Regulation of mTOR signaling depends on an intricate interplay of post-translational protein modi... more Regulation of mTOR signaling depends on an intricate interplay of post-translational protein modification. Recently, mEAK-7 (mTOR associated protein, eak-7 homolog) was identified as a positive activator of mTOR signaling via an alternative mTOR complex. However, the upstream regulation of mEAK-7 in human cells is not known. Because microRNAs are capable of modulating protein translation of RNA in eukaryotes, we conducted a bioinformatic search for relevant mEAK-7 targeting microRNAs using the Exiqon miRSearch V3.0 algorithm. Based on the score obtained through miRSearch V3.0, the top predicted miRNA (miR-1911-3p) was studied. miR-1911-3p mimics decreased protein levels of both mEAK-7 and mTORC1 downstream effectors p-S6 and p-4E-BP1 in non-small cell lung carcinoma (NSCLC) cell lines H1975 and H1299. miR-1911-3p levels and MEAK7 mRNA/ mEAK-7/mTOR signaling levels were negatively correlated between normal lung and NSCLC cells. miR-1911-3p directly interacted with MEAK7 mRNA at the 3 0-UTR to negatively regulate mEAK-7 and significantly decreased mTOR localization to the lysosome. Furthermore, miR-1911-3p significantly decreased cell proliferation and migration in both H1975 and H1299 cells. Thus, miR-1911-3p functions as a suppressor of mTOR signaling through the regulation of MEAK7 mRNA translation in human cancer cells.
and cell replacement therapy. For example, recent demonstration that hESCs can be induced to diff... more and cell replacement therapy. For example, recent demonstration that hESCs can be induced to differentiate and become motor neurons (MNs) offers unprecedented opportunity for studying MN development/function and developing cell-based therapies. [2] However, current MN differentiation protocols, based on soluble factors and small molecules that inhibit and/ or stimulate particular signaling pathways in defined culture conditions, not only are limited by low differentiation purity and yield, but also require prolonged cell culture that can take several weeks. [3] Mechanical forces are generated and transmitted across multiple scales, affecting cell fate during early embryonic development. [4] It has been increasingly recognized that besides chemical factors, biomechanical and topographical cues also play critical roles in differentiation and self-renewal of hESCs. [5] Thus new
MTOR associated protein, eak-7 homolog (mEAK-7), activates mechanistic target of rapamycin (mTOR)... more MTOR associated protein, eak-7 homolog (mEAK-7), activates mechanistic target of rapamycin (mTOR) signaling in human cells through an alternative mTOR complex to regulate S6K2 and 4E-BP1. However, the role of mEAK-7 in human cancer has not yet been identified. We demonstrate that mEAK-7 and mTOR signaling are strongly elevated in tumor and metastatic lymph nodes of patients with non-small-cell lung carcinoma compared with those of patients with normal lung or lymph tissue. Cancer stem cells, CD44+/CD90+ cells, yield elevated mEAK-7 and activated mTOR signaling. mEAK-7 is required for clonogenic potential and spheroid formation. mEAK-7 associates with DNAdependent protein kinase catalytic subunit isoform 1 (DNA-PKcs), and this interaction is increased in response to X-ray irradiation to regulate S6K2 signaling. DNA-PKcs pharmacologic inhibition or genetic knockout reduced S6K2, mEAK-7, and mTOR binding with DNA-PKcs, resulting in loss of S6K2 activity and mTOR signaling. Therefore, mEAK-7 forms an alternative mTOR complex with DNA-PKcs to regulate S6K2 in human cancer cells.
Human embryonic stem cells subjected to a one-time uniaxial stretch for as short as 30-min on a f... more Human embryonic stem cells subjected to a one-time uniaxial stretch for as short as 30-min on a flexible substrate coated with Matrigel experienced rapid and irreversible nuclear-to-cytoplasmic translocation of NANOG and OCT4, but not Sox2. Translocations were directed by intracellular transmission of biophysical signals from cell surface integrins to nuclear CRM1 and were independent of exogenous soluble factors. On E-CADHERIN-coated substrates, presumably with minimal integrin engagement, mechanical strain-induced rapid nuclear-to-cytoplasmic translocation of the three transcription factors. These findings might provide fundamental insights into early developmental processes and may facilitate mechanotransductionmediated bioengineering approaches to influencing stem cell fate determination. Insight, innovation, integration How do mechanical stimuli, sensed through different types of cell adhesions, alter embryonic stem cell fate? This paper employs a stretchable device coated with different cell adhesion molecules to apply stretch to human embryonic stem cells in a manner that might mimic differentiation of cell lineages present at the blastocyst stage of development. The work reveals that stretch induces an unexpectedly rapid export of a subset of pluripotent transcription factors from the nucleus to the cytoplasm before significant decreases in overall levels. Interestingly, the subset of transcription factors exported differs between cells stretched on Matrigel-versus E-CADHERIN-coated devices. These results have physiological relevance to embryonic development where some cells experience mechanical forces predominantly through cell-cell contact while other cells also experience cell-ECM interactions leading to different cell-fate specification. These insights might enhance our understanding of early development and may also guide bioengineering approaches where stretching is used to accelerate or regulate the direction of differentiation.
Craniosynostosis is defined as congenital premature fusion of one or more cranial sutures. While ... more Craniosynostosis is defined as congenital premature fusion of one or more cranial sutures. While the genetic basis for about 30% of cases is known, the causative genes for the diverse presentations of the remainder of cases are unknown. The recently discovered cranial suture stem cell population affords an opportunity to identify early signaling pathways that contribute to craniosynostosis. We previously demonstrated that enhanced BMP signaling in neural crest cells (caA3 mutants) leads to premature cranial suture fusion resulting in midline craniosynostosis. Since enhanced mTOR signaling in neural crest cells leads to craniofacial bone lesions, we investigated the extent to which mTOR signaling is involved in the pathogenesis of BMP-mediated craniosynostosis by affecting the suture stem cell population. Our results demonstrate a loss of suture stem cells in the caA3 mutant mice by the newborn stage. We have found increased activation of mTOR signaling in caA3 mutant mice during emb...
Mechanical forces play critical roles in influencing human embryonic stem cell (hESC) fate. Howev... more Mechanical forces play critical roles in influencing human embryonic stem cell (hESC) fate. However, it remains largely uncharacterized how local mechanical forces influence hESC behavior in vitro. Here, we used an ultrasound (US) technique, acoustic tweezing cytometry (ATC), to apply targeted cyclic subcellular forces to hESCs via integrin-bound microbubbles (MBs). We found that ATC-mediated cyclic forces applied for 30 min to hESCs near the edge of a colony induced immediate global responses throughout the colony, suggesting the importance of cell-cell connection in the mechanoresponsiveness of hESCs to ATC-applied forces. ATC application generated increased contractile force, enhanced calcium activity, as well as decreased expression of pluripotency transcription factors Oct4 and Nanog, leading to rapid initiation of hESC differentiation and characteristic epithelial-mesenchymal transition (EMT) events that depend on focal adhesion kinase (FAK) activation and cytoskeleton (CSK) t...
The advent of adaptive manufacturing techniques supports the vision of cell-instructive materials... more The advent of adaptive manufacturing techniques supports the vision of cell-instructive materials that mimic biological tissues. 3D jet writing, a modified electrospinning process reported herein, yields 3D structures with unprecedented precision and resolution offering customizable pore geometries and scalability to over tens of centimeters. These scaffolds support the 3D expansion and differentiation of human mesenchymal stem cells in vitro. Implantation of these constructs leads to the healing of critical bone defects in vivo without exogenous growth factors. When applied as a metastatic target site in mice, circulating cancer cells home in to the osteogenic environment simulated on 3D jet writing scaffolds, despite implantation in an anatomically abnormal site. Through 3D jet writing, the formation of tessellated microtissues is demonstrated, which serve as a versatile 3D cell culture platform in a range of biomedical applications including regenerative medicine, cancer biology,...
There is evidence that cancer stem-like cells (CSC) and neuroendocrine behavior play critical rol... more There is evidence that cancer stem-like cells (CSC) and neuroendocrine behavior play critical roles in the pathogenesis and clinical course of metastatic castration-resistant prostate cancer (m-CRPC). However, there is limited mechanistic understanding of how CSC and neuroendocrine phenotypes impact the development of m-CRPC. In this study, we explored the role of the intracellular chemokine CXCL12γ in CSC induction and neuroendocrine differentiation and its impact on m-CRPC. CXCL12γ expression was detected in small-cell carcinoma of metastatic tissues and circulating tumor cells from m-CRPC patients and in prostate cancer cells displaying an neuroendocrine phenotype. Mechanistic investigations demonstrated that overexpression of CXCL12γ induced CSC and neuroendocrine phenotypes in prostate cancer cells through CXCR4-mediated PKCα/NFκB signaling, which promoted prostate tumor outgrowth, metastasis, and chemoresistance Together, our results establish a significant function for CXCL12...
Therapeutic strategies targeting both cancer cells and associated cells in the tumor microenviron... more Therapeutic strategies targeting both cancer cells and associated cells in the tumor microenvironment offer significant promise in cancer therapy. We previously reported that generation 5 (G5) dendrimers conjugated with cyclic-RGD peptides target cells expressing integrin alpha V beta 3. In this study, we report a novel dendrimer conjugate modified to deliver the mammalian target of rapamycin (mTOR) inhibitor, rapamycin. In vitro analyses demonstrated that this drug conjugate, G5-FI-RGD-rapamycin, binds to prostate cancer (PCa) cells and fibroblasts to inhibit mTOR signaling and VEGF expression. In addition, G5-FI-RGD-rapamycin inhibits mTOR signaling in cancer cells more efficiently under proinflammatory condition compared to free rapamycin. In vivo studies established that G5-FI-RGD-rapamycin significantly inhibits fibroblast-mediated PCa progression and metastasis. Thus, our results suggest the potential of new rapamycin-conjugated multifunctional nanoparticles for PCa therapy. T...
Dental graduates of 2040 will face new and complex challenges. If they are to meet these challeng... more Dental graduates of 2040 will face new and complex challenges. If they are to meet these challenges, dental schools must develop a research and discovery mission that will equip graduates with the new knowledge required to function in a modern health care environment. The dental practitioner of 2040 will place greater emphasis on risk assessment, disease prevention, and health maintenance; and the emerging discipline of precision medicine and systems biology will revolutionize disease diagnosis and reveal new targeted therapies. The dental graduate of 2040 will be expected to function effectively in a collaborative, learning health care system and to understand the impact of health care policy on local, national, and global communities. Emerging scientific fields such as big data analytics, stem cell biology, tissue engineering, and advanced biomimetics will impact dental practice. Despite all the warning signs indicating how the changing scientific and heath care landscape will dramatically alter dental education and dental practice, dental schools have yet to reconsider their research and educational priorities and clinical practice objectives. Until dental schools and the practicing community come to grips with these challenges, this persistent attitude of complacency will likely be at the dental profession's peril. This article was written as part of the project "Advancing Dental Education in the 21 st Century."
