[Show abstract][Hide abstract] ABSTRACT: The epidermis is mainly composed of keratinocytes forming a protective barrier. It is perpetually subjected to mechanical stress and strain during development, homeostasis and disease. Perturbation of the normal strain with alteration of its biological response may lead to severe diseases such as psoriasis and epidermolysis bullosa. To date, most of the studies about skin response to mechanical stress used immor-talized cell lines (i.e. HaCaT) or primary cells from donors, which suffer issues of limited physiological relevance and inter-donor variability. It is therefore necessary to develop a new human model for the study of normal skin physiology and response to mechanical stress. In this study, we investi-gated the use of keratinocytes derived from human embryonic stem cells (hESCs) as a reliable alternative model to HaCaT for study of the effects of mechanical tension. With compar-ison to HaCaT, hESC-derived keratinocytes (hESC-Kert) were exposed to up to 3 days of cyclic mechanical stress, and gene expression changes were analyzed. Dynamic expres-sion of several key mechanical stress related-genes was stud-ied at mRNA level using qPCR. The expression of matrix-metallopeptidase9 was studied at protein level using ELISA. The two cell types displayed similar gene expression kinetics for most of the genes including E-cadherin, cateninβ1, connexin43, desmoglein1, endothelin1, integrinα6, interleukinα1, keratin1, 6, and 10, keratinocyte growth-factor-receptor and lamininα5. Unlike HaCaT, hESC-Kert displayed early gene and protein expression of matrix metallopeptidase 9 following mechanical stimulation, sug-gesting that these cells have remodeling capacity that resem-bles that of normal human skin. Our study confirmed the use of hESC-Kert as a good model for study of skin response to mechanical stress.
Stem Cell Reviews and Reports 10/2014; · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To engineer constructs of the periodontal ligament (PDL), human PDL cells were incorporated into a matrix of hyaluronan, gelatin and type I collagen in sample holders (13 mm x 1mm) of 6-well Biopress culture plates. The loading dynamics of the PDL were mimicked by applying a cyclic compressive strain of 33.4 kPa (340.6 gm/cm2) to the constructs for 1.0 sec every 60 sec, for 6, 12 and 24h in a Flexercell FX-4000C Strain Unit. Compression significantly increased the number of nonviable cells and increased the expression of several apoptosis-related genes, including initiator and executioner caspases. Of the 15 extracellular matrix genes screened, most were upregulated at some point after 6‒12h deformation, but all were down-regulated at 24h except for MMPs1‒3 and CTGF. In culture supernatants, MMP-1 and TIMP-1 protein levels were upregulated at 24h; RANKL, OPG and FGF-2 were unchanged; and CTGF not detected. The low modulus of elasticity of the constructs was a disadvantage ‒ future mechanobiology studies and tissue engineering applications will require constructs with much higher stiffness. Since the major structural protein of the PDL is type I collagen, a more rational approach would be to permeabilize preformed type I collagen scaffolds with PDL-populated matricies.
[Show abstract][Hide abstract] ABSTRACT: Human embryonic stem cells (hESCs) have the ability to self-renew and differentiate into any cell lineage of the three germ layers, therefore holding great promise for regenerative applications in dentistry and medicine. We previously described a micromass culture system as a model system to induce and study the chondrogenic commitment of hESCs. Using this system, chondrogenic cells can be further isolated and expanded under specific growth factor conditions. When encapsulated in hyaluronic acid (HA)-based hydrogels and cultured under appropriate growth factor and medium conditions, these chondrogenic cells synthesized and deposited extracellular matrix (ECM) characteristic of neocartilage. Here, we describe the micromass culture of hESCs, the isolation and expansion of hESC-derived chondrogenic cells, and the three-dimensional (3-D) culture of the chondrogenic cells in hydrogels for cartilage tissue engineering. We will also describe the various tools and techniques used for characterizing the tissue-engineered cartilage.
