Efficacy of adjunctive modalities during tooth extraction for the prevention of osteoradionecrosis: A systematic review and meta-analysis.

BACKGROUND: Jaw osteoradionecrosis (ORN) is a complication in patients with previous head and neck radiotherapy. Its incidence increases with dental extractions. Hence, this review aimed to evaluate the efficacy of adjunctive treatment modalities undertaken at the time of extraction in previous head and neck radiotherapy patients in preventing ORN. METHODS: A systematic review was conducted, where studies with data on ORN incidence after extraction with or without adjunctive interventions were included. Meta-analyses were conducted to estimate the pooled prevalence of ORN per intervention and the pooled odds ratio for incidence of ORN between interventions. RESULTS: In total, 1520 patients in 29 studies were included. Interventions identified were hyperbaric oxygen (HBO), pentoxifylline-tocopherol (PENTO), antibiotics (ABX), platelet-rich fibrin and photobiomodulation. The pooled prevalence of ORN for HBO (4.6%), PENTO (3.4%) and ABX (3.8%) was significantly lower than the Control (17.6%). For studies with direct comparisons between groups, HBO had lower but not significant odds of developing ORN than the Control (OR 0.27) and ABX (OR 0.57). CONCLUSIONS: HBO, PENTO and ABX may reduce the incidence of ORN compared to no intervention. Given that all three have similar incidences of ORN, ABX may be the most cost-effective and accessible adjunctive modality.

Deep Convolutional Clustering-Based Time Series Anomaly Detection.

This paper presents a novel approach for anomaly detection in industrial processes. The system solely relies on unlabeled data and employs a 1D-convolutional neural network-based deep autoencoder architecture. As a core novelty, we split the autoencoder latent space in discriminative and reconstructive latent features and introduce an auxiliary loss based on k-means clustering for the discriminatory latent variables. We employ a Top-K clustering objective for separating the latent space, selecting the most discriminative features from the latent space. We use the approach to the benchmark Tennessee Eastman data set to prove its applicability. We provide different ablation studies and analyze the method concerning various downstream tasks, including anomaly detection, binary and multi-class classification. The obtained results show the potential of the approach to improve downstream tasks compared to standard autoencoder architectures.

Efficient derivation of dopaminergic neurons from SOX1⁻ floor plate cells under defined culture conditions.

BACKGROUND: Parkinson's disease (PD) is a severe neurodegenerative disease associated with loss of dopaminergic neurons. Derivation of dopaminergic neurons from human embryonic stem cells (hESCs) could provide new therapeutic options for PD therapy. Dopaminergic neurons are derived from SOX(-) floor plate (FP) cells during embryonic development in many species and in human cell culture in vitro. Early treatment with sonic hedgehog (Shh) has been reported to efficiently convert hESCs into FP lineages. METHODS: In this study, we attempted to utilize a Shh-free approach in deriving SOX1(-) FP cells from hESCs in vitro. Neuroectoderm conversion from hESCs was achieved with dual inhibition of the BMP4 (LDN193189) and TGF-ß signaling pathways (SB431542) for 24 h under defined culture conditions. RESULTS: Following a further 5 days of treatment with LDN193189 or LDN193189 + SB431542, SOX1(-) FP cells constituted 70-80 % of the entire cell population. Upon treatment with Shh and FGF8, the SOX1(-) FP cells were efficiently converted to functional Nurr1(+) and TH(+) dopaminergic cells (patterning), which constituted more than 98 % of the entire cell population. However, when the same growth factors were applied to SOX1(+) cells, only less than 4 % of the cells became Nurr1(+), indicating that patterning was effective only if SOX1 expression was down-regulated. After transplanting the Nurr1(+) and TH(+) cells into a hemiparkinsonian rat model, significant improvements were observed in amphetamine induced ipslateral rotations, apomorphine induced contra-lateral rotations and Rota rod motor tests over a duration of 8 weeks. CONCLUSIONS: Our findings thus provide a convenient approach to FP development and functional dopaminergic neuron derivation.

Pluripotent stem cells: An in vitro model for nanotoxicity assessments.

