemble breast epithelium. Until now, it has been difficult to identify the cell type(s) in the human breast that gives rise to these various forms of breast cancer. Here we report that transformation of EpCAM(+) epithelial cells results in the formation of common forms of human breast cancer, including estrogen receptor-positive and estrogen receptor-negative tumors with luminal and basal-like characteristics, respectively, whereas transformation of CD10(+) cells results in the development of rare metaplastic tumors reminiscent of the claudin-low subtype. We also demonstrate the existence of CD10(+) breast cells with metaplastic traits that can give rise to skin and epidermal tissues. Furthermore, we show that the development of metaplastic breast cancer is attributable, in part, to the transformation of these metaplastic breast epithelial cells. These findings identify normal cellular precursors to human breast cancers and reveal the existence of a population of cells with epidermal progenitor activity within adult human breast tissues.
Proceedings of the National Academy of Sciences 09/2011; 109(8):2772-7. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Advanced stages of epithelial carcinogenesis involve the loss of intercellular adhesion, but it remains unclear how proteins that regulate alterations in cell-cell and cell-matrix adhesion are deregulated to promote the early stages of cancer development. To address this, a three-dimensional human tissue model that mimics the incipient stages of squamous cell carcinoma (SCC) was used to study how E-cadherin suppression promotes tumor progression in Ras-expressing human keratinocytes. We found that E-cadherin suppression triggered elevated mRNA and protein expression levels of focal adhesion kinase (FAK), and increased FAK and Src activities above the level seen in Ras-expressing E-cadherin-competent keratinocytes. The short hairpin RNA (shRNA)-mediated depletion of FAK and Src restored E-cadherin expression levels by increasing its stability in the membrane, and blocked tumor cell invasion in tissues. Surface transplantation of these tissues to mice resulted in reversion of the tumor phenotype to low-grade tumor islands in contrast to control tissues that manifested an aggressive, high-grade SCC. These findings suggest that the tumor-promoting effect of E-cadherin suppression, a common event in SCC development, is exacerbated by enhanced E-cadherin degradation induced by elevated FAK and Src activities. Furthermore, they imply that targeting FAK or Src in human epithelial cells with neoplastic potential may inhibit the early stages of SCC.
Journal of Investigative Dermatology 06/2011; 131(11):2306-15. · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic ulcerative stomatitis is a condition characterized by chronic, painful oral ulcers, whose pathogenesis is unknown. Patients demonstrate specific IgG autoantibodies against ΔNp63α, an epithelial nuclear transcription factor. The aim of this study was to investigate the role of patient autoantibodies in the disease pathogenesis.
Three-dimensional in vitro tissues consisting of a fully differentiated, multilayer epithelium that mimics its in vivo counterpart were incubated with serum from patients with chronic ulcerative stomatitis.
Our results show a subepithelial detachment of the epithelium at the basement membrane interface, mimicking the oral ulcerations that are seen clinically. Expression of basement membrane proteins Type IV collagen and laminin-5 was unaltered, whereas the expression of α6β4 integrins, hemidesmosome components that attach basal keratinocytes to the basement membrane, was reduced, as determined by immunohistochemistry.
These results give evidence that patient autoantibodies are pathogenic; and support an autoimmune pathogenesis in chronic ulcerative stomatitis.
[Show abstract][Hide abstract] ABSTRACT: Pluripotent, 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.
In 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.
While 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.
Our 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.
[Show abstract][Hide abstract] ABSTRACT: A large body of evidence has shown that stromal cells play a significant role in determining the fate of neighboring tumor cells through the secretion of various cytokines. How cytokine secretion by stromal cells is regulated in this context is poorly understood. In this study, we used a bioengineered human tissue model of skin squamous cell carcinoma progression to reveal that RalA function in dermal fibroblasts is required for tumor progression of neighboring neoplastic keratinocytes. This conclusion is based on the observations that suppression of RalA expression in dermal fibroblasts blocked tumorigenic keratinocytes from invading into the dermal compartment of engineered tissues and suppressed more advanced tumor progression after these tissues were transplanted onto the dorsum of mice. RalA executes this tumor-promoting function of dermal fibroblasts, at least in part, by mediating hepatocyte growth factor (HGF) secretion through its effector proteins, the Sec5 and Exo84 subunits of the exocyst complex. These findings reveal a new level of HGF regulation and highlight the RalA signaling cascade in dermal fibroblasts as a potential anticancer target.
