K. HEWITT

Tufts University, Бостон, Georgia, United States

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Publications (20)32.97 Total impact

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    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.
    12/2014; 3(12):742-750. DOI:10.1089/wound.2013.0480
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    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 · 3.53 Impact Factor
  • Stem Cells in Craniofacial Development and Regeneration, 03/2013: pages 204-222; , ISBN: 9781118279236
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    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
  • Kyle J Hewitt, Jonathan A Garlick
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    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 · 10.30 Impact Factor
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    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). Methods: 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). Results: 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. Conclusion: 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.
    AADR Annual Meeting 2012; 03/2012
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    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.
    AADR Annual Meeting 2012; 03/2012
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    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.
    Journal of Cell Science 02/2012; 125(Pt 9):2276-87. DOI:10.1242/jcs.099192 · 5.33 Impact Factor
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    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.
    In Vitro Cellular & Developmental Biology - Animal 02/2012; 48(2):112-22. DOI:10.1007/s11626-011-9478-4 · 1.00 Impact Factor
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    ABSTRACT: OBJECTIVES: Human induced pluripotent stem(hiPS) cells have potential to generate patient-specific cells that can be differentiated into multiple cell fates. However, the biological potential of iPS-derived cells with features of stromal fibroblasts is not known. The objective of our study was to characterize the functional properties of fibroblasts derived from iPS and to compare their fibrogenic properties to cells derived from human embryonic stem cells(hESC). METHODS: We derived two iPS cell lines with features of stromal fibroblasts (iPDK2 and iPDK4) and hESC (H9) were used to differentiate cell lines (EDK6 and EDK7). To analyze gene expression, RNA was extracted and RT-PCR was performed on EDK and iPDK cells to compare expression of extracellular matrix (ECM) proteins and flow cytometry was performed to measure CD surface markers. To assess the fibrogenic potential of EDK and iPDK lines, we used a tissue engineering approach to assess production of fibroblast-derived, self-assembled ECM, that was deposited after directly seeding cells onto a porous membrane in the presence and absence of Ascorbic Acid (AA). RESULTS: ESC and iPDK cells demonstrated a fibroblastic, morphology during the sequential stages of their differentiation. EDK and iPDK demonstrated a similar expression profile for CD10, 73, 90 and 166 in over 90% of cells, demonstrating that these cell lines maintained their fibroblastic phenotype throughout the construction of self-assembled tissues. Significantly, tissues derived from iPDK acquired a highly synthetic phenotype characterized by elevated ECM synthesis, deposition and organization into a stromal tissue harboring highly synthetic firboblasts. CONCLUSION: Fibroblasts derived from iPS showed de novo production of a thick, self-assembled 3D, collagen matrix. This demonstrates the fibrogenic potential of iPS-derived cells and shows the developmental capacity of iPS to form functional cell types that will pave the way for the derivation of patient-specific fibroblasts for future regenerative therapies. Supported by NIDCR-DE017413.
    IADR General Session 2011; 03/2011
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    ABSTRACT: OBJECTIVE: Pluripotent, human embryonic stem (hES) cells and induced pluripotent stem (iPS) cells may provide plentiful sources of primary cells for regenerative medicine due to their ability to differentiate into cell types required to form complex tissues. However, it is not known if functional lineages can be derived from these cells that can be used for wound repair and regeneration. The goal of this study was to characterize the production and secretion of soluble factors and extracellular matrix (ECM) proteins that may mediate such repair processes. METHODS: FIbroblast-like cells were derived from hES (EDK) and iPS (iPDK) using a protocol developed in our lab (Hewitt et al. 2009). RNA was extracted from confluent cultures of EDK, iPDK and control fibroblasts (HFF) and relative levels of gene expression for IL-8, vimentin (VIM), platelet-derived growth factor receptor- (PDGFR), Type I collagen (COLIA, COLIA2), alpha smooth muscle actin (a-SMA) and Tenascin C (TENC) were determined using three technical replicates. Levels of soluble factors produced by these cells were measured by ELISA for HGF, KGF, IGF-1, IL-8. IL-1a and PDFG-BB. RESULTS: Expression of genes associated with fibroblast phenotype and matrix production, including VIM, PDGFR, TENC, COLIA and COLIA2, were found in iPDK and EDK at comparable levels to those seen in mature fibroblasts while the presence of a-SMA suggested a myofibroblast fate. ELISA revealed dramatically elevated secretion of HGF and KGF in EDK and IDK cell lines when compared to control fibroblasts and increased levels of IGF-1 and IL-8. PDGF-BB and IL-1a were produced at levels similar to control fibroblasts. CONCLUSIONS: These results demonstrate the presence of paracrine-acting factors and ECM proteins that can play a role in the actiavtion of wound repair. Both iPS and ES cells can therefore differentiate into functionally-relevant cell populations displaying properties useful in regenerative medicine. Supported by NIDCR-DE017413.
    IADR General Session 2011; 03/2011
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    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 02/2011; 6(2):e17128. DOI:10.1371/journal.pone.0017128 · 3.53 Impact Factor
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    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.
    Stem Cell Research & Therapy 02/2011; 2(1):10. DOI:10.1186/scrt51 · 4.63 Impact Factor
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    ABSTRACT: Objective: Human embryonic stem cells (hESCs) can undergo directed differentiation to generate specific cell lineages, making them a potential source for cell therapy. The objective of this study was to characterize two distinct populations of mesenchymal progenitor cell lines (MPCs) derived from hESCs (EDK1 and H9-MSC) by determining their capacity to direct 3D tissue normalization through production of paracrine-acting soluble factors involved in epithelial-mesenchymal cross-talk. Methods: We generated two populations of MPCs from hESCs (hES-MPC) that we previously shown to either differentiate to osteogenic and adipogenic lineages (H9-MSC) or to fibroblasts (EDK1) that could support formation of stratified epithelial tissues. We analyzed production of soluble factors by hES-MPCs in 2D cultures and 3D skin equivalent tissues using ELISA and RT-PCR. Results: We found that EDK1 cells produced higher levels of KGF and HGF while H9-MSCs produced higher levels of IL-6. To investigate whether paracrine interactions between hES-MPCs and keratinocytes were linked to 3D tissue development, we assessed the production of KGF, HGF, and IL-6 in response to stimulation by IL-1 and IL-1, that are known to activate these growth factors in 3D epithelial tissues. The stimulation by IL-1 and IL-1 induced dose-dependent production of KGF and HGF in EDK1 cells, and IL-6 in H9-MSCs. Both hES-MPC lines expressed high levels of IL-1 cytokine receptor (IL1R1) in 2D cultures and 3D skin equivalent tissues. Conclusions: We found that although EDK1 and H9-MSC lines had similar mesenchymal morphology and surface protein expression but varied greatly in their behavior in 3D tissues. EDK1 cells were restricted to fibroblast lineages that provided paracrine-acting crosstalk to support epithelial normalization. These results demonstrate that directed differentiation of hESCs can generate distinct MPCs producing specific growth factors linked to distinct mesenchymal cell fates and tissue outcomes. Supported by Grant #DE017413 from the NIDCR.
    AADR Annual Meeting 2010; 03/2010
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    ABSTRACT: Objectives: MicroRNAs play an important regulatory role in the development and differentiation of stem cells. Specifically, we have recently found that miR-21 is elevated in human embryonic stem cell (hESC)-derived mesenchymal cells (EDK1) that are able to support the development and normalization of engineered, 3D human epithelial tissue equivalents. The objective of this study was to examine how expression of cytokines that are essential for development of 3D epithelial tissues by knocking down expression of miR-21. Methods: Knockdown of miR-21 epxression was performed using siRNA delivered using lentiviral vectors and levels of miR-21 were measured by RT-PCR. Expression of cytokines was measured using RT-PCR with gene specific primers and SYBR green for detection. Results: miR-21 knockdown was directly related to the amount of siRNA used for knockdown and resulted in 100%, 90% and 40% decrease in miR-21 expression. miR-21 knockdown resulted in 2-8 fold increase in IL-1A, IL-1B, CSF, IL-6, IL-1R2 and HGF while KGF was not altered. Both AP-1 and PTEN, known targets of miR- 21 were also elevated in response to loss of miR-21 expression. Conclusions: Our results demonstrate that miR-21 expression is associated with lower levels of cytokines important in epithelial-mesenchymal cross-talk. Since loss of this miR results in increased growth factor production, further study will be required to further understand how tissue growth and repair are modulated by targets associated with mIR-21 regulation. Supported by Grant #DE017413 from the NIDCR.