Stem cells have the ability to self-renew and differentiate into specialized cell types, and, in ... more Stem cells have the ability to self-renew and differentiate into specialized cell types, and, in the human body, they reside in specialized microenvironments called Bstem cell niches.^Although several niches have been described and studied in vivo, their functional replication in vitro is still incomplete. The in vitro culture of pluripotent stem cells may represent one of the most advanced examples in the effort to create an artificial or synthetic stem cell niche. A focus has been placed on the development of human stem cell microenvironments due to their significant clinical implications, in addition to the potential differences between animal and human cells. In this concise review, we describe the advances in human pluripotent stem cell culture and explore the idea that the knowledge gained from this model could be replicated to create synthetic niches for other human stem cell populations, which have proven difficult to maintain in vitro.
Scientific data presented at the meeting contributed to the knowledge and basic understanding of ... more Scientific data presented at the meeting contributed to the knowledge and basic understanding of the events involved in the formation, maintenance and repair/regeneration of mineralized tissues. Through the exchange of information at the meeting, scientists are working to improve the diagnosis, treatment therapies and ultimately the prevention of diseases of these tissues (for example, in osteoporosis, periodontal disease, osteoarthritis and osteogenesis as well as dentinogenesis imperfecta and other collagen diseases) as well as tissues that undergo pathologic mineralization. The knowledge gained should also lead to the development of improved biomaterials for dental implants and orthopedic prostheses and of new tissue-engineered devices for dental and skeletal applications. This meeting would not be possible without the generous support from our sponsors. A big thank you goes to the National Institutes of Dental and Craniofacial Research (NIDCR, NIH), with additional funding provided from the NIH, including NIAMS, NIBIB and the Office of Rare Diseases, to the Orthopaedic Research and Educational Foundation (OREF) and to the University of Missouri Kansas City School of Dentistry. We would also like to acknowledge the generosity of the Alliance for Better Bone Health, Proctor and Gamble and the Réseau de recherche en santé buccodentaire et osseuse (RSBO) of Canada. Thanks also go to Wyeth, Eli Lilly, Institute Straumann and Medtronic. Lynda F. Bonewald, Kansas City, Mo., USA Co-chair, 9th ICCMBT Paul H. Krebsbach, Ann Arbor, Mich., USA Co-chair, 9th ICCMBT This special issue of Cells, Tissues, Organs highlights the science presented at the 9th International Conference on the Chemistry and Biology of Mineralized Tissues (ICCBMT). The 9th ICCBMT took place between November 4 and 8, 2007, at the Lakeway Inn Conference Resort in Austin, Tex., USA. This conference, which has been held every 3 years since 1980, emphasizes physicochemical, biological and clinical topics concerning the mineralization process. These include, for example, aspects of the cell and molecular biology of mineralized tissue formation, hormone and cytokine regulation of mineralized tissues, signaling pathways, structure and function of extracellular components of mineralized tissues, model systems of biomineralization, disorder and pathology of mineralized tissues, therapeutic approaches, and new technologies for studying and analyzing mineralization. The goal of the ICCBMT meetings is to disseminate state-of-the-art information about the structure, function and interactions of the mineral, matrix and cellular components in bones, teeth and other mineralized vertebrate and invertebrate tissues, while encouraging collaboration among investigators in different disciplines. This meeting, which attracted a multidisciplinary international audience, promoted and advanced the exchange of new ideas in several ways: by supporting participation of young investigators through travel awards, by providing a free and open forum for both oral and poster presentation of results of new scientific studies as well as by encouraging interaction between senior investigators and new investigators during informal discussions at meals, afternoon breaks and focused workshops. Published online: August 18, 2008
A new method for generating and modeling reduced-scale copolymer gradients by CVD is reported. By... more A new method for generating and modeling reduced-scale copolymer gradients by CVD is reported. By exploiting diffusion through confined channels, functionalized [2.2]paracyclophanes are copolymerized into their poly(p-xylylene) (PPX) analogues as a composition gradient. Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS) are used to verify the gradient composition profiles. Gradients are deposited on both flat substrates and 3-dimensional cylinders. Both the thickness and compositional profiles are fitted to a diffusion-based model using realistic physical parameters. The derived equation can be generalized and optimized for any copolymerization gradient through a confined geometry, thus allowing for broad applicability to other copolymer systems.
Human embryonic stem cells (hESCs) hold promise for tissue regeneration therapies by providing a ... more Human embryonic stem cells (hESCs) hold promise for tissue regeneration therapies by providing a potentially unlimited source of cells capable of undergoing differentiation into specified cell types. Several preclinical studies and a few clinical studies use human bone marrow stromal cells (hBMSCs) to treat skeletal diseases and repair damaged tissue. However, hBMSCs have limited proliferation and differentiation capacity, suggesting that an alternate cell source is desirable, and hESCs may serve this purpose. Here we describe a protocol for the reproducible derivation of mesenchymal stem cells from hESCs (hES-MSCs). The hES-MSCs have a similar immunophenotype to hBMSCs, specifically they are CD73+, STRO-1+ and CD45–, and are karyotypically stable. The derived hES-MSCs are also capable of differentiating into osteoblasts and adipocytes. When the hES-MSCs were genetically modified with the lineage-specific Col2.3-GFP lentivirus and cultured in osteogenic medium, increased GFP express...