[Show abstract][Hide abstract] ABSTRACT: This study evaluated human embryonic stem cells (hESC) and their differentiated fibroblastic progenies as cellular models for genotoxicity screening. The DNA damage response of hESCs and their differentiated fibroblastic progenies were compared to a fibroblastic cell line (HEPM, CRL1486) and primary cultures of peripheral blood lymphocytes (PBL), upon exposure to Mitomycin C, gamma irradiation and H2O2. It was demonstrated that hESC-derived fibroblastic progenies (H1F) displayed significantly higher chromosomal aberrations, micronuclei formation and double strand break (DSB) formation, as compared to undifferentiated hESC upon exposure to genotoxic stress. Nevertheless, H1F cell types displayed comparable sensitivities to genotoxic challenge as HEPM and PBL, both of which are representative of somatic cell types commonly used for genotoxicity screening. Subsequently, transcriptomic and pathways analysis identified differential expression of critical genes involved in cell death and DNA damage response upon exposure to gamma irradiation. The results thus demonstrate that hESC-derived fibroblastic progenies are as sensitive as commonly-used somatic cell types for genotoxicity screening. Moreover, hESCs have additional advantages, such as their genetic normality compared to immortalized cell lines, as well as their amenability to scale-up for producing large, standardized quantities of cells for genotoxicity screening on an industrial scale, something which can never be achieved with primary cell cultures.
Journal of Biotechnology 05/2014; · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction: The induced pluripotent stem cells (iPSCs) have characteristics similar to embryonic stem cells, including the capability of self-renewal and large-scale expansion and the ability to differentiate into all types of cells including germ cells, which defines pluripotency. Using iPSC avoids problems of immunological rejection and ethical controversy. The possible future uses of iPSC are diverse and go beyond the differentiation into somatic cells for regeneration of damaged tissues. Areas covered: A unique feature of iPSC is the potential to generate patient disease-specific tissues. Thus, cells from patients can be differentiated into relevant cells of interest for drug screening, characterization of drug effects and cytotoxic assays. This review presents key aspects related to iPSC, such as their generation, potential for disease modeling, treatment, drug development and future contributions to the craniofacial complex. Expert opinion: It is undisputable that the evolution in iPSC knowledge will improve the approaches for drug screening and development, help to understand and treat disease origins and mechanisms and provide new strategies to clinical treatment. However, it is necessary to fine-tune protocols to establish iPSCs that are cost-effective and safe for clinical use.
[Show abstract][Hide abstract] ABSTRACT: Scaffolds based on decellularized adipose tissue (DAT) are gaining popularity in adipose tissue engineering due to their high biocompatibility and adipogenic inductive property. However, previous studies involving DAT-derived scaffolds have not fully revealed their potentials for in vivo adipose tissue construction. With the aim of developing a more efficient adipose tissue engineering technique based on DAT, in this study, we investigated the in vivo adipogenic potential of a basic fibroblast growth factor (bFGF) delivery system based on heparinized DAT (Hep-DAT). To generate this system, heparins were cross-linked to mouse DATs by using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide and N-Hydroxysuccinimide. The bFGF-binding Hep-DATs were first tested for controlled release ability in vitro and then transplanted subcutaneously. Highly vascularized adipose tissues were formed 6weeks after transplantation. Histology and gene expression analysis revealed that majority of the Hep-DAT scaffolds were infiltrated with host-derived adipose tissues that possessed similar adipogenic and inflammatory gene expression as endogenous adipose tissues. Additionally, strong de novo adipogenesis could also be induced when bFGF-binding Hep-DATs were thoroughly minced and injected subcutaneously. In conclusion, our study demonstrated that bFGF-binding Hep-DAT could be an efficient, biocompatible and injectable adipogenic system for in vivo adipose tissue engineering.
Journal of Controlled Release 11/2013; · 7.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To understand, in greater detail, the molecular mechanisms regulating the complex relationship between mechanical strain and alveolar bone metabolism during orthodontic treatment, passive cross-arch palatal springs were bonded to the maxillary molars of 6-wk-old rats, which were killed after 4 and 8 d. Outcome measures included serum assays for markers of bone formation and resorption and for the multifunctional hormone leptin, and histomorphometry of the inter-radicular bone. The concentration of the bone-formation marker alkaline phosphatase (ALP) was significantly reduced at both time points in the appliance group, accompanied by a 50% reduction in inter-radicular bone volume; however, osteocalcin (bone Gla protein) levels remained unaffected. Bone collagen deoxypyridinoline (DPD) crosslinks increased 2.3-fold at 4 d only, indicating a transient increase in bone resorption; in contrast, the level of the osteoclast-specific marker, tartrate-resistant acid phosphatase 5b (TRACP 5b), was unchanged. Leptin levels closely paralleled ALP reductions at both time points, suggesting an important role in the mechanostat negative-feedback loop required to normalize bone mass. These data suggest that an orthodontic appliance, in addition to remodeling the periodontal ligament (PDL)-bone interface, may exert unexpected side-effects on the tooth-supporting alveolar bone, and highlights the importance of recognizing that bone strains can have negative, as well as positive, effects on bone mass.
European Journal Of Oral Sciences 09/2013; · 1.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to differentiate human embryonic stem cells (hESCs) into odontoblastic lineage in an optimized culture milieu.
In Phase 1, hESCs were differentiated into mesenchymal stem cells (H9-MSCs). In Phase 2, H9-MSCs were then differentiated into odontoblast-like cells (H9-Odont) under the stimulation of FGF-8 and BMP-4. Alternatively, H9-MSCs were differentiated into osteogenic lineage (H9-Osteo). In Phase 3, H9-Odont were seeded on 17% EDTA-treated dentine substrates in the presence of FGF-8 and BMP-4 for further differentiation. All experiments were performed in triplicate (n = 3). One-way anova was used to test hESC differentiation into different cell types. Post hoc Tukey's test was used to compare between groups. P < 0.05 was considered statistically significant.
H9-Odont expressed the odontoblastic marker DSPP gene 125.47 ± 0.1 (SD)-folds higher compared with H9-MSCs at mRNA level (real-time RT-PCR). Additionally, the flow cytometry results revealed 53.1 ± 3.4 (SD) % of DSP (+) cells in H9-Odont. Alternatively, H9-Osteo expressed 5.9 ± 2.2 (SD) % of DSP (+) cells. Moreover, the SEM results demonstrated that H9-Odont were found to undergo morphological changes from a fibroblast-like shape into more rounded shapes with cytoplasmic extensions into the dentinal tubules when seeded on 17% EDTA-treated dentine substrate in the presence of FGF-8 and BMP-4. However, H9-Osteo and H9-MSCs did not show similar morphological changes under similar culture milieu.
This study supports the potential of hESCs as a stable, consistent, unlimited and 'off-the-shelf' cell source to obtain odontoblastic cells for future clinical and research applications.
International Endodontic Journal 08/2013; · 2.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Excessive bone marrow adipocytes formation is tightly associated with development of osteoporosis. Considering the high heterogeneity of bone marrow stromal cells (BMSCs), identification of bone marrow adipocyte progenitors (BMAPs) within heterogeneous BMSCs may provide better cellular models for researches concerning osteoporosis development and therapy. However, currently there is no efficient method or specific surface makers available for BMAPs isolation. In the current study, we developed a novel BMAPs isolation method based on silica microbeads incubation and subsequent centrifugation in ficoll-paque. The "Sca-1+CD73-CD90-CD105+" subpopulation selected by this method exhibited significantly stronger adipogenic potential than non-selected BMSCs in vitro and could homogeneously differentiate to mature adipocytes within 4 days. Moreover, these cells also highly expressed a series of adipogenesis related genes even before differentiation. After long-term culture, however, BMAPs would gradually lose high adipogenic ability, but sorting CD105+ cells from BMAPs in later passages was able to retrieve the highly adipogenic subpopulation. In conclusion, this study demonstrated that BMAPs subpopulation could be effectively isolated from heterogeneous BMSCs by a special silica microbeads incubation method and re-enriched by sorting CD105+ cells. These findings offer convenient and repeatable approaches to obtain pure BMAPs for researches concerning pathogenic mechanisms and therapeutics development of osteoporosis.