Cancer Research 02/2011; 71(3):758-67. · 9.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It is unknown if epidermal stem cells are maintained during the commercial-scale manufacture of Apligraf, a bilayered living cellular construct (BLCC). To answer this question, we genetically marked replicating keratinocytes, derived from production-scale expansion of working cell banks, in two-dimensional culture with a beta-galactosidase-expressing retrovirus and monitored their fate after incorporation into BLCC and subsequent in vivo transplantation to a nude mouse. Histological analysis of BLCCs showed distinct beta-galactosidase-positive clusters similar to clonal proliferation units visible 8-32 weeks after grafting. Keratinocytes recovered from grafts at week 32 were expanded in vitro in two-dimensional culture, and clonal growth of recovered cells was then compared to the original pregraft population of keratinocytes by colony-forming efficiency (CFE) assays. The CFE of the cells regrown from the grafts was similar to pregraft CFEs (45% and 40%, respectively). Cells regrown from the grafts were then used to produce a second BLCC and generated a well-differentiated epithelium that was histologically similar to pregraft BLCC. These findings provide clear evidence that epidermal stem cells were sustained during the process of large-scale tissue fabrication and that the process of isolation and expansion of cells in BLCC construction retains viable stem cells.
Tissue Engineering Part A 02/2011; 17(3-4):487-93. · 4.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human induced pluripotent stem (hiPS) cells offer a novel source of patient-specific cells for regenerative medicine. However, the biological potential of iPS-derived cells and their similarities to cells differentiated from human embryonic stem (hES) cells remain unclear. We derived fibroblast-like cells from two hiPS cell lines and show that their phenotypic properties and patterns of DNA methylation were similar to that of mature fibroblasts and to fibroblasts derived from hES cells. iPS-derived fibroblasts (iPDK) and their hES-derived counterparts (EDK) showed similar cell morphology throughout differentiation, and patterns of gene expression and cell surface markers were characteristic of mature fibroblasts. Array-based methylation analysis was performed for EDK, iPDK and their parental hES and iPS cell lines, and hierarchical clustering revealed that EDK and iPDK had closely-related methylation profiles. DNA methylation analysis of promoter regions associated with extracellular matrix (ECM)-production (COL1A1) by iPS- and hESC-derived fibroblasts and fibroblast lineage commitment (PDGFRβ), revealed promoter demethylation linked to their expression, and patterns of transcription and methylation of genes related to the functional properties of mature stromal cells were seen in both hiPS- and hES-derived fibroblasts. iPDK cells also showed functional properties analogous to those of hES-derived and mature fibroblasts, as seen by their capacity to direct the morphogenesis of engineered human skin equivalents. Characterization of the functional behavior of ES- and iPS-derived fibroblasts in engineered 3D tissues demonstrates the utility of this tissue platform to predict the capacity of iPS-derived cells before their therapeutic application.