Nematode EAK-7 (enhancer-of--1-7) regulates dauer formation and controls life span; however, the ... more Nematode EAK-7 (enhancer-of--1-7) regulates dauer formation and controls life span; however, the function of the human ortholog mammalian EAK-7 (mEAK-7) is unknown. We report that mEAK-7 activates an alternative mechanistic/mammalian target of rapamycin (mTOR) signaling pathway in human cells, in which mEAK-7 interacts with mTOR at the lysosome to facilitate S6K2 activation and 4E-BP1 repression. Despite interacting with mTOR and mammalian lethal with SEC13 protein 8 (mLST8), mEAK-7 does not interact with other mTOR complex 1 (mTORC1) or mTOR complex 2 (mTORC2) components; however, it is essential for mTOR signaling at the lysosome. This phenomenon is distinguished by S6 and 4E-BP1 activity in response to nutrient stimulation. Conventional S6K1 phosphorylation is uncoupled from S6 phosphorylation in response to mEAK-7 knockdown. mEAK-7 recruits mTOR to the lysosome, a crucial compartment for mTOR activation. Loss of mEAK-7 results in a marked decrease in lysosomal localization of mT...
Human embryonic stem cells subjected to a one-time uniaxial stretch for as short as 30-min on a f... more Human embryonic stem cells subjected to a one-time uniaxial stretch for as short as 30-min on a flexible substrate coated with Matrigel experienced rapid and irreversible nuclear-to-cytoplasmic translocation of NANOG and OCT4, but not Sox2. Translocations were directed by intracellular transmission of biophysical signals from cell surface integrins to nuclear CRM1 and were independent of exogenous soluble factors. On E-CADHERIN-coated substrates, presumably with minimal integrin engagement, mechanical strain-induced rapid nuclear-to-cytoplasmic translocation of the three transcription factors. These findings might provide fundamental insights into early developmental processes and may facilitate mechanotransduction-mediated bioengineering approaches to influencing stem cell fate determination.
Here, we used an ultrasound (US) technique, acoustic tweezing cytometry (ATC), to apply targeted ... more Here, we used an ultrasound (US) technique, acoustic tweezing cytometry (ATC), to apply targeted cyclic subcellular forces to hESCs via integrin-bound microbubbles (MBs). We found that ATCmediated cyclic forces applied for 30 min to hESCs near the edge of a colony induced immediate global responses throughout the colony, suggesting the importance of cell-cell connection in the mechanoresponsiveness of hESCs to ATC-applied forces. ATC application generated increased contractile force, enhanced calcium activity, as well as decreased expression of pluripotency transcription factors Oct4 and Nanog, leading to rapid initiation of hESC differentiation and characteristic epithelial-mesenchymal transition (EMT) events that depend on focal adhesion kinase (FAK) activation and cytoskeleton (CSK) tension. These results reveal a unique, rapid mechanoresponsiveness and community behavior of hESCs to integrin-targeted cyclic forces.
Self-renewal of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs)-know... more Self-renewal of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs)-known as pluripotent stem cells (PSC)-is influenced by culture conditions, including the substrate on which they are grown. However, details of the molecular mechanisms interconnecting the substrate and self-renewal of these cells remain unclear. We describe a signaling pathway in hPSCs linking self-renewal and expression of pluripotency transcription factors to integrin α6β1 and inactivation of focal adhesion kinase (FAK). Disruption of this pathway results in hPSC differentiation. In hPSCs, α6β1 is the dominant integrin and FAK is not phosphorylated at Y397, and thus, it is inactive. During differentiation, integrin α6 levels diminish and Y397 FAK is phosphorylated and activated. During reprogramming of fibroblasts into iPSCs, integrin α6 is upregulated and FAK is inactivated. Knockdown of integrin α6 and activation of β1 integrin lead to FAK phosphorylation and reduction of Nanog, Oct4, a...
Bioactive glasses are potentially useful as bone defect fillers, and vascular endothelial growth ... more Bioactive glasses are potentially useful as bone defect fillers, and vascular endothelial growth factor (VEGF) has demonstrated benefit in bone regeneration as well. We hypothesized that the specific combination of prolonged localized VEGF presentation from a matrix coated with a bioactive glass may enhance bone regeneration. To test this hypothesis, the capacity of VEGF-releasing polymeric scaffolds with a bioactive glass coating was examined in vitro and in vivo using a rat critical-sized defect model. In the presence of a bioactive glass coating, we did not detect pronounced differences in the differentiation of human mesenchymal stem cells in vitro. However, we observed significantly enhanced mitogenic stimulation of endothelial cells in the presence of the bioactive glass coating, with an additive effect with VEGF release. This trend was maintained in vivo, where coated VEGF-releasing scaffolds demonstrated significant improvements in blood vessel density at 2 weeks versus coated control scaffolds. At 12 weeks, bone mineral density was significantly increased in coated VEGF-releasing scaffolds versus coated controls, while only a slight increase in bone volume fraction was observed. The results of this study suggest that a bioactive glass coating on a polymeric substrate participates in bone healing through indirect processes which enhance angiogenesis and bone maturation and not directly on osteoprogenitor differentiation and bone formation. The mass of bioactive glass used in this study provides a comparable and potentially additive, response to localized VEGF delivery over early time points. These studies demonstrate a materials approach to achieve an angiogenic response formerly limited to the delivery of inductive growth factors.
Regulation of mTOR signaling depends on an intricate interplay of post-translational protein modi... more Regulation of mTOR signaling depends on an intricate interplay of post-translational protein modification. Recently, mEAK-7 (mTOR associated protein, eak-7 homolog) was identified as a positive activator of mTOR signaling via an alternative mTOR complex. However, the upstream regulation of mEAK-7 in human cells is not known. Because microRNAs are capable of modulating protein translation of RNA in eukaryotes, we conducted a bioinformatic search for relevant mEAK-7 targeting microRNAs using the Exiqon miRSearch V3.0 algorithm. Based on the score obtained through miRSearch V3.0, the top predicted miRNA (miR-1911-3p) was studied. miR-1911-3p mimics decreased protein levels of both mEAK-7 and mTORC1 downstream effectors p-S6 and p-4E-BP1 in non-small cell lung carcinoma (NSCLC) cell lines H1975 and H1299. miR-1911-3p levels and MEAK7 mRNA/ mEAK-7/mTOR signaling levels were negatively correlated between normal lung and NSCLC cells. miR-1911-3p directly interacted with MEAK7 mRNA at the 3 0-UTR to negatively regulate mEAK-7 and significantly decreased mTOR localization to the lysosome. Furthermore, miR-1911-3p significantly decreased cell proliferation and migration in both H1975 and H1299 cells. Thus, miR-1911-3p functions as a suppressor of mTOR signaling through the regulation of MEAK7 mRNA translation in human cancer cells.
and cell replacement therapy. For example, recent demonstration that hESCs can be induced to diff... more and cell replacement therapy. For example, recent demonstration that hESCs can be induced to differentiate and become motor neurons (MNs) offers unprecedented opportunity for studying MN development/function and developing cell-based therapies. [2] However, current MN differentiation protocols, based on soluble factors and small molecules that inhibit and/ or stimulate particular signaling pathways in defined culture conditions, not only are limited by low differentiation purity and yield, but also require prolonged cell culture that can take several weeks. [3] Mechanical forces are generated and transmitted across multiple scales, affecting cell fate during early embryonic development. [4] It has been increasingly recognized that besides chemical factors, biomechanical and topographical cues also play critical roles in differentiation and self-renewal of hESCs. [5] Thus new
MTOR associated protein, eak-7 homolog (mEAK-7), activates mechanistic target of rapamycin (mTOR)... more MTOR associated protein, eak-7 homolog (mEAK-7), activates mechanistic target of rapamycin (mTOR) signaling in human cells through an alternative mTOR complex to regulate S6K2 and 4E-BP1. However, the role of mEAK-7 in human cancer has not yet been identified. We demonstrate that mEAK-7 and mTOR signaling are strongly elevated in tumor and metastatic lymph nodes of patients with non-small-cell lung carcinoma compared with those of patients with normal lung or lymph tissue. Cancer stem cells, CD44+/CD90+ cells, yield elevated mEAK-7 and activated mTOR signaling. mEAK-7 is required for clonogenic potential and spheroid formation. mEAK-7 associates with DNAdependent protein kinase catalytic subunit isoform 1 (DNA-PKcs), and this interaction is increased in response to X-ray irradiation to regulate S6K2 signaling. DNA-PKcs pharmacologic inhibition or genetic knockout reduced S6K2, mEAK-7, and mTOR binding with DNA-PKcs, resulting in loss of S6K2 activity and mTOR signaling. Therefore, mEAK-7 forms an alternative mTOR complex with DNA-PKcs to regulate S6K2 in human cancer cells.
Human embryonic stem cells subjected to a one-time uniaxial stretch for as short as 30-min on a f... more Human embryonic stem cells subjected to a one-time uniaxial stretch for as short as 30-min on a flexible substrate coated with Matrigel experienced rapid and irreversible nuclear-to-cytoplasmic translocation of NANOG and OCT4, but not Sox2. Translocations were directed by intracellular transmission of biophysical signals from cell surface integrins to nuclear CRM1 and were independent of exogenous soluble factors. On E-CADHERIN-coated substrates, presumably with minimal integrin engagement, mechanical strain-induced rapid nuclear-to-cytoplasmic translocation of the three transcription factors. These findings might provide fundamental insights into early developmental processes and may facilitate mechanotransductionmediated bioengineering approaches to influencing stem cell fate determination. Insight, innovation, integration How do mechanical stimuli, sensed through different types of cell adhesions, alter embryonic stem cell fate? This paper employs a stretchable device coated with different cell adhesion molecules to apply stretch to human embryonic stem cells in a manner that might mimic differentiation of cell lineages present at the blastocyst stage of development. The work reveals that stretch induces an unexpectedly rapid export of a subset of pluripotent transcription factors from the nucleus to the cytoplasm before significant decreases in overall levels. Interestingly, the subset of transcription factors exported differs between cells stretched on Matrigel-versus E-CADHERIN-coated devices. These results have physiological relevance to embryonic development where some cells experience mechanical forces predominantly through cell-cell contact while other cells also experience cell-ECM interactions leading to different cell-fate specification. These insights might enhance our understanding of early development and may also guide bioengineering approaches where stretching is used to accelerate or regulate the direction of differentiation.