The advent of technology has led to an established range of engineered nanoparticles that are used in diverse applications, such as cell-cell interactions, cell-material interactions, medical therapies and the target modulation of cellular processes. The exponential increase in the utilization of nanomaterials and the growing number of associated criticisms has highlighted the potential risks of nanomaterials to human health and the ecosystem. The existing in vivo and in vitro platforms show limitations, with fluctuations being observed in the results of toxicity assessments. Pluripotent stem cells (PSCs) are viable source of cells that are capable of developing into specialized cells of the human body. PSCs can be efficiently used to screen new biomaterials/drugs and are potential candidates for studying impairments of biophysical morphology at both the cellular and tissue levels during interactions with nanomaterials and for diagnosing toxicity. Three-dimensional in vitro models obtained using PSC-derived cells would provide a realistic, patient-specific platform for toxicity assessments and in drug screening applications. The current review focuses on PSCs as an alternative in vitro platform for assessing the hazardous effects of nanomaterials on health systems and highlights the importance of PSC-derived in vitro platforms. Copyright © 2016 John Wiley & Sons, Ltd.

Antibiotic prophylaxis prescribing practices of dentists in Singapore.

BACKGROUND: Infective endocarditis is a serious complication that results in significant morbidity and mortality in susceptible patients. The guidelines for antibiotic prophylaxis have been updated by the American Heart Association and National Institute for Health and Clinical Excellence. The antibiotic prophylaxis prescribing practices among dentists in Singapore are unknown. AIM: To determine the specific infective endocarditis antibiotic prophylaxis prescribing practices of dentists in Singapore. METHODS: A questionnaire survey was sent through an email link and by postal mail. Statistical analysis was carried out using SPSS 19.0. RESULTS: Responses were received from 458 dentists (34.3% response rate), of which 278 (65.9%) were general practitioners. The majority of respondents (39.8%) followed the American Heart Association 2007 guidelines and 30.2% followed cardiologists' recommendations. The accuracy of prescriptions for 13 cardiac conditions and 12 dental procedures were evaluated. The median number of accurate answers for cardiac conditions was eight for the American Heart Association 1999 guidelines, and four for the American Heart Association 2007 and National Institute for Health and Clinical Excellence guidelines, respectively. The median number of accurate answers for dental procedures was generally high, both for dentists who followed the American Heart Association 1999 guidelines (median = 10) and American Heart Association 2007 (median = 9) guidelines. Majority of respondents (82.8%) felt that developing a local guideline would be beneficial to the local dental community. CONCLUSION: Dentists were accurate in their prescriptions of antibiotic prophylaxis for dental procedures, but not for cardiac conditions. It may be helpful to attain a consensus among local cardiologists and dentists to unify the antibiotic prophylaxis prescription practices in Singapore.

Implant presence-triggered osteonecrosis: A scoping review.

Unlike medication-related osteonecrosis of the jaw (MRONJ), implant presence-triggered osteonecrosis of the jaw (IPTO) is not well appreciated. Recent reports have suggested a mechanical aetiology unique to osseointegrated dental implants that may be responsible for this phenomenon. A scoping review was performed to consolidate the available evidence. Two reviewers independently searched the PubMed, EMBASE, CINAHL, and Cochrane Library databases. The study was performed according to the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) extension for scoping reviews. Studies that described or evaluated the development of IPTO in osseointegrated implants, which were placed prior to the commencement of anti-resorptive or anti-angiogenic agents, were included. Twenty-three (23) articles were included in this study. Patient characteristics, aetiopathogenesis, presentation, and treatment of the disease were evaluated. Most studies suggested a 6-month period between dental implant insertion and the commencement of anti-resorptive therapy as a criterion for IPTO. Both infective and mechanical processes were reported to be involved in the pathogenesis of IPTO. Most patients required surgical intervention to achieve resolution. While there are several knowledge gaps regarding IPTO, the evidence points towards a continuum in the pathogenesis of the disease, whereby there is a mechanical cause followed by secondary infection. Similar to typical MRONJ, the severity and treatment required also vary. Persistent peri­implantitis features around a dental implant should alert the clinician to the possibility of IPTO in patients taking anti-resorptive or anti-angiogenic agents. Prompt identification of the disease may play a role in timely management or appropriate referrals.

Oral Cancer Awareness of Tertiary Education Students and General Public in Singapore.