    AADR Annual Meeting 2010; 03/2010
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    ABSTRACT: Objectives: While Human Embryonic Stem Cells (hESCs) hold great potential as a source of cells for regenerative therapies of the oral and maxillofacial complex, it is unclear how to direct these pluripotent cells to oral tissue fates. Recently, we have derived mesenchymal progenitor cell lines (MPCs) from hESCs (H9 cell line) that differentiate into bone and fat. However, the potential of these cells to differentiate into dentin-producing cells remains unknown. Our goal was to characterize the phenotype of three, hESC-derived MPCs as a first step towards elucidating the potential of MPCs to differentiate into dentin-producing lineages. Methods: Three distinct MPC cell lines were derived from hESCs (H9-MSC, H1-MSC, and EDK) by sequentially growing cells on mouse embryonic fibroblasts and Type I collagen-coated plates (EDK1), or by expanding FACS-sorted, CD73+ cells (H9- and H1-MSC). Cultured human dental pulp stem cells (hDPSCs) isolated from extracted wisdom teeth were used as controls. MPCs were compared to DPSCs using FACS analysis for surface markers including CD117, CD146, CD34, CD105, CD31, CD45, CD34 and STRO-1. FACS-sorted populations of STRO-1 positive cells from DPSCs and H1-MSC and H9-MSC cells were expanded for further analysis. Results: FACS analysis revealed that cell surface markers CD-105 and CD-146 were found in DPSCs, H1-MSC and H9-MSC but not in EDK cells, which demonstrated properties of mature fibroblasts. STRO-1 was found in DPSCs and in all MPCs tested. Conclusions: Overlapping surface marker expression between DPSCs and specific MSC lines suggests that the differentiation potential of human DPSC and MPC may be similar. Future studies are needed to asses the biological potential for dentin production by these hESC-derived cells, and to establish if they can be used as a source of dental mesenchymal cell-derived tissue regeneration for applications in endodontics and restorative dentistry. Supported by NIH/NIDCR Grants DE017413 (JAG) and DE016132 (PCY).
    AADR Annual Meeting 2010; 03/2010
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    ABSTRACT: Objectives: Induced pluripotent stem (iPS) cells hold significant therapeutic potential as an alternative to human embryonic stem cells (hESC). However, it is unknown if iPS and hESC share similar biological potencies and differentiation potential. To address this, we compared patterns of gene expression in iPS cells and hESC at different stages of differentiation towards mesenchymal progenitor cells (MPCs). Methods: iPS derived from foreskin fibroblasts and hESCs (H9) were directed towards differentiation using our protocol to direct cells to mesenchymal fate. RNA was extracted and RT-PCR was performed for markers of mesenchymal differentiation. Results: The morphologic appearances of iPS- and hESC-derived differentiated cells were identical. We found that the fibroblast marker Thy-1 (CD-90) was expressed in iPS-derived cells at an earlier stage than in hESC-derived cells, suggesting that iPS manifest a cellular memory that facilitates their differentiation to a fate from which they were initially derived. Elastin and fibrillin-1 were expressed in MPCs, EDK and iPDK cells, derived from hESC and iPS, respectively. EDK cells did not express the markers of mesodermal differentiation but did express the neuroectodermal marker PDGFRβ, suggesting an ectomesenchymal origin for these cells. Importantly, we found that lentiviral Oct-4 used to reprogram iPS cells was re-expressed upon differentiation of iPDK cells. Conclusions: We show that iPS and hESC can be similarly directed to mesenchymal fate. However, differences in timing of expression of Thy-1 in iPS-derived cells suggest that they may possess an epigenetic memory that enables rapid differentiation to mesenchymal lineage. The neuroectodermal lineage of these cells suggests that craniofacial structures may be derived from these cells which could have broad utility for maxillofacial reconstruction. However, clinical applications of these cells will require screening due to rapid re-expression of Oct-4 in differentiated iPDK cells. Supported by Grant #DE017413 from the NIDCR
    AADR Annual Meeting 2010; 03/2010