Craniosynostosis is defined as congenital premature fusion of one or more cranial sutures. While ... more Craniosynostosis is defined as congenital premature fusion of one or more cranial sutures. While the genetic basis for about 30% of cases is known, the causative genes for the diverse presentations of the remainder of cases are unknown. The recently discovered cranial suture stem cell population affords an opportunity to identify early signaling pathways that contribute to craniosynostosis. We previously demonstrated that enhanced BMP signaling in neural crest cells (caA3 mutants) leads to premature cranial suture fusion resulting in midline craniosynostosis. Since enhanced mTOR signaling in neural crest cells leads to craniofacial bone lesions, we investigated the extent to which mTOR signaling is involved in the pathogenesis of BMP-mediated craniosynostosis by affecting the suture stem cell population. Our results demonstrate a loss of suture stem cells in the caA3 mutant mice by the newborn stage. We have found increased activation of mTOR signaling in caA3 mutant mice during emb...
Mechanical forces play critical roles in influencing human embryonic stem cell (hESC) fate. Howev... more Mechanical forces play critical roles in influencing human embryonic stem cell (hESC) fate. However, it remains largely uncharacterized how local mechanical forces influence hESC behavior in vitro. Here, we used an ultrasound (US) technique, acoustic tweezing cytometry (ATC), to apply targeted cyclic subcellular forces to hESCs via integrin-bound microbubbles (MBs). We found that ATC-mediated cyclic forces applied for 30 min to hESCs near the edge of a colony induced immediate global responses throughout the colony, suggesting the importance of cell-cell connection in the mechanoresponsiveness of hESCs to ATC-applied forces. ATC application generated increased contractile force, enhanced calcium activity, as well as decreased expression of pluripotency transcription factors Oct4 and Nanog, leading to rapid initiation of hESC differentiation and characteristic epithelial-mesenchymal transition (EMT) events that depend on focal adhesion kinase (FAK) activation and cytoskeleton (CSK) t...
The advent of adaptive manufacturing techniques supports the vision of cell-instructive materials... more The advent of adaptive manufacturing techniques supports the vision of cell-instructive materials that mimic biological tissues. 3D jet writing, a modified electrospinning process reported herein, yields 3D structures with unprecedented precision and resolution offering customizable pore geometries and scalability to over tens of centimeters. These scaffolds support the 3D expansion and differentiation of human mesenchymal stem cells in vitro. Implantation of these constructs leads to the healing of critical bone defects in vivo without exogenous growth factors. When applied as a metastatic target site in mice, circulating cancer cells home in to the osteogenic environment simulated on 3D jet writing scaffolds, despite implantation in an anatomically abnormal site. Through 3D jet writing, the formation of tessellated microtissues is demonstrated, which serve as a versatile 3D cell culture platform in a range of biomedical applications including regenerative medicine, cancer biology,...
There is evidence that cancer stem-like cells (CSC) and neuroendocrine behavior play critical rol... more There is evidence that cancer stem-like cells (CSC) and neuroendocrine behavior play critical roles in the pathogenesis and clinical course of metastatic castration-resistant prostate cancer (m-CRPC). However, there is limited mechanistic understanding of how CSC and neuroendocrine phenotypes impact the development of m-CRPC. In this study, we explored the role of the intracellular chemokine CXCL12γ in CSC induction and neuroendocrine differentiation and its impact on m-CRPC. CXCL12γ expression was detected in small-cell carcinoma of metastatic tissues and circulating tumor cells from m-CRPC patients and in prostate cancer cells displaying an neuroendocrine phenotype. Mechanistic investigations demonstrated that overexpression of CXCL12γ induced CSC and neuroendocrine phenotypes in prostate cancer cells through CXCR4-mediated PKCα/NFκB signaling, which promoted prostate tumor outgrowth, metastasis, and chemoresistance Together, our results establish a significant function for CXCL12...
Therapeutic strategies targeting both cancer cells and associated cells in the tumor microenviron... more Therapeutic strategies targeting both cancer cells and associated cells in the tumor microenvironment offer significant promise in cancer therapy. We previously reported that generation 5 (G5) dendrimers conjugated with cyclic-RGD peptides target cells expressing integrin alpha V beta 3. In this study, we report a novel dendrimer conjugate modified to deliver the mammalian target of rapamycin (mTOR) inhibitor, rapamycin. In vitro analyses demonstrated that this drug conjugate, G5-FI-RGD-rapamycin, binds to prostate cancer (PCa) cells and fibroblasts to inhibit mTOR signaling and VEGF expression. In addition, G5-FI-RGD-rapamycin inhibits mTOR signaling in cancer cells more efficiently under proinflammatory condition compared to free rapamycin. In vivo studies established that G5-FI-RGD-rapamycin significantly inhibits fibroblast-mediated PCa progression and metastasis. Thus, our results suggest the potential of new rapamycin-conjugated multifunctional nanoparticles for PCa therapy. T...
Dental graduates of 2040 will face new and complex challenges. If they are to meet these challeng... more Dental graduates of 2040 will face new and complex challenges. If they are to meet these challenges, dental schools must develop a research and discovery mission that will equip graduates with the new knowledge required to function in a modern health care environment. The dental practitioner of 2040 will place greater emphasis on risk assessment, disease prevention, and health maintenance; and the emerging discipline of precision medicine and systems biology will revolutionize disease diagnosis and reveal new targeted therapies. The dental graduate of 2040 will be expected to function effectively in a collaborative, learning health care system and to understand the impact of health care policy on local, national, and global communities. Emerging scientific fields such as big data analytics, stem cell biology, tissue engineering, and advanced biomimetics will impact dental practice. Despite all the warning signs indicating how the changing scientific and heath care landscape will dramatically alter dental education and dental practice, dental schools have yet to reconsider their research and educational priorities and clinical practice objectives. Until dental schools and the practicing community come to grips with these challenges, this persistent attitude of complacency will likely be at the dental profession's peril. This article was written as part of the project "Advancing Dental Education in the 21 st Century."
