An in vivo Like Micro-Carcinoma Model.

We here present a novel micro-system which allows to reconstitute an in vivo lung carcinoma where the various constituting epithelial and/or stromal structural and/or cellular components can be incorporated at will. In contrast to various "organs on a chip" the model is based on the observation that in nature, epithelial cells are always supported by a connective tissue or stroma. The model is based on acellular micro-scaffolds of microscopic dimensions which enable seeded cells to obtain gases and nutrients through diffusion thus avoiding the need for vascularization. As a proof of concept, we show that in this model, Calu-3 cells can form a well-organized, continuous, polarized, one-layer epithelium lining the stromal derived alveolar cavities, and express a different pattern of tumor-related genes than when grown as standard monolayer cultures on plastic culture dishes. To our knowledge, this model, introduces for the first time a system where the function of carcinogenic cells can be tested in vitro in an environment that closely mimics the natural in vivo situation.

Maternal and zygotic Zfp57 modulate NOTCH signaling in cardiac development.

Zfp57 is a maternal-zygotic effect gene that maintains genomic imprinting. Here we report that Zfp57 mutants exhibited a variety of cardiac defects including atrial septal defect (ASD), ventricular septal defect (VSD), thin myocardium, and reduced trabeculation. Zfp57 maternal-zygotic mutant embryos displayed more severe phenotypes with higher penetrance than the zygotic ones. Cardiac progenitor cells exhibited proliferation and differentiation defects in Zfp57 mutants. ZFP57 is a master regulator of genomic imprinting, so the DNA methylation imprint was lost in embryonic heart without ZFP57. Interestingly, the presence of imprinted DLK1, a target of ZFP57, correlated with NOTCH1 activation in cardiac cells. These results suggest that ZFP57 may modulate NOTCH signaling during cardiac development. Indeed, loss of ZFP57 caused loss of NOTCH1 activation in embryonic heart with more severe loss observed in the maternal-zygotic mutant. Maternal and zygotic functions of Zfp57 appear to play redundant roles in NOTCH1 activation and cardiomyocyte differentiation. This serves as an example of a maternal effect that can influence mammalian organ development. It also links genomic imprinting to NOTCH signaling and particular developmental functions.

Strategies for Oral Mucosal Repair by Engineering 3D Tissues with Pluripotent Stem Cells.

Significance: Human-induced pluripotent stem cells (iPSC) can be differentiated into patient-specific cells with a wide spectrum of cellular phenotypes and offer an alternative source of autologous cells for therapeutic use. Recent studies have shown that iPSC-derived fibroblasts display enhanced cellular functions suggesting that iPSC may eventually become an important source of stem cells for regenerative therapies. Recent Advances: The discovery of approaches to reprogram somatic cells into pluripotent cells opens exciting avenues for their use in personalized, regenerative therapies. The controlled differentiation of functional cell types from iPSC provides a replenishing source of fibroblasts. There is intriguing evidence that iPSC reprogramming and subsequent differentiation to fibroblast lineages may improve cellular functional properties. Augmenting the biological potency of iPSC-derived fibroblasts may enable the development of novel, personalized stem cell therapies to treat oral disease. Critical Issues: Numerous questions need to be addressed before iPSC-derived cells can be used as a practical oral therapy. This will include understanding why iPSC-derived cells are predisposed towards differentiation pathways along lineages related to their cell of origin, screening iPSC-derived cells to ensure their safety and phenotypic stability and developing engineered, three-dimensional tissue models to optimize their function and efficacy for future therapeutic transplantation. Future Directions: Future research will need to address how to develop efficient methods to deliver and integrate iPSC-derived fibroblasts into the oral mucosa. This will require an improved understanding of how to harness their biological potency for regenerative therapies that are specifically targeted to the oral mucosa.

Genomic imprinting is variably lost during reprogramming of mouse iPS cells.

Derivation of induced pluripotent stem (iPS) cells is mainly an epigenetic reprogramming process. It is still quite controversial how genomic imprinting is reprogrammed in iPS cells. Thus, we derived multiple iPS clones from genetically identical mouse somatic cells. We found that parentally inherited imprint was variably lost among these iPS clones. Concurrent with the loss of DNA methylation imprint at the corresponding Snrpn and Peg3 imprinted regions, parental origin-specific expression of the Snrpn and Zim1 imprinted genes was also lost in these iPS clones. This loss of parental genomic imprinting in iPS cells was likely caused by the reprogramming process during iPS cell derivation because extended culture of iPS cells did not lead to significant increase in the loss of genomic imprinting. Intriguingly, one to several paternal chromosomes appeared to have acquired de novo methylation at the Snrpn and Zac1 imprinted regions in a high percentage of iPS clones. These results might have some implications for future therapeutic applications of iPS cells. Since DNA methylation imprint can be completely erased in some iPS clones at multiple imprinted regions, iPS cell reprogramming may also be employed to dissect the underlying mechanisms of erasure, reacquisition and maintenance of genomic imprinting in mammals.