OBJECTIVES: Oral cancer confers high morbidity and mortality rates. Late diagnosis of oral cancer is linked to a lack of awareness of its existence and known risk factors. The purpose of this survey was to examine the knowledge and awareness of oral cancer amongst different groups in Singapore. METHODS: A self-administered questionnaire (including questions on awareness, risk factor knowledge, and health beliefs about oral cancer) was distributed to undergraduate students from the medical and dental schools and other faculties at the National University of Singapore, as well as the general public. RESULTS: A total of 470 responses were analysed. Both medical and dental students were almost universally aware of the disease and correctly identified recognised risk factors for oral cancers. Dental students had a significantly higher level of knowledge of chewing betel quid as a risk factor than medical students (98% vs 74%; P < .0001), although 1 in 10 dental students did not identify alcohol as a risk factor. In contrast, undergraduate students from other faculties were the least aware of oral cancer (62%). Within the general public, knowledge of the risk factors of oral cancer aside from smoking was low, with only 41% aware of viruses as a possible aetiology. However, the younger population group, aged 18 to 34 years old, in general had better knowledge of the risk factors of oral cancer compared with older participants. CONCLUSIONS: There is a general lack of awareness about oral cancer and its associated risk factors amongst certain cohorts of the Singapore population. There exists room for further targeted education.

Fabrication of vascularized tissue constructs under chemically defined culture conditions.

Three-dimensional (3D) biofabrication techniques that enable the production of multicellular tissue equivalents for applications in basic biology, drug screening and regenerative medicne. Fabrication of these tissue constructs with in-built microvasculature enables recapitulation of the biological environment of the native tissues. Here, we present the fabrication of 3D vascularized tissue constructs containing microvascular networks using human embryonic stem cell (hESC)-derived endothelial cells (ECs) and pericytes encapsulated within a fibrin-based matrix and cultured under chemically defined conditions. Firstly, by manipulating the developmental signaling pathways under chemically defined culture conditions, hESCs were efficiently differentiated to hESC-ECs and hESC-pericytes through intermediate stages of lateral plate and paraxial mesoderm respectively. Next, encapsulation of these hESC-derived vascular cells within fibrin-based matrix and culture under chemically defined conditions, result in self-assembly of hESC-ECs into a network of microvessels within a period of 6-9 d. With the supporting influence of hESC-pericytes, the microvascular network with lumen was stable for at least 3 weeks. Quantification of the fractal dimensions of the microvascular networks demonstrate the increasing complexity of the vascular network with increasing endothelial cell densities. Dextran permeation studies in the presence or absence of vasodilating agent (histamine) showed the presence of hollow lumen, modulation of barrier properties of the microvasculature and its functional response to histamine. Hence, this versatile in vitro 3D model of vascularized constructs generated under chemically defined conditions is well suited to study early angiogenesis for in vitro drug testing applications and provide a clinically amenable, fundamental step towards fabrication of complex and functional tissues for regenerative applications in the future.

CVD-grown monolayer graphene induces osteogenic but not odontoblastic differentiation of dental pulp stem cells.

OBJECTIVE: The objective was to investigate the potential of graphene (Gp) to induce odontogenic and osteogenic differentiation in dental pulp stem cells (DPSC). METHODS: Gp was produced by chemical vapor deposition. DPSC were seeded on Gp or glass (Gl). Cells were maintained in culture medium for 28 days. Every two days, culture medium from Gp was used to treat cells on Gl and vice versa. Mineralization and differentiation of DPSC on all substrates were evaluated after 14 and 28 days by alizarin red S staining, qPCR, immunofluorescence and FACS. Statistics were performed with two-way ANOVA and multiple comparisons were performed using Tukey's post hoc test at a pre-set significance level of 5%. RESULTS: After 14 and 28 days, Gp induced higher levels of mineralization as compared to Gl. Odontoblastic genes (MSX-1, PAX and DMP) were down-regulated and osteogenic genes and proteins (RUNX2, COL and OCN) were significantly upregulated on Gp comparing to Gl (p<0.05 for all cases). Medium from Gp induced downregulation of odontoblastic genes and increased bone-related gene and protein on Gl. SIGNIFICANCE: Graphene induced osteogenic and not odontoblastic differentiation of DPSC without the use of chemical inducers for osteogenesis. Graphene has the potential to be used as a substrate for craniofacial bone tissue engineering research.

Comparison of the physical and mechanical properties of MTA and portland cement.