Stem cells have the ability to self-renew and differentiate into specialized cell types, and, in ... more Stem cells have the ability to self-renew and differentiate into specialized cell types, and, in the human body, they reside in specialized microenvironments called Bstem cell niches.^Although several niches have been described and studied in vivo, their functional replication in vitro is still incomplete. The in vitro culture of pluripotent stem cells may represent one of the most advanced examples in the effort to create an artificial or synthetic stem cell niche. A focus has been placed on the development of human stem cell microenvironments due to their significant clinical implications, in addition to the potential differences between animal and human cells. In this concise review, we describe the advances in human pluripotent stem cell culture and explore the idea that the knowledge gained from this model could be replicated to create synthetic niches for other human stem cell populations, which have proven difficult to maintain in vitro.
Scientific data presented at the meeting contributed to the knowledge and basic understanding of ... more Scientific data presented at the meeting contributed to the knowledge and basic understanding of the events involved in the formation, maintenance and repair/regeneration of mineralized tissues. Through the exchange of information at the meeting, scientists are working to improve the diagnosis, treatment therapies and ultimately the prevention of diseases of these tissues (for example, in osteoporosis, periodontal disease, osteoarthritis and osteogenesis as well as dentinogenesis imperfecta and other collagen diseases) as well as tissues that undergo pathologic mineralization. The knowledge gained should also lead to the development of improved biomaterials for dental implants and orthopedic prostheses and of new tissue-engineered devices for dental and skeletal applications. This meeting would not be possible without the generous support from our sponsors. A big thank you goes to the National Institutes of Dental and Craniofacial Research (NIDCR, NIH), with additional funding provided from the NIH, including NIAMS, NIBIB and the Office of Rare Diseases, to the Orthopaedic Research and Educational Foundation (OREF) and to the University of Missouri Kansas City School of Dentistry. We would also like to acknowledge the generosity of the Alliance for Better Bone Health, Proctor and Gamble and the Réseau de recherche en santé buccodentaire et osseuse (RSBO) of Canada. Thanks also go to Wyeth, Eli Lilly, Institute Straumann and Medtronic. Lynda F. Bonewald, Kansas City, Mo., USA Co-chair, 9th ICCMBT Paul H. Krebsbach, Ann Arbor, Mich., USA Co-chair, 9th ICCMBT This special issue of Cells, Tissues, Organs highlights the science presented at the 9th International Conference on the Chemistry and Biology of Mineralized Tissues (ICCBMT). The 9th ICCBMT took place between November 4 and 8, 2007, at the Lakeway Inn Conference Resort in Austin, Tex., USA. This conference, which has been held every 3 years since 1980, emphasizes physicochemical, biological and clinical topics concerning the mineralization process. These include, for example, aspects of the cell and molecular biology of mineralized tissue formation, hormone and cytokine regulation of mineralized tissues, signaling pathways, structure and function of extracellular components of mineralized tissues, model systems of biomineralization, disorder and pathology of mineralized tissues, therapeutic approaches, and new technologies for studying and analyzing mineralization. The goal of the ICCBMT meetings is to disseminate state-of-the-art information about the structure, function and interactions of the mineral, matrix and cellular components in bones, teeth and other mineralized vertebrate and invertebrate tissues, while encouraging collaboration among investigators in different disciplines. This meeting, which attracted a multidisciplinary international audience, promoted and advanced the exchange of new ideas in several ways: by supporting participation of young investigators through travel awards, by providing a free and open forum for both oral and poster presentation of results of new scientific studies as well as by encouraging interaction between senior investigators and new investigators during informal discussions at meals, afternoon breaks and focused workshops. Published online: August 18, 2008
A new method for generating and modeling reduced-scale copolymer gradients by CVD is reported. By... more A new method for generating and modeling reduced-scale copolymer gradients by CVD is reported. By exploiting diffusion through confined channels, functionalized [2.2]paracyclophanes are copolymerized into their poly(p-xylylene) (PPX) analogues as a composition gradient. Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS) are used to verify the gradient composition profiles. Gradients are deposited on both flat substrates and 3-dimensional cylinders. Both the thickness and compositional profiles are fitted to a diffusion-based model using realistic physical parameters. The derived equation can be generalized and optimized for any copolymerization gradient through a confined geometry, thus allowing for broad applicability to other copolymer systems.
Human embryonic stem cells (hESCs) hold promise for tissue regeneration therapies by providing a ... more Human embryonic stem cells (hESCs) hold promise for tissue regeneration therapies by providing a potentially unlimited source of cells capable of undergoing differentiation into specified cell types. Several preclinical studies and a few clinical studies use human bone marrow stromal cells (hBMSCs) to treat skeletal diseases and repair damaged tissue. However, hBMSCs have limited proliferation and differentiation capacity, suggesting that an alternate cell source is desirable, and hESCs may serve this purpose. Here we describe a protocol for the reproducible derivation of mesenchymal stem cells from hESCs (hES-MSCs). The hES-MSCs have a similar immunophenotype to hBMSCs, specifically they are CD73+, STRO-1+ and CD45–, and are karyotypically stable. The derived hES-MSCs are also capable of differentiating into osteoblasts and adipocytes. When the hES-MSCs were genetically modified with the lineage-specific Col2.3-GFP lentivirus and cultured in osteogenic medium, increased GFP express...
Nematode EAK-7 (enhancer-of--1-7) regulates dauer formation and controls life span; however, the ... more Nematode EAK-7 (enhancer-of--1-7) regulates dauer formation and controls life span; however, the function of the human ortholog mammalian EAK-7 (mEAK-7) is unknown. We report that mEAK-7 activates an alternative mechanistic/mammalian target of rapamycin (mTOR) signaling pathway in human cells, in which mEAK-7 interacts with mTOR at the lysosome to facilitate S6K2 activation and 4E-BP1 repression. Despite interacting with mTOR and mammalian lethal with SEC13 protein 8 (mLST8), mEAK-7 does not interact with other mTOR complex 1 (mTORC1) or mTOR complex 2 (mTORC2) components; however, it is essential for mTOR signaling at the lysosome. This phenomenon is distinguished by S6 and 4E-BP1 activity in response to nutrient stimulation. Conventional S6K1 phosphorylation is uncoupled from S6 phosphorylation in response to mEAK-7 knockdown. mEAK-7 recruits mTOR to the lysosome, a crucial compartment for mTOR activation. Loss of mEAK-7 results in a marked decrease in lysosomal localization of mT...
Human embryonic stem cells subjected to a one-time uniaxial stretch for as short as 30-min on a f... more Human embryonic stem cells subjected to a one-time uniaxial stretch for as short as 30-min on a flexible substrate coated with Matrigel experienced rapid and irreversible nuclear-to-cytoplasmic translocation of NANOG and OCT4, but not Sox2. Translocations were directed by intracellular transmission of biophysical signals from cell surface integrins to nuclear CRM1 and were independent of exogenous soluble factors. On E-CADHERIN-coated substrates, presumably with minimal integrin engagement, mechanical strain-induced rapid nuclear-to-cytoplasmic translocation of the three transcription factors. These findings might provide fundamental insights into early developmental processes and may facilitate mechanotransduction-mediated bioengineering approaches to influencing stem cell fate determination.
Here, we used an ultrasound (US) technique, acoustic tweezing cytometry (ATC), to apply targeted ... more Here, we used an ultrasound (US) technique, acoustic tweezing cytometry (ATC), to apply targeted cyclic subcellular forces to hESCs via integrin-bound microbubbles (MBs). We found that ATCmediated cyclic forces applied for 30 min to hESCs near the edge of a colony induced immediate global responses throughout the colony, suggesting the importance of cell-cell connection in the mechanoresponsiveness of hESCs to ATC-applied forces. ATC application generated increased contractile force, enhanced calcium activity, as well as decreased expression of pluripotency transcription factors Oct4 and Nanog, leading to rapid initiation of hESC differentiation and characteristic epithelial-mesenchymal transition (EMT) events that depend on focal adhesion kinase (FAK) activation and cytoskeleton (CSK) tension. These results reveal a unique, rapid mechanoresponsiveness and community behavior of hESCs to integrin-targeted cyclic forces.
Self-renewal of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs)-know... more Self-renewal of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs)-known as pluripotent stem cells (PSC)-is influenced by culture conditions, including the substrate on which they are grown. However, details of the molecular mechanisms interconnecting the substrate and self-renewal of these cells remain unclear. We describe a signaling pathway in hPSCs linking self-renewal and expression of pluripotency transcription factors to integrin α6β1 and inactivation of focal adhesion kinase (FAK). Disruption of this pathway results in hPSC differentiation. In hPSCs, α6β1 is the dominant integrin and FAK is not phosphorylated at Y397, and thus, it is inactive. During differentiation, integrin α6 levels diminish and Y397 FAK is phosphorylated and activated. During reprogramming of fibroblasts into iPSCs, integrin α6 is upregulated and FAK is inactivated. Knockdown of integrin α6 and activation of β1 integrin lead to FAK phosphorylation and reduction of Nanog, Oct4, a...
Bioactive glasses are potentially useful as bone defect fillers, and vascular endothelial growth ... more Bioactive glasses are potentially useful as bone defect fillers, and vascular endothelial growth factor (VEGF) has demonstrated benefit in bone regeneration as well. We hypothesized that the specific combination of prolonged localized VEGF presentation from a matrix coated with a bioactive glass may enhance bone regeneration. To test this hypothesis, the capacity of VEGF-releasing polymeric scaffolds with a bioactive glass coating was examined in vitro and in vivo using a rat critical-sized defect model. In the presence of a bioactive glass coating, we did not detect pronounced differences in the differentiation of human mesenchymal stem cells in vitro. However, we observed significantly enhanced mitogenic stimulation of endothelial cells in the presence of the bioactive glass coating, with an additive effect with VEGF release. This trend was maintained in vivo, where coated VEGF-releasing scaffolds demonstrated significant improvements in blood vessel density at 2 weeks versus coated control scaffolds. At 12 weeks, bone mineral density was significantly increased in coated VEGF-releasing scaffolds versus coated controls, while only a slight increase in bone volume fraction was observed. The results of this study suggest that a bioactive glass coating on a polymeric substrate participates in bone healing through indirect processes which enhance angiogenesis and bone maturation and not directly on osteoprogenitor differentiation and bone formation. The mass of bioactive glass used in this study provides a comparable and potentially additive, response to localized VEGF delivery over early time points. These studies demonstrate a materials approach to achieve an angiogenic response formerly limited to the delivery of inductive growth factors.
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