PLoS ONE 01/2011; 6(2):e17128. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The co-evolution of tumors and their microenvironment involves bidirectional communication between tumor cells and tumor-associated stroma. Various cell types are present in tumor-associated stroma, of which fibroblasts are the most abundant. The Rac exchange factor Tiam1 is implicated in multiple signaling pathways in epithelial tumor cells and lack of Tiam1 in tumor cells retards tumor growth in Tiam1 knockout mouse models. Conversely, tumors arising in Tiam1 knockout mice have increased invasiveness. We have investigated the role of Tiam1 in tumor-associated fibroblasts as a modulator of tumor cell invasion and metastasis, using retroviral delivery of short hairpin RNA to suppress Tiam1 levels in three different experimental models. In spheroid co-culture of mammary epithelial cells and fibroblasts, Tiam1 silencing in fibroblasts led to increased epithelial cell outgrowth into matrix. In tissue-engineered human skin, Tiam1 silencing in dermal fibroblasts led to increased invasiveness of epidermal keratinocytes with pre-malignant features. In a model of human breast cancer in mice, co-implantation of mammary fibroblasts inhibited tumor invasion and metastasis, which was reversed by Tiam1 silencing in co-injected fibroblasts. These results suggest that stromal Tiam1 may have a role in modulating the effects of the tumor microenvironment on malignant cell invasion and metastasis. This suggests a set of pathways for further investigation, with implications for future therapeutic targets.
[Show abstract][Hide abstract] ABSTRACT: This report assesses the ability of intrinsic two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) imaging to characterize features associated with the motility and invasive potential of epithelial tumor cells engineered in tissues. Distinct patterns of organization are found both within the cells and the matrix that depend on the adhesive properties of the cells as well as factors attributed to adjacent fibroblasts. TPEF images are analyzed using automated algorithms that reveal unique features in subcellular organization and cell spacing that correlate with the invasive potential. We expect that such features have significant diagnostic potential for basic in vitro studies that aim to improve our understanding of cancer development or response to treatments, and, ultimately can be applied in prognostic evaluation.
[Show abstract][Hide abstract] ABSTRACT: The alpha6beta4 integrin plays a significant role in tumor growth, angiogenesis and metastasis through modulation of growth factor signaling, and is a potentially important therapeutic target. However, alpha6beta4-mediated cell-matrix adhesion is critical in normal keratinocyte attachment, signaling and anchorage to the basement membrane through its interaction with laminin-5, raising potential risks for targeted therapy. Bioengineered Human Skin Equivalent (HSE), which have been shown to mimic their normal and wounded counterparts, have been used here to investigate the consequences of targeting beta4 to establish toxic effects on normal tissue homeostasis and epithelial wound repair. We tested two antibodies directed to different beta4 epitopes, one adhesion-blocking (ASC-8) and one non-adhesion blocking (ASC-3), and determined that these antibodies were appropriately localized to the basal surface of keratinocytes at the basement membrane interface where beta4 is expressed. While normal tissue architecture was not altered, ASC-8 induced a sub-basal split at the basement membrane in non-wounded tissue. In addition, wound closure was significantly inhibited by ASC-8, but not by ASC-3, as the epithelial tongue only covered 40 percent of the wound area at 120 hours post-wounding. These results demonstrate beta4 adhesion-blocking antibodies may have adverse effects on normal tissue, whereas antibodies directed to other epitopes may provide safer alternatives for therapy. Taken together, we conclude that these three-dimensional tissue models provide a biologically relevant platform to identify toxic effects induced by candidate therapeutics, which will allow generation of findings that are more predictive of in vivo responses early in the drug development process.
PLoS ONE 01/2010; 5(5):e10528. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human skin equivalents (HSEs) are in vitro tissues in which a fully differentiated, stratified squamous epithelium is grown at an air-liquid interface on a Type I collagen gel harboring human dermal fibroblasts. HSEs now provide experimental human tissue models to study factors that direct re-epithelialization and epithelial-mesenchymal cross-talk following wounding. This chapter describes the fabrication of HSEs from human keratinocytes and fibroblasts and how HSEs can be modified to characterize the response of the human epithelium during wound repair. The protocols outlined first describe techniques for the generation of human tissues that closely approximate the architectural features, differentiation, and growth of human skin. This will be followed by a description of a protocol that enables HSEs to be adapted to monitor their response following wounding. These engineered human tissues provide powerful tools to study biological process in tissues that mimic the healing of human skin and of the epithelial tissue.