This study evaluated and compared the pH, radiopacity, setting time, solubility, dimensional change, and compressive strength of ProRoot MTA (PMTA), ProRoot MTA (tooth colored formula) (WMTA), white Portland cement (WP), and ordinary Portland cement (OP). The results showed that PMTA and Portland cement have very similar physical properties. However, the radiopacity of Portland cement is much lower than that of PMTA. The compressive strength of PMTA was greater than Portland cement at 28 days. The major constituent of PMTA is Portland cement. Given the low cost of Portland cement and similar properties when compared to PMTA, it is reasonable to consider Portland cement as a possible substitute for PMTA in endodontic applications. However, industrially manufactured Portland cement is not approved currently for use in the United States and therefore no clinical recommendation can be made for its use in the human body. Further in vitro and in vivo tests, especially with regards its biocompatibility, should be conducted to ascertain if it meets the FDA requirements for use as a medical device.

Pluripotency of Stem Cells from Human Exfoliated Deciduous Teeth for Tissue Engineering.

Stem cells from human exfoliated deciduous teeth (SHED) are highly proliferative pluripotent cells that can be retrieved from primary teeth. Although SHED are isolated from the dental pulp, their differentiation potential is not limited to odontoblasts only. In fact, SHED can differentiate into several cell types including neurons, osteoblasts, adipocytes, and endothelial cells. The high plasticity makes SHED an interesting stem cell model for research in several biomedical areas. This review will discuss key findings about the characterization and differentiation of SHED into odontoblasts, neurons, and hormone secreting cells (e.g., hepatocytes and islet-like cell aggregates). The outcomes of the studies presented here support the multipotency of SHED and their potential to be used for tissue engineering-based therapies.

In Vitro Osteogenic Potential of Green Fluorescent Protein Labelled Human Embryonic Stem Cell-Derived Osteoprogenitors.

Cellular therapy using stem cells in bone regeneration has gained increasing interest. Various studies suggest the clinical utility of osteoprogenitors-like mesenchymal stem cells in bone regeneration. However, limited availability of mesenchymal stem cells and conflicting evidence on their therapeutic efficacy limit their clinical application. Human embryonic stem cells (hESCs) are potentially an unlimited source of healthy and functional osteoprogenitors (OPs) that could be utilized for bone regenerative applications. However, limited ability to track hESC-derived progenies in vivo greatly hinders translational studies. Hence, in this study, we aimed to establish hESC-derived OPs (hESC-OPs) expressing green fluorescent protein (GFP) and to investigate their osteogenic differentiation potential in vitro. We fluorescently labelled H9-hESCs using a plasmid vector encoding GFP. The GFP-expressing hESCs were differentiated into hESC-OPs. The hESC-OPsGFP+ stably expressed high levels of GFP, CD73, CD90, and CD105. They possessed osteogenic differentiation potential in vitro as demonstrated by increased expression of COL1A1, RUNX2, OSTERIX, and OPG transcripts and mineralized nodules positive for Alizarin Red and immunocytochemical expression of osteocalcin, alkaline phosphatase, and collagen-I. In conclusion, we have demonstrated that fluorescently labelled hESC-OPs can maintain their GFP expression for the long term and their potential for osteogenic differentiation in vitro. In future, these fluorescently labelled hESC-OPs could be used for noninvasive assessment of bone regeneration, safety, and therapeutic efficacy.

Innate Immune Response of Human Embryonic Stem Cell-Derived Fibroblasts and Mesenchymal Stem Cells to Periodontopathogens.

Periodontitis involves complex interplay of bacteria and host immune response resulting in destruction of supporting tissues of the tooth. Toll-like receptors (TLRs) play a role in recognizing microbial pathogens and eliciting an innate immune response. Recently, the potential application of multipotent stem cells and pluripotent stem cells including human embryonic stem cells (hESCs) in periodontal regenerative therapy has been proposed. However, little is known about the impact of periodontopathogens on hESC-derived progenies. This study investigates the effects of heat-killed periodontopathogens, namely, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, on TLR and cytokine expression profile of hESC-derived progenies, namely, fibroblasts (hESC-Fib) and mesenchymal stem cells (hESC-MSCs). Additionally, the serotype-dependent effect of A. actinomycetemcomitans on hESC-derived progenies was explored. Both hESC-Fib and hESC-MSCs constitutively expressed TLR-2 and TLR-4. hESC-Fib upon exposure to periodontopathogens displayed upregulation of TLRs and release of cytokines (IL-1ß, IL-6, and IL-8). In contrast, hESC-MSCs were largely nonresponsive to bacterial challenge, especially in terms of cytokine production. Further, exposure of hESC-Fib to A. actinomycetemcomitans serotype c was associated with higher IL-8 production than serotype b. In contrast, the hESC-MSCs displayed no serotype-dependent response. Differential response of the two hESC progenies implies a phenotype-dependent response to periodontopathogens and supports the concept of immunomodulatory properties of MSCs.