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, PDGFRß, 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.

PDGFRß expression and function in fibroblasts derived from pluripotent cells is linked to DNA demethylation.

Platelet-derived growth factor receptor-beta (PDGFRß) 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 PDGFRß 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 PDGFRß 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 PDGFRß 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.

iPSC-derived fibroblasts demonstrate augmented production and assembly of extracellular matrix proteins.

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.

The 3D tissue microenvironment modulates DNA methylation and E-cadherin expression in squamous cell carcinoma.

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.

Suppression of E-cadherin function drives the early stages of Ras-induced squamous cell carcinoma through upregulation of FAK and Src.

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.

Organ-specific scaffolds for in vitro expansion, differentiation, and organization of primary lung cells.

In light of the increasing need for differentiated primary cells for cell therapy and the rapid dedifferentiation occurring during standard in vitro cultivation techniques, there is an urgent need for developing three-dimensional in vitro systems in which expanded cells display in vivo-like differentiated phenotypes. It is becoming clear that the natural microenvironment provides the optimal conditions for achieving this aim. To this end, we prepared natural decellularized scaffolds of microscopic dimensions that would allow appropriate diffusion of gases and nutrients to all seeded cells. Scaffolds from either the lung or the liver were analyzed for their ability to support growth and differentiation of progenitor alveolar cells and hepatocytes. We observed that progenitor alveolar cells that have been expanded on plastic culture and thus dedifferentiated grew within the lung-derived scaffolds into highly organized structures and regained differentiation markers classical for type I and type II alveolar cells. The cells generated proper alveolar structures, and only 15%-30% of them secreted surfactant proteins in a localized manner for extended periods. Vice versa, liver-derived scaffolds supported the differentiation state of primary hepatocytes. We further demonstrate that the natural scaffolds are organ specific, that is, only cells derived from the same organ become properly differentiated. A proteomic analysis shows significant different composition of lung and liver scaffolds, for example, decorin, thrombospondin 1, vimentin, and various laminin isoforms are especially enriched in the lung. Altogether, our data demonstrate that complex interactions between the seeded cells and a highly organized, organ-specific stroma are required for proper localized cell differentiation. Thus, our novel in vitro culture system can be used for ex vivo differentiation and organization of expanded primary cells.

Epigenetic and phenotypic profile of fibroblasts derived from induced pluripotent stem cells.

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 (PDGFRß), 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.

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

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Epidermal stem cells are preserved during commercial-scale manufacture of a bilayered, living cellular construct (Apligraf®).

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.

RalA function in dermal fibroblasts is required for the progression of squamous cell carcinoma of the skin.

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.

Three-dimensional human tissue models of wounded skin.

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.

Three-dimensional epithelial tissues generated from human embryonic stem cells.

The use of pluripotent human embryonic stem (hES) cells for tissue engineering may provide advantages over traditional sources of progenitor cells because of their ability to give rise to multiple cell types and their unlimited expansion potential. We derived cell populations with properties of ectodermal and mesenchymal cells in two-dimensional culture and incorporated these divergent cell populations into three-dimensional (3D) epithelial tissues. When grown in specific media and substrate conditions, two-dimensional cultures were enriched in cells (EDK1) with mesenchymal morphology and surface markers. Cells with a distinct epithelial morphology (HDE1) that expressed cytokeratin 12 and beta-catenin at cell junctions became the predominant cell type when EDK1 were grown on surfaces enriched in keratinocyte-derived extracellular matrix proteins. When these cells were incorporated into the stromal and epithelial tissue compartments of 3D tissues, they generated multilayer epithelia similar to those generated with foreskin-derived epithelium and fibroblasts. Three-dimensional tissues demonstrated stromal cells with morphologic features of mature fibroblasts, type IV collagen deposition in the basement membrane, and a stratified epithelium that expressed cytokeratin 12. By deriving two distinct cell lineages from a common hES cell source to fabricate complex tissues, it is possible to explore environmental cues that will direct hES-derived cells toward optimal tissue form and function.

E-cadherin suppression directs cytoskeletal rearrangement and intraepithelial tumor cell migration in 3D human skin equivalents.

The link between loss of cell-cell adhesion, the activation of cell migration, and the behavior of intraepithelial (IE) tumor cells during the early stages of skin cancer progression is not well understood. The current study characterized the migratory behavior of a squamous cell carcinoma cell line (HaCaT-II-4) upon E-cadherin suppression in both 2D, monolayer cultures and within human skin equivalents that mimic premalignant disease. The migratory behavior of tumor cells was first analyzed in 3D tissue context by developing a model that mimics transepithelial tumor cell migration. We show that loss of cell adhesion enabled migration of single, IE tumor cells between normal keratinocytes as a prerequisite for stromal invasion. To further understand this migratory behavior, E-cadherin-deficient cells were analyzed in 2D, monolayer cultures and displayed altered cytoarchitecture and enhanced membrane protrusive activity that was associated with circumferential actin organization and induction of the nonmuscle, beta actin isoform. These features were associated with increased motility and random, individual cell migration in response to scrape-wounding. Thus, loss of E-cadherin-mediated adhesion led to the acquisition of phenotypic properties that augmented cell motility and directed the transition from the precancer to cancer in skin-like tissues.

Denatured collagen modulates the phenotype of normal and wounded human skin equivalents.

Epithelial-mesenchymal interactions are known to play an important role in modulating homeostasis and repair. However, it remains unclear how the composition of the extracellular matrix may regulate the ability of dermal fibroblasts to engage in such cross talk. To address this, we studied how fibroblast phenotype was linked to the behavior of normal and wounded human skin equivalents (HSE) by comparing human dermal fibroblasts (HDF) incorporated into the three-dimensional tissues to those extensively cultivated in two-dimensional (2D) monolayer culture on denatured collagen (DC) matrix, native collagen, or tissue culture plastic before incorporation into HSEs. We first established that prolonged passage and growth of HDF on DC increased their migratory potential in a 2D monolayer culture. When HDF variants were grown in HSEs, we found that extended passage on DC and incorporation of DC directly into the collagen gel enhanced proliferation of both HDF and basal keratinocytes in HSEs. By adapting HSEs to study wound reepithelialization, we found that the extended passage of HDF on DC accelerated the rate of wound healing by 38%. Thus, extensive ex vivo expansion on DC was able to modify the phenotype of skin fibroblasts by augmenting their reparative properties in skin-like HSEs.

Skin-derived microorgan autotransplantation as a novel approach for therapeutic angiogenesis.

Despite promising preclinical results, transient single-factor-based therapeutic angiogenesis has shown no definitive benefits in clinical trials. The use of skin-derived microorgans (SMOs), capable of sustained expression of angiogenic factors and sustained viability with their cellular and extracellular elements, constitutes an attractive alternative. We sought to evaluate the efficacy of SMO implantation in a porcine model of chronic myocardial ischemia. Eighteen pigs underwent placement of an ameroid constrictor on the proximal circumflex artery. Three weeks later, split-thickness skin biopsies were harvested and pigs were randomized to lateral wall implantation of either 8 or 16 SMOs or blank injections. The procedure was safe and resulted in no adverse events. Three weeks after treatment, SMO implantation resulted in significant improvement of lateral wall perfusion during pacing, assessed by isotope-labeled microspheres [post- vs. pretreatment ratios of lateral/anterior wall blood flow were 1.31 +/- 0.09 (SMOs) and 1.04 +/- 0.06 (controls); P = 0.03]. No significant difference in angiographic scores was observed. Microvascular relaxation in response to VEGF was impaired in the ischemic territory of the control group but returned to normal after SMO implantation, indicating restoration of endothelial function. Molecular studies showed significant increases in VEGF and CD31 expression in the ischemic area of treated animals. Morphometric analysis showed increased neovascularization with SMO treatment. Autotransplantation of SMOs constitutes a novel approach for safe and effective therapeutic angiogenesis with improvement in perfusion, normalization of microvascular reactivity, and increased expression of VEGF and CD31.

E-cadherin loss promotes the initiation of squamous cell carcinoma invasion through modulation of integrin-mediated adhesion.

Much remains to be learned about how cell-cell and cell-matrix interactions are coordinated to influence the earliest development of neoplasia. We used novel 3D human tissue reconstructs that mimic premalignant disease in normal epidermis, to directly investigate how loss of E-cadherin function directs conversion to malignant disease. We used a genetically tagged variant of Ha-Ras-transformed human keratinocytes (II-4) expressing dominant-interfering E-cadherin fusion protein (H-2k(d)-Ecad). These cells were admixed with normal human keratinocytes and tumor cell fate was monitored in 3D reconstructed epidermis upon transplantation to immunodeficient mice. Tumor initiation was suppressed in tissues harboring control- and mock-infected II-4 cells that lost contact with the stromal interface. By contrast, H-2k(d)-Ecad-expressing cells persisted at this interface, thus enabling incipient tumor cell invasion upon in vivo transplantation. Loss of intercellular adhesion was linked to elevated cell surface expression of alpha2, alpha3 and beta1 integrins and increased adhesion to laminin-1 and Types I and IV collagen that was blocked with beta1-integrin antibodies, suggesting that invasion was linked to initial II-4 cell attachment at the stromal interface. Collectively, these results outline a novel aspect to loss of E-cadherin function that is linked to the mutually interdependent regulation of cell-cell and cell-matrix adhesion and has significant consequences for the conversion of premalignancy to cancer.