Stem cells and development 06/2013; · 4.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human embryonic stem cells (hESCs) are uniquely endowed with a capacity for both self-renewal and multi-lineage differentiation. The aim of this investigation was to determine if short periods of cyclic mechanical strain enhanced dexamethasone, ascorbic acid and β-glycerophosphate (triple supplement) induced osteogenesis and bone nodule formation by hESCs. Colonies were cultured for 21 days and divided into control (no stretch) and three treatment groups; these were subjected to in-plane deformation of 2% for 5 sec (0.2 Hertz) every 60 sec for 1 hr on alternate days in BioFlex plates linked to a Flexercell strain unit over the following periods (Day 7-13), (D15-21) and (D7-21). Numerous bone nodules were formed which stained positively for osteocalcin and type I collagen; in addition, MTS assays for cell number as well as total collagen assays showed a significant increase in the D7-13 group compared to controls and other treatment groups. Alizarin red staining further showed that cyclic mechanical stretching significantly increased nodule size and mineral density between days 7-13 compared to control cultures and the other two experimental groups. We then performed a real time-PCR microarray on the D7-13 treatment group to identify mechanoresponsive osteogenic genes. Up-regulated genes included the transcription factors RUNX2 and SOX9, bone morphogenetic proteins BMP1, BMP4, BMP5, BMP6, transforming growth factor-β family members TGFB1, TGFB2 and TGFB3, and three genes involved in mineralization - ALPL, BGLAP and VDR. In conclusion, this investigation has demonstrated that four one hour episodes of cyclic mechanical strain acted synergistically with triple supplement to enhance osteogenesis and bone nodule formation by cultured hESCs. This suggests the development of methods to engineer 3-dimensional constructs of mineralized bone in vitro, could offer an alternative approach to osseous regeneration by producing a biomaterial capable of providing stable surfaces for osteoblasts to synthesize new bone, while at the same time able to be resorbed by osteoclastic activity - in other words, one that can recapitulate the remodelling dynamics of naturally occurring bone matrix.
Tissue Engineering Part A 04/2013; · 4.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND AND OBJECTIVE: Periodontal ligament (PDL) cells in stationary two-dimensional culture systems are in a double default state. Our aim therefore was to engineer and characterize three-dimensional constructs, by seeding PDL cells into hyaluronan-gelatin hydrogel films (80-100 μm) in a format capable of being mechanically deformed. MATERIAL AND METHODS: Human PDL constructs were cultured with and without connective tissue growth factor (CTGF) and fibroblast growth factor (FGF)-2 in (i) stationary cultures, and (ii) mechanically active cultures subjected to cyclic strains of 12% at 0.2 Hz each min, 6 h/d, in a Flexercell FX-4000 Strain Unit. The following parameters were measured: cell number and viability by laser scanning confocal microscopy; cell proliferation with the MTS assay; the expression of a panel of 18 genes using real-time RT-PCR; matrix metalloproteinases (MMPs) 1-3, TIMP-1, CTGF and FGF-2 protein levels in supernatants from mechanically activated cultures with Enzyme-linked immunosorbent assays. Constructs from stationary cultures were also examined by scanning electron microscopy and immunostained for actin and vinculin. RESULTS: Although initially randomly distributed, the cells became organized into a bilayer by day 7; apoptotic cells remained constant at approximately 5% of the total. CTGF/FGF-2 stimulated cell proliferation in stationary cultures, but relative quantity values suggested modest effects on gene expression. Two transcription factors (RUNX2 and PPARG), two collagens (COL1A1, COL3A1), four MMPs (MMP-1-3, TIMP-1), TGFB1, RANKL, OPG and P4HB were detected by gel electrophoresis and Ct values < 35. In mechanically active cultures, with the exception of P4HB, TGFB1 and RANKL, each was upregulated at some point in the time scale, as was the synthesis of MMPs and TIMP-1. SOX9, MYOD, SP7, BMP2, BGLAP or COL2A1 were not detected in either stationary or mechanically active cultures. CONCLUSION: Three-dimensional tissue constructs provide additional complexity to monolayer culture systems, and suggest some of the assumptions regarding cell growth, differentiation and matrix turnover based on two-dimensional cultures may not apply to cells in three-dimensional matrices. Primarily developed as a transitional in vitro model for studying cell-cell and cell-matrix interactions in tooth support, the system is also suitable for investigating the pathogenesis of periodontal diseases, and importantly from the clinical point of view, in a mechanically active environment.