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    ABSTRACT: Objectives: Mesenchymal stem cells (MSCs) hold promise for human therapies, but their heterogeneity and scarcity necessitate the search for alternative sources of cells. Human embryonic stem cells (hESC) provide advantages over traditional sources of MSCs due to their pluripotency and expansion potential. Our research goal was to characterize the phenotype and function of two distinct, hESC-derived, MSC lines (EDK1 and H9-MSC) in 2D cultures and 3D epithelial tissues. Methods: EDK1 were differentiated sequentially on mouse embryonic fibroblasts and on Type I collagen-coated plates, while H9-MSC were differentiated by expanding FACS-sorted, CD73+ cells. MSC lines were analyzed by immunohistochemistry for expression of cytokeratin 12 and 18, vimentin, p63, keratin 14 and β-catenin and for surface antigens CD105, CD73, CD90 and CD49a by FACS analysis. To establish the biological potential of MPC lines, we performed osteogenic and adipogenic differentiation assays in 2D cultures. To establish function of MSC lines in human tissues, cells were incorporated into collagen gels of human skin equivalents to test their potential to direct morphogenesis of keratinocytes grown on their surface. Results: Although EDK1 and H9-MSC showed similar mesenchymal morphology and overlapping CD marker expression, they demonstrated divergent cell fates in 2D culture and different 3D tissue outcomes. H9-MSCs could differentiate into fat and bone but did not support 3D tissue development. In contrast, EDK1 were restricted to fibroblast-like lineages that provided paracrine-acting crosstalk enabling functional support of tissue development, but did not generate other mesenchymal cell types or products. Conclusions: For the first time, we have shown that hESC-derived MSCs can be directed to divergent and specified MSC fates by establishing their functional properties in 3D, engineered tissues. Our findings provide a critical step towards predicting the stability, safety and efficacy of hESC-derived cells for future therapeutic transplantation. Supported by Grant #DE017413 from the NIDCR.
    AADR Annual Meeting 2010; 03/2010
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    ABSTRACT: 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.
    Tissue Engineering Part A 05/2009; 15(11):3417-26. DOI:10.1089/ten.TEA.2009.0060 · 4.64 Impact Factor
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    ABSTRACT: Objective: Generation of functional tissues that mimic their in vivo counterparts holds great promise to treat oral diseases. Pluripotent Human Embryonic Stem cells (hESC) provide a potential source of oral cells that would have significant advantages over other stem cell sources for tissue engineering. However, these cells have not been used successfully to construct complex, multilayer tissues. Our objective was to derive multiple cell types from hESC that will provide progenitor cells to fabricate stratified epithelial tissues. Methods: We derived multiple cell lineages needed for fabrication of stratified epithelial tissues from the H9, hES cell line. This was accomplished by growing hESC in specific media and substrate conditions to enrich for cells with either mesenchymal or ectodermal phenotypes. These cells were then used to fabricate 3D tissues by incorporating mesenchymal cell-enriched progenitors into collagen gels (EDK1) and growing ectodermal cell-enriched progenitors (EDKH1)on them at an air-liquid interface. Results: hESC differentiated into an ectodermal lineage (EDK1) and expressed CK18 and Vimentin, but not hESC markers Oct-4 or SSEA4. EDK1 also demonstrated a population of cells characteristic of mesenchymal lineage. EDK1 were then grown on laminin 5-enriched surfaces to generate CK18-negative and CK12-positive cells (EDKH1). EDKH1 cells generated a multi-layer epithelium when grown at an air-liquid interface on collagen gels populated with EDK1 cells. Tissues demonstrated deposition of basement membrane components at the epithelial-stromal interface and EDK1 acquired features of fibroblast-like cells that secreted paracrine-acting factors. Conclusions: For the first time, we have generated 3D tissues from hESC by committing these pluripotent cells to ectodermal and mesenchymal fates and then by using cues from their microenvironment to generate stratified epithelial tissues. By further modifying microenvironmental cues that direct hESC cell fate, we can now optimize epithelial tissue organization and function. Supported by NIH Grant#R01-DE017413-01
    IADR General Session 2009; 04/2009

Publication Stats

71 Citations
32.97 Total Impact Points

Institutions

  • 2009–2014
    • Tufts University
      • • Sackler School of Graduate Biomedical Sciences
      • • Department of Oral and Maxillofacial Pathology
      Бостон, Georgia, United States
  • 2012
    • University of Wisconsin–Madison
      Madison, Wisconsin, United States