Graphene oxide-based substrate: physical and surface characterization, cytocompatibility and differentiation potential of dental pulp stem cells.

OBJECTIVE: The aim of this study was to evaluate the cytotoxicity and differentiation potential of a graphene oxide (GO)-based substrate using dental pulp stem cell (DPSC). METHODS: GO was obtained via chemical exfoliation of graphite using the modified Hummer's method and dispersed in water-methanol solution. 250µL of 1.5mg/mL solution were added to a cover slip and allowed to dry (25°C, 24h). GO-based substrate was characterized by Raman spectroscopy, AFM and contact angle. DPSC were seeded on GO and glass (control). Cell attachment and proliferation were evaluated by polymeric F-actin staining, SEM and MTS assay for five days. mRNA expression of MSX-1, PAX-9, RUNX2, COL I, DMP-1 and DSPP were evaluated by qPCR (7 and 14 days). Statistical analyses were performed by either Mann-Whitney, one or two-way Anova followed by and Tukey's post hoc analysis (α=0.05). RESULTS: Peaks at 1587cm(-1) and 1340cm(-1) (G and D band) and ID/IG of 0.83 were observed for GO with Raman. AFM showed that GO was randomly deposited and created a rougher surface comparing to the control. Cells successfully adhered on both substrates. There was no difference in cell proliferation after 5 days. Cells on GO presented higher expression for all genes tested except MSX-1 and RUNX2 for 7 days. SIGNIFICANCE: GO-based substrate allowed DPSC attachment, proliferation and increased the expression of several genes that are upregulated in mineral-producing cells. These findings open opportunities to the use of GO alone or in combination with dental materials to improve their bioactivity and beyond.

Properties of a new root-end filling material.

The purposes of this investigation were 2-fold: to study the physical properties and sealing ability of Viscosity Enhanced Root Repair Material (VERRM); and, to compare them with Mineral Trioxide Aggregate (MTA). VERRM has a composition similar to mineral trioxide aggregate, with handling characteristics and consistency similar to commercially available materials such as IRM and Super EBA. The pH, setting times, solubility, radiopacity, dimensional change upon setting, and apical sealing ability of VERRM were evaluated and compared to that of ProRoot MTA (GMTA) and ProRoot MTA (Tooth Colored Formula) (WMTA). The results showed that VERRM had physical properties similar to WMTA. VERRM and WMTA showed significantly greater dye penetration than GMTA (p < 0.05) when used as a root-end filling material. There was no significant difference in depth of dye penetration between VERRM and WMTA. Further development of VERRM is indicated to produce a biocompatible root-end filling material with superior handling characteristics.

Comparison of the root-end sealing ability of MTA and Portland cement.

The aim of this study was to compare the in vitro sealing ability of ProRoot MTA, ProRoot MTA (Tooth-Coloured Formula), ordinary Portland cement and white Portland cement when used as root-end filling materials. Twenty-four single-rooted human premolars were prepared and obturated using standard techniques, then retrofilled with the test materials. The prepared teeth were immersed in 1% methylene blue dye for 72 hours and then assessed for dye leakage. The depth of dye penetration was measured and expressed as a percentage of the length of the retrofilling. Data was analysed using ANOVA and Fisher's Least Significant Test (LSD) (p < 0.05). None of the teeth in any of the test groups showed leakage beyond the retrofillings. Given the low cost and apparently similar sealing ability of the cements, it is reasonable to consider Portland cement as a possible substitute for MTA as a root-end filling material. However, further tests, especially in vivo biocompatibility tests, need to be conducted before Portland cement can be recommended for clinical use.

Efficient differentiation of human embryonic stem cells to arterial and venous endothelial cells under feeder- and serum-free conditions.

BACKGROUND: Heterogeneity of endothelial cells (ECs) is a hallmark of the vascular system which may impact the development and management of vascular disorders. Despite the tremendous progress in differentiation of human embryonic stem cells (hESCs) towards endothelial lineage, differentiation into arterial and venous endothelial phenotypes remains elusive. Additionally, current differentiation strategies are hampered by inefficiency, lack of reproducibility, and use of animal-derived products. METHODS: To direct the differentiation of hESCs to endothelial subtypes, H1- and H9-hESCs were seeded on human plasma fibronectin and differentiated under chemically defined conditions by sequential modulation of glycogen synthase kinase-3 (GSK-3), basic fibroblast growth factor (bFGF), bone morphogenetic protein 4 (BMP4) and vascular endothelial growth factor (VEGF) signaling pathways for 5 days. Following the initial differentiation, the endothelial progenitor cells (CD34(+)CD31(+) cells) were sorted and terminally differentiated under serum-free conditions to arterial and venous ECs. The transcriptome and secretome profiles of the two distinct populations of hESC-derived arterial and venous ECs were characterized. Furthermore, the safety and functionality of these cells upon in vivo transplantation were characterized. RESULTS: Sequential modulation of hESCs with GSK-3 inhibitor, bFGF, BMP4 and VEGF resulted in stages reminiscent of primitive streak, early mesoderm/lateral plate mesoderm, and endothelial progenitors under feeder- and serum-free conditions. Furthermore, these endothelial progenitors demonstrated differentiation potential to almost pure populations of arterial and venous endothelial phenotypes under serum-free conditions. Specifically, the endothelial progenitors differentiated to venous ECs in the absence of VEGF, and to arterial phenotype under low concentrations of VEGF. Additionally, these hESC-derived arterial and venous ECs showed distinct molecular and functional profiles in vitro. Furthermore, these hESC-derived arterial and venous ECs were nontumorigenic and were functional in terms of forming perfused microvascular channels upon subcutaneous implantation in the mouse. CONCLUSIONS: We report a simple, rapid, and efficient protocol for directed differentiation of hESCs into endothelial progenitor cells capable of differentiation to arterial and venous ECs under feeder-free and serum-free conditions. This could offer a human platform to study arterial-venous specification for various applications related to drug discovery, disease modeling and regenerative medicine in the future.

Graphene: A Versatile Carbon-Based Material for Bone Tissue Engineering.

The development of materials and strategies that can influence stem cell attachment, proliferation, and differentiation towards osteoblasts is of high interest to promote faster healing and reconstructions of large bone defects. Graphene and its derivatives (graphene oxide and reduced graphene oxide) have received increasing attention for biomedical applications as they present remarkable properties such as high surface area, high mechanical strength, and ease of functionalization. These biocompatible carbon-based materials can induce and sustain stem cell growth and differentiation into various lineages. Furthermore, graphene has the ability to promote and enhance osteogenic differentiation making it an interesting material for bone regeneration research. This paper will review the important advances in the ability of graphene and its related forms to induce stem cells differentiation into osteogenic lineages.

Differential effects of the extracellular microenvironment on human embryonic stem cell differentiation into keratinocytes and their subsequent replicative life span.

Culture microenvironment plays a critical role in the propagation and differentiation of human embryonic stem cells (hESCs) and their differentiated progenies. Although high efficiency of hESC differentiation to keratinocytes (hESC-Kert) has been achieved, little is known regarding the effects of early culture microenvironment and pertinent extracellular matrix (ECM) interactions during epidermal commitment on subsequent proliferative capacity of hESC-Kert. The aim of this study is to evaluate the effects of the different ECM microenvironments during hESC differentiation on subsequent replicative life span of hESC-Kert. In doing so, H1-hESCs were differentiated to keratinocytes (H1-Kert) in two differentiation systems. The first system employed autologous fibroblast feeder support, in which keratinocytes (H1-Kert(ACC)) were derived by coculture of hESCs with hESC-derived fibroblasts (H1-ebFs). The second system employed a novel decellularized matrix from H1-ebFs to create a dermoepidermal junction-like (DEJ) matrix. H1-Kert(AFF) were derived by differentiation of hESCs on the feeder-free system employing the DEJ matrix. Our study indicated that the feeder-free system with the use of DEJ matrix was more efficient in differentiation of hESCs toward epidermal progenitors. However, the feeder-free system was not sufficient to support the subsequent replicative capacity of differentiated keratinocytes. Of note, H1-Kert(AFF) showed limited replicative capacity with reduced telomere length and early cellular senescence. We further showed that the lack of cell-cell interactions during epidermal commitment led to heightened production of TGF-ß1 by hESC-Kert during extended culture, which in turn was responsible for resulting in the limited replicative life span with cellular senescence of hESC-Kert derived under the feeder-free culture system. This study highlights for the first time the importance of the culture microenvironment and cell-ECM interactions during differentiation of hESCs on subsequent replicative life span and cellular senescence of the differentiated keratinocytes, with implications for use of these cells for applications in tissue engineering and regenerative medicine.