Journal of Periodontal Research 04/2013; · 1.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: The innate immune response (IMR) is critical for the oral mucosa due to their continuous exposure to various oral pathogens. Keratinocytes play important role in IMR. Therefore, to date, keratinocytes from different sources have been used as in vitro research model for the study of IMR. However, current keratinocyte research models are hampered by the limited supply, patients' dependency and batch to batch variation. Therefore, in this study, we demonstrated the use of human embryonic stem cells (hESCs) derived keratinocytes (H9-Kert) as an alternative research model for the study of IMR. METHODS: The expression kinetics of toll-like receptor (TLR) 2, TLR 4, interleukin (IL) -6, IL-8, inducible nitric oxide synthase (iNOS) and tumour necrosis factor-alpha (TNF-α), in H9-Kert and immortalized human keratinocyte cell line (HaCaT) were analysed at mRNA levels by both reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. The activation of the inflammatory transcription factor nuclear factor kappa-b (NFĸB) was assayed in these cells by transiently transfecting the cells with NFĸB reporter plasmid. Activation of NFĸB following treatment with heat-killed Porphyromonas gingivalis (P. gingivalis), an oral pathogen, was determined by assaying for the reporter, secreted alkaline phosphatase activity. RESULTS: The expression of TLRs, cytokines and activation of NFĸB following bacterial stimulation showed in both H9-Kert and the widely used HaCaT keratinocyte cell line was similar. CONCLUSION: Overall, our results support the potential application of hESCs as an alternative limitless cell source for primary keratinocytes which can be used as consistent and dependable research tool with minimum variations and no donor's dependency.
Journal of Oral Pathology and Medicine 03/2013; · 2.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The current international standard for toxicity screening of biomedical devices and materials recommend the use of immortalized cell lines because of their homogeneous morphologies and infinite proliferation which provide good reproducibility for in vitro cytotoxicity screening. However, most of the widely used immortalized cell lines are derived from animals and may not be representative of normal human cell behavior in vivo, in particular in terms of the cytotoxic and genotoxic response. Therefore, It is vital to develop a model for toxicity evaluation. In our studies, two Chinese human embryonic stem cell (hESC) lines as toxicity model were established. hESC derived tissue/organ cell model for tissue/organ specific toxicity evaluation were developed. The efficiency and accuracy of using hESC model for cytoxicity, embryotoxicity and genotoxicity evaluation were confirmed. The results indicated that hESCs might be good tools for toxicity testing and biosafety evaluation in vitro.
Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences 02/2013; 45(1):9-11.
[Show abstract][Hide abstract] ABSTRACT: The vertebrae mesoderm is a source of cells that forms a variety of tissues including the heart, vasculature and blood. Consequently, the derivation of various mesoderm-specific cell types from human embryonic stem cells (hESCs) has attracted the interest of many investigators owing to their therapeutic potential in clinical applications. However, the need for efficient and reliable methods of differentiation into mesoderm lineage cell types remains a significant challenge. Here, we demonstrated that inhibition of glycogen synthase kinase-3 (GSK-3) is an essential first step towards efficient generation of the mesoderm. Under chemically defined conditions without additional growth factors/cytokines, short-term GSKi (GSK inhibitor) treatment effectively drives differentiation of hESCs into the primitive streak (PS), which can potentially commit towards the mesoderm when further supplemented with BMP4. Further analysis confirmed that the PS-like cells derived from GSKi treatment are bipotential, being able to specify towards the endoderm as well. Our findings suggest that the bipotential, PS/mesendoderm-like cell population exists only at the initial stages of GSK-3 inhibition whereas long term inhibition results in an endodermal fate. Lastly, we demonstrated that our differentiation approach could efficiently generate lateral plate (CD34+KDR+) and paraxial (CD34-PDGFRα+) mesoderm subsets that can be further differentiated along the endothelial and smooth muscle lineages respectively. In conclusion, our study presents a unique approach for generating early mesoderm progenitors in a chemically directed fashion through the use of small molecule GSK-3 inhibitor, which may be useful for future applications in regenerative medicine.
