Increased DMT1 and FPN1 expression with enhanced iron absorption in ulcerative colitis human colon.

Iron deficiency anemia is a common complication of ulcerative colitis (UC) that can profoundly impact quality of life. Most iron absorption occurs in the duodenum via divalent metal transporter 1 (DMT1)-mediated uptake and ferroportin-1 (FPN1)-mediated export across the apical and basolateral membranes, respectively. However, the colon also contains iron transporters and can participate in iron absorption. Studies have shown increased duodenal DMT1 and FPN1 in patients with UC, but there is conflicting evidence about whether expression is altered in UC colon. We hypothesized that expression of colonic DMT1 and FPN1 will also increase to compensate for iron deficiency. Quantitative RT-PCR and Western blot analyses were performed on duodenal and colonic segmental (right colon, transverse colon, left colon, and rectum) biopsies obtained during colonoscopy. DMT1 mRNA and protein abundances in colonic segments were approximately equal to those in the duodenum, whereas colonic FPN1 mRNA and protein abundances of colonic segments were about one-quarter of those of the duodenum. DMT1 specific mRNA and protein abundances were increased twofold, whereas FPN1 mRNA and protein expressions were increased fivefold in UC distal colon. Immunofluorescence studies revealed enhanced expression of apical membrane- and basolateral membrane-localized DMT1 and FPN1 in UC human colon, respectively. Increased DMT1 expression was associated with enhanced 2-(3-carbamimidoylsulfanylmethyl-benzyl)-isothiourea (CISMBI, DMT1 specific inhibitor)-sensitive 59Fe uptake in UC human colon. We conclude from these results that patients with active UC have increased expression of colonic iron transporters and increased iron absorption, which may be targeted in the treatment of UC-related anemia.

Parallel intermediate conductance K+ and Cl- channel activity mediates electroneutral K+ exit across basolateral membranes in rat distal colon.

Transepithelial K+ absorption requires apical K+ uptake and basolateral K+ exit. In the colon, apical H+-K+-ATPase mediates cellular K+ uptake, and it has been suggested that electroneutral basolateral K+ exit reflects K+-Cl- cotransporter-1 (KCC1) operating in parallel with K+ and Cl- channels. The present study was designed to identify basolateral transporter(s) responsible for K+ exit in rat distal colon. Active K+ absorption was determined by measuring 86Rb+ (K+ surrogate) fluxes across colonic epithelia under voltage-clamp conditions. With zero Cl- in the mucosal solution, net K+ absorption was reduced by 38%, indicating that K+ absorption was partially Cl--dependent. Serosal addition of DIOA (KCC1 inhibitor) or Ba2+ (nonspecific K+ channel blocker) inhibited net K+ absorption by 21% or 61%, respectively, suggesting that both KCC1 and K+ channels contribute to basolateral K+ exit. Clotrimazole and TRAM34 (IK channel blockers) added serosally inhibited net K+ absorption, pointing to the involvement of IK channels in basolateral K+ exit. GaTx2 (CLC2 blocker) added serosally also inhibited net K+ absorption, suggesting that CLC2-mediated Cl- exit accompanies IK channel-mediated K+ exit across the basolateral membrane. Net K+ absorption was not inhibited by serosal addition of either IbTX (BK channel blocker), apamin (SK channel blocker), chromanol 293B (KV7 channel blocker), or CFTRinh172 (CFTR blocker). Immunofluorescence studies confirmed basