[Show abstract][Hide abstract] ABSTRACT: Diabetes Mellitus (DM) is a chronic, severe disease rapidly increasing in incidence and prevalence and is associated with numerous complications. Patients with DM are at high risk of developing diabetic foot ulcers (DFU) that often lead to lower limb amputations, long term disability, and a shortened lifespan. Despite this, the effects of DM on human foot skin biology are largely unknown. Thus, the focus of this study was to determine whether DM changes foot skin biology predisposing it for healing impairment and development of DFU. Foot skin samples were collected from 20 patients receiving corrective foot surgery and, using a combination of multiple molecular and cellular approaches, we performed comparative analyses of non-ulcerated non-neuropathic diabetic foot skin (DFS) and healthy non-diabetic foot skin (NFS). MicroRNA (miR) profiling of laser captured epidermis and primary dermal fibroblasts from both DFS and NFS samples identified 5 miRs de-regulated in the epidermis of DFS though none reached statistical significance. MiR-31-5p and miR-31-3p were most profoundly induced. Although none were significantly regulated in diabetic fibroblasts, miR-29c-3p showed a trend of up-regulation, which was confirmed by qPCR in a prospective set of 20 skin samples. Gene expression profiling of full thickness biopsies identified 36 de-regulated genes in DFS (>2 fold-change, unadjusted p-value ≤ 0.05). Of this group, three out of seven tested genes were confirmed by qPCR: SERPINB3 was up-regulated whereas OR2A4 and LGR5 were down-regulated in DFS. However no morphological differences in histology, collagen deposition, and number of blood vessels or lymphocytes were found. No difference in proliferative capacity was observed by quantification of Ki67 positive cells in epidermis. These findings suggest DM causes only subtle changes to foot skin. Since morphology, mRNA and miR levels were not affected in a major way, additional factors, such as neuropathy, vascular complications, or duration of DM, may further compromise tissue's healing ability leading to development of DFUs.
PLoS ONE 08/2015; 10(8):e0137133. DOI:10.1371/journal.pone.0137133 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Diabetic foot ulcers (DFU) are a major, debilitating complication of diabetes mellitus. Unfortunately, many DFUs are refractory to existing treatments and frequently lead to amputation. The development of more effective therapies has been hampered by the lack of predictive in vitro methods to investigate the mechanisms underlying impaired healing. To address this need for realistic wound healing models, we established patient-derived fibroblasts from DFUs and site-matched controls and used them to construct three-dimensional (3D) models of chronic wound healing. Incorporation of DFU-derived fibroblasts into these models accurately recapitulated the following key aspects of chronic ulcers: reduced stimulation of angiogenesis, increased keratinocyte proliferation, decreased re-epithelialization, and impaired extracellular matrix (ECM) deposition. In addition to reflecting clinical attributes of DFUs, the wound healing potential of DFU fibroblasts demonstrated in this suite of models correlated with in vivo wound closure in mice. Thus, the reported panel of 3D DFU models provides a more biologically-relevant platform for elucidating the cell-cell and cell-matrix related mechanisms responsible for chronic wound pathogenesis and may improve translation of in vitro findings into efficacious clinical applications.
Tissue Engineering Part C Methods 10/2014; 21(5). DOI:10.1089/ten.TEC.2014.0414 · 4.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Diabetic foot ulcers (DFUs) are a serious complication of diabetes. Previous exposure to hyperglycemic conditions accelerates a decline in cellular function through metabolic memory despite normalization of glycemic control. Persistent, hyperglycemia-induced epigenetic patterns are considered a central mechanism that activates metabolic memory; however, this has not been investigated in patient-derived fibroblasts from DFUs. We generated a cohort of patient-derived lines from DFU fibroblasts (DFUF), and site- and age-matched diabetic foot fibroblasts (DFF) and non-diabetic foot fibroblasts (NFF) to investigate global and genome-wide DNA methylation patterns using liquid chromatography/mass spectrometry and the Illumina Infinium HumanMethylation450K array. DFFs and DFUFs demonstrated significantly lower global DNA methylation compared to NFFs (p = 0.03). Hierarchical clustering of differentially methylated probes (DMPs, p = 0.05) showed that DFFs and DFUFs cluster together and separately from NFFs. Twenty-five percent of the same probes were identified as DMPs when individually comparing DFF and DFUF to NFF. Functional annotation identified enrichment of DMPs associated with genes critical to wound repair, including angiogenesis (p = 0.07) and extracellular matrix assembly (p = 0.035). Identification of sustained DNA methylation patterns in patient-derived fibroblasts after prolonged passage in normoglycemic conditions demonstrates persistent metabolic memory. These findings suggest that epigenetic-related metabolic memory may also underlie differences in wound healing phenotypes and can potentially identify therapeutic targets.
Epigenetics: official journal of the DNA Methylation Society 10/2014; 9(10):1339-49. DOI:10.4161/15592294.2014.967584 · 4.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective: Signaling between fibroblasts and keratinocytes is essential for proper wound healing but is thought to be dysregulated in the diabetic foot ulcer (DFU) environment. An engineered 3D human skin model was used to examine differences in the ability of DFU fibroblasts to support the differentiation and proliferation of human keratinocytes. The long-term goal is to develop improved therapeutic approaches for DFU repair.
Method: Fibroblasts were harvested from diabetic foot ulcers (DFU) and non-ulcerated diabetic feet (DFF) from Type II diabetic patients as well as from site-matched non-diabetic feet (NFF). DFU, DFF or NFF fibroblasts were embedded in collagen gels, normal human keratinocytes (NHKs) were seeded on them and tissues grown at air-liquid interface. Tissues were pulsed with BrdU for 6 hours, formalin-fixed and paraffin embedded. Sections of these tissues were stained by H&E to visualize tissue development and for BrdU to identify proliferating cells. The number of BrdU-positive, basal keratinocytes were counted and expressed as a percentage of the total number of basal keratinocytes per 20x image.
Result: H&E staining showed that all three fibroblast types (DFU, DFF and NFF) were able to support keratinocyte development into a fully differentiated epidermal layer. However, keratinocytes grown on DFU fibroblasts were more proliferative than those grown on DFF fibroblasts (p < .05). The number of proliferating keratinocytes grown on NFF fibroblasts was less than DFUs and greater than DFFs, although not statistically significant.
Conclusion: Our findings indicate that DFU fibroblasts stimulated increased keratinocyte proliferation in 3D tissues when compared with DFF fibroblasts, suggesting that the ulcer environment may direct paracrine signaling between fibroblasts and keratinocytes. This suggests a possible mechanism for the hyperproliferative keratinocytes found in the callus surrounding DFUs. This marks an important step towards understanding mechanisms that will help develop future regenerative therapies to treat oral and cutaneous wounds.
[Show abstract][Hide abstract] ABSTRACT: Objective: Altered growth factor-mediated communication between fibroblasts and other cell types in chronic wounds are linked to a failure to heal. The aim of our study was to characterize growth factor secretion in 3D tissues harboring chronic wound fibroblasts grown with and without keratinocytes. The long-term objective of this study is to develop stem cell therapies that can effectively treat non-healing, diabetic foot ulcers (DFU).
Method: Biopsies were obtained from DFUs and site-matched normal skin (NFF). Fibroblasts were isolated and 3D, skin-like tissues were generated. Tissues grown with fibroblasts in collagen gel were compared to those grown with fibroblasts and keratinocytes (NHKs). Supernatants were collected and growth factor secretion was measured from tissues on a per cell basis.
Result: Both NFF and DFU fibroblasts increased in number when co-cultured with NHKs compared to fibroblasts alone. NFFs were more responsive than DFU in secretion of HGF and IL-8. DFUs showed a 8 to 10 fold increase in IL-8 versus a 20 fold increase in NFF. Similarly, HGF showed a 6 to 10 fold increase in NFF. In contrast, IL-6 did not show a conclusive pattern while KGF secretion was below detection range. In general, growth factor secretion was increased in tissues harboring NFF when compared to those harboring NFF fibroblasts.
Conclusion: This study determined that DFU fibroblasts were deficient in secretion of growth factors in 3D skin like tissues. This helps explain why these cells are repair-deficient. Our next step will be to reprogram these cells to induced pluripotent stem cells, in the hope that this may improve healing through enhanced growth factor signaling.
[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 12/2013; 8(12):e83755. DOI:10.1371/journal.pone.0083755 · 3.23 Impact Factor
[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. DOI:10.1016/j.jphotobiol.2013.07.006 · 2.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Stem cell biology focuses on an understanding of a full spectrum of cell behaviors from the stem cell state to the fully differentiated condition. It examines the dynamics of cell physiology as to how stem cells maintain their stemness and give rise to specialized cells. Furthermore, this field has become a fundamental underpinning for the establishment of regenerative medicine and cell-based therapies. The differentiation approach to induced pluripotent stem cells (iPSCs) provides a more controlled environment to direct cells to specific lineage fates and to monitor cell differentiation toward a desired germ layer or cell type. While pluripotent cell types have been shown to be remarkably resistant to chromosomal abnormalities, both embryonic stem cells (ESCs) and iPSCs have been shown to acquire aberrant karyotypes following prolonged culture.
Stem Cells in Craniofacial Development and Regeneration, 03/2013: pages 204-222; , ISBN: 9781118279236
[Show abstract][Hide abstract] ABSTRACT: Objectives: Diabetic foot ulceration impairs quality of life and leads to prolonged hospitalizations, demonstrating the need to develop novel sources of repair-competent, autologous cells. Our lab is developing a novel therapeutic approach, based on induced pluripotent stem cell (iPSC) technologies, whose goal is to reverse chronic wound fibroblasts from a non-healing to a healing phenotype. As a first step towards this goal, we characterized the gene expression profile of fibroblasts cultured from ulcers and normal skin from diabetic patients.
Methods: Fibroblasts were harvested from non-healing ulcers or normal skin from the feet of diabetic patients (DFU 1, 3, 6) as well as from normal plantar skin and palatal mucosa from non-diabetic patients. Microarray analysis was performed on diabetic fibroblasts and control oral fibroblasts, to identify genes from diabetic patients that were altered. Microarrays were then used to select RNA targets to compare plantar skin vs. oral fibroblasts from non-diabetic patients by RT-PCR to establish how site-specific expression patterns could compare to those seen in diabetic ulcer fibroblasts.
Results: Genes selected from the microarray relevant to wound repair included upregulated LOXL4, SFRP1, and IGF2 and downregulated CDH6. These microarray results were confirmed when DFU 1, 3, 6 were compared to oral fibroblast controls by RT-PCR analysis. However, DFU cells showed different patterns of gene expression when compared to plantar skin control fibroblasts, suggesting the existence of site-specific patterns of gene expression.
Conclusions: Site-matched, plantar fibroblasts displayed different patterns of gene expression compared to non-site matched, oral fibroblasts. Therefore, comparisons between DFU fibroblasts and those from different sites, such as the oral cavity, are of limited use to predict patterns of expression. These studies lay the groundwork for ongoing studies whose goal is to improve the repair potency of DFU fibroblasts following their reprogramming to iPSC and subsequent differentiation to repair-competent fibroblasts.
IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
[Show abstract][Hide abstract] ABSTRACT: Objective: Efforts to better understand interactions between dental epithelium and mesenchyme during odontogenesis have focused on recreating the three dimensional (3D) tissue microenvironments that can mimic this process in vitro. We have developed a 3D co-culture system using epithelial spheroids to study cellular morphology and patterns of gene expression characteristic of epithelial-mesenchymal cross-talk.
Methods: Human epithelial cells (HEP) were cultured on low adhesion plates to generate 3D spheroids that were then plated on confluent monolayers (2D) of dental pulp stem cells (DPSC19 or DPSC23) or human foreskin fibroblasts (HFF) (3D-2D approach). HEP cells (2D) were also seeded as single cells onto confluent layers of DPSCs or HFFs (2D-2D approach). Co-cultures were grown using osteogenic medium for up to three weeks. RNA extracted from these cells were analyzed by RT-PCR or fixed with 4% PFA for immunohistochemical staining.
Results: Cultures grown as spheroids in 3D-2D co-cultures demonstrated epithelial cells with a distinct border while the 2D-2D cultures demonstrated single cell growth when cultured on confluent mesenchymal monolayers. After 3 weeks, calcified structures were detected in 3D-2D cultures when grown on DPSC but not on control HFF cells or in 2D-2D cultures. RT-PCR analysis of genes expressed during early and late odontogenesis are currently underway.
Conclusion: We found that generation of epithelial spheroids with 3D architecture induced differentiation when grown on monolayers of DPSCs, but not when grown as a monolayer in a 2D co-culture system. We expect that ongoing analysis of these 3D co-culture systems will help improve our understanding of factors essential to stem cell commitment to odontogenic pathways that will be broadly applicable to eventual translation for clinical applications in regenerative therapies.
IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
[Show abstract][Hide abstract] ABSTRACT: Objective: Loss of the tumor suppressor E-cadherin is a hallmark of the advanced stages of squamous cell carcinoma (SCC). Nevertheless, how loss of cell-cell contacts promotes cell invasion in the onset of SCC is only partially understood. Disabled-2 (Dab2) is a highly expressed adaptor protein that is involved in endocytosis of surface proteins, such as beta1 integrins. We recently found that E-cadherin loss is associated with Dab2 down-regulation and beta1 integrins up-regulation in SCC cells. Here we studied the correlation between E-cadherin and the expression of key endocytic proteins in SCC, cells and determined the consequence of Dab2 silencing on tumor cell behavior.
Method: Dab2, Eps15, Intersectin, EEA1, and Rab5 protein levels were determined in lysates of E-cadherin competent (II-4) and E-cadherin suppressed (II-4-Ecad-) skin SCC cells by western blot (WB) analysis. Cultures were transfected with si-Dab2-RNA and imaged to analyze morphological changes. Dab2 depletion was confirmed by WB analysis.
Result: II-4-Ecad- cells demonstrated reduced expression of Dab2, Intersectin, EEA1, and Rab5 proteins in comparison to E-cadherin competent II-4 cells. Transfection of both cell types with si-Dab2 resulted in effective reduction of Dab2, Eps15 and Rab5 in II-4-Ecad- cells, while it caused an opposing increase of Eps15 in II-4 cells. Moreover, Dab2 depletion had a dramatic effect on E-cadherin competent II-4 cells; it reverted the phenotype of well-organized colonies and led to cell separation from each other -- a characteristic of E-cadherin suppressed II-4-Ecad- cell morphology.
Conclusion: Our study revealed that Dab2 plays a key role in the behavior of skin SCC cells, and that its expression correlates with E-cadherin levels. These findings imply that Dab2 may be a potential biomarker for other epithelial SCC, such as oral cancer, and may represent a novel target for SCC therapy.
IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
[Show abstract][Hide abstract] ABSTRACT: Objectives: Advanced stages of Squamous Cell Carcinoma (SCC) are linked to loss of E-cadherin-mediated cell-cell contacts; however, the mechanisms that direct the transition of premalignant lesions to carcinomas are still elusive. Disabled-2 (Dab2) protein is a candidate to regulate this transition by controlling the endocytosis of surface proteins involved in cell adhesion and proliferation. We have shown that Dab2 is down-regulated in human bioengineered epithelial tissues and tumors harboring E-cadherin-suppressed SCC cells. Here we investigated the effect of E-cadherin loss on the localization and expression of key proteins in Dab2-mediated endocytosis.
Methods: The cellular distribution and expression of endocytic proteins in fixed human E-cadherin-competent- and E-cadherin-suppressed SCC cell cultures was determined by immunofluorescence analysis. Localization of acidified vesicular compartments was visualized by Lysotracker Red-fluorescent probe in live tumor cells.
Results: We first examined the expression and localization of critical proteins involved in endocytosis of surface proteins, prior to integration with lysosomal compartments. Dab2, Rab5 and Rab11 showed a weak perinuclear staining in E-cadherin-suppressed SCC cells, and intense staining throughout the cytoplasm of E-cadherin-competent cells. We next analyzed the distribution of acidified vesicular compartments in live cell cultures. These compartments were found in proximity to the nuclei of E-cadherin-suppressed cells, whereas they spread from the plasma membrane to the perinuclear area in E-cadherin-competent cells.
Conclusions: This study provides new insights into the consequences of E-cadherin loss in epithelial cells with malignant potential. It indicates that the tumor-promoting effect of E-cadherin suppression is associated with altered Dab2-mediated endocytosis and trafficking of acidified compartments that can affect, at least in part, tumor cell adhesion, motility and invasion. Thus, Dab2 may be a biomarker for SCC development in the human skin and in life-threatening epithelial cancers, such as oral cancer.
IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
[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; 34(4). DOI:10.1016/j.mam.2012.08.002 · 10.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objectives: Loss of E-cadherin-mediated cell-cell contact is linked to advanced stages of Squamous Cell Carcinoma (SCC), however, the mechanisms that regulate the transition of a premalignant lesion to SCC are still elusive. Disabled-2 (Dab-2) is a candidate to regulate this transition by controlling the trafficking of surface proteins that are involved in cell adhesion and growth factor signaling. Furthermore, decreased Dab-2 expression is found in prostate, lung, breast and nasopharyngeal carcinomas. Here we investigated the effect of E-cadherin loss on Dab-2 expression in SCC cells in three-dimensional (3D) tissue models of skin and oral precancer.
Methods: Bioengineered human skin-equivalents were generated by seeding E-cadherin-suppressed or E-cadherin-competent SCC cells onto fibroblasts-populated collagen gels. Dab-2 expression levels were determined by immunoblotting. Tissue morphology and Dab-2 expression were analyzed by H&E, immunohistochemistry and immunofluorescence staining of tissues and SCC tumors generated following surface transplantation of these tissues to mice.
Results: In 2D cultures and 3D tissues, Dab-2 expression is decreased in E-cadherin-suppressed cells when compared to E-cadherin-competent cells. In SCC tumors, Dab-2 expression is closely associated with the degree of differentiation of these tumors. Low-grade tumors harboring E-cadherin-competent cells demonstrated Dab-2-positive, well differentiated sheets of cells. In contrast, high-grade tumors harboring E-cadherin-suppressed cells demonstrated Dab-2-negative, poorly differentiated tumor cell sheets and invasive cell clusters in the mouse stroma.
Conclusions: This study provides new insights into the functional consequences of altered E-cadherin-mediated cell-cell adhesion in the early stages of SCC development. It suggests that the tumor-promoting effect of E-cadherin suppression is associated with decreased Dab-2 expression, an event that can in turn promote, at least in part, the tumorigenesity of E-cadherin-suppressed SCC tumor cells. These findings imply that manipulating Dab-2 expression in epithelial cells with neoplastic potential may inhibit SCC development.
A Tufts Collaborates! grant awarded to Drs. Alt-Holland and Baleja funded this study.
[Show abstract][Hide abstract] ABSTRACT: Objectives:
Advances in human induced pluripotent stem (iPSC) technology have raised new questions regarding the effect of reprogramming on the phenotype and epigenetic profile of cells differentiated from them. We recently derived cells from iPSC showing characteristic properties of fibroblasts (iPDK) that were similar to human embryonic stem cell- (hESC) derived fibroblasts (EDK). The goal of the current study was to compare the DNA methylation profile of fibroblasts derived from iPSC to fibroblasts from both normal and chronic wound tissues, to determine if there is a DNA methylation signature characteristic of chronic wound fibroblasts (CWF).
CWFs were harvested from excess tissues procured during wound debridement, expanded in culture and banked. Methylation analysis was performed using Infinium Human Methylation27 BeadChip (Illumina Inc.), analyzing 27,578 individual CpG sites. DNA was extracted using the DNeasy kit and 1 μg of purified genomic DNA was bisulfite-converted using the EZ DNA Methylation Kit and hybridized to the Infinium Methylation Chip. Immunoprecipitation of methylated DNA was performed using a MeDIP methylation kit (Diagenode).
We demonstrate that EDK and iPDK fibroblasts shared a similar DNA methylation profile with adult fibroblasts, but distinct from other cell lineages. Initial analysis indicated that we can enrich methylated DNA using immunoprecipitation, to allow for screening of CWF samples. We are currently analyzing primary fibroblasts from non-healing wounds using an immunoprecipitation-based method to enrich for methylated DNA, and using NimbleGen technology to determine their DNA methylation profile.
When CWFs are compared to ESC- and iPSC-derived fibroblasts, we expect to find significant differences between their methylation profile at specific promoter sites of CWFs that may partially account for their non-healing phenotype. These studies will be an important step towards understanding the underlying epigenetic mechanisms of chronic wounds that will have implications for oral disease processes characterized by failure to heal.
[Show abstract][Hide abstract] ABSTRACT: Objective: Replicative senescence shortens the lifespan of mammalian cells and limits their potential for regenerative therapies. Senescent cells secrete factors, known as senescence-associated secretory phenotype (SASP), that are linked to loss of growth potential. We have recently generated fibroblasts with improved biological potency from human embryonic stem cells (EDK6) and induced pluripotent stem cells (iPDK2). However, it is not known if iPDK cells acquire an increased proliferative lifespan when compared to the fibroblasts from which they were reprogrammed. The goal of this study was to compare SASP profiles between hESC- and iPSC-derived fibroblasts by comparing them to the parental fibroblasts (BJ) from which iPSC were derived.
Method: Replicative senescence of EDK6, iPDK2 and BJ fibroblasts was induced by serial passaging of these cells over three months. RNA was extracted from early (p8) and late (p19-20) passaged cells and relative levels of gene expression for SASP including IL-8, IL-6, IGF-1, HGF, and VEGF were determined by RT-PCR.
Result: In vitro replicative capacity of EDK6, iPDK2 and BJ fibroblasts was assessed by calculating the number of population doublings (PDs) during serial subculture. While a decline in PDs of EDK6 and iPDK2 was observed at p19, BJ underwent an earlier decrease in PDs (p13). As assessed by RT-PCR, levels of SASP RNA expression increased with passage number in EDK and iPDK, while a decrease in SASP RNA expression was seen with increasing passage of BJ fibroblasts.
Conclusion: These results demonstrate prolonged growth potential and stable secretory profile of EDK6 and iPDK2 when compared to BJ. This indicates that fibroblasts derived from iPSC may acquire replicative properties that exceed those of the somatic cells from which they were derived. The sustained growth potential and secretory microenvironment of iPSC-derived fibroblasts demonstrate that these cells may have applications for future regenerative therapies.
[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.