Stem cells and development 02/2013; · 4.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human embryonic stem cells (hESCs)-derived keratinocytes hold great clinical and research potential. However, the current techniques are hampered by the use of xenogenic components that limits their clinical application. Here we demonstrated an efficient differentiation of H9 hESCs (H9-hESCs) into keratinocytes (H9-Kert) with the minimum use of animal-derived materials. For differentiation, we established two microenvironment systems originated from H9-hESCs (autogenic microenvironment). These autogenic microenvironment systems consist of an autogenic coculture system (ACC) and an autogenic feeder-free system (AFF). In addition, we showed a stage-specific effect of Activin in promoting keratinocyte differentiation from H9-hESCs while repressing the expression of early neural markers in the ACC system. Furthermore, we also explained the effect of Activin in construction of the AFF system made up of extracellular matrix similar to basement membrane extracted from H9-hESC-derived fibroblasts. H9-Kert differentiated in both systems expressed keratinocyte markers at mRNA and protein levels. H9-Kert were also able to undergo terminal differentiation in high Ca(2+) medium. These findings support the transition toward the establishment of an animal-free microenvironment for successful differentiation of hESCs into keratinocytes for potential clinical application.Journal of Investigative Dermatology advance online publication, 13 December 2012; doi:10.1038/jid.2012.384.
Journal of Investigative Dermatology 12/2012; · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cell fate determination and development is a biology question that has yet to be fully answered. During embryogenesis and in vivo stem cell differentiation, cells/tissues deploy epigenetic mechanisms to accomplish differentiation and give rise to the fully developed organism. Although a biochemistry description of cellular genetics and epigenetics is important, additional mechanisms are necessary to completely solve the problem of embryogenesis, especially differentiation and the spatiotemporal coordination of cells/tissues during morphogenesis. The cell state splitter and differentiation wave working-model was initially proposed to explain the homeostatic primary neural induction in amphibian embryos. Here the model is adopted to explain experimental findings on in vitro embryonic stem cell, pluripotency and differentiation. Moreover, since somatic cells can be reverted to a stem-cell-like pluripotent state through the laboratory procedure called epigenetic reprogramming, erection of a cell state splitter could be a key event in their successful reprogramming. Overall, the cell state splitter working-model introduces a bistable cytoskeletal mechanism that partially explains cell fate determination and biological development. It offers an interdisciplinary framework that bridges the gap between molecular epigenetics and embryogenesis.
Bio Systems 06/2012; 109(3):390-6. · 1.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the use of differentiated fibroblasts from human embryonic stem cells as a cellular model for cytotoxicity and genotoxicity screening. The EBf-H9 cells were derived from human embryonic stem cells (H9) via embryonic body (EB) and treated with Sodium fluoride (NaF) and Formaldehyde (FA). Proliferation, specific gene and protein expression and karyotype of cells were analyzed by MTT assay, RT-PCR, immunocytochemistry and karyotype analysis, respectively. Cytotoxicity was detected by MTT assay and flow cytometry, and genotoxicity was studied by micronucleus test (MNT), sister chromatid exchange (SCE) and comet assay. EBf-H9s were spindle-shaped with a diploid karyotype. They expressed the fibroblast markers prolyl 4-hydroxylase β and vimentin but did not express Oct-4 and Sox-2, and decreased expression of Nanog. The proliferation of EBf-H9 and murine L929 cells was inhibited by sodium fluoride (NaF) and formaldehyde (FA), and the cell cycle was arrested in different phases with the treatments. In genotoxicity assays with NaF and FA, positive responses were detected in human EBf-H9s comparable to those in the murine L929 cell line. EBf-H9 may be a suitable new cell source for toxicity research on biomaterials and other agents.
Toxicology in Vitro 04/2012; 26(6):1056-63. · 2.65 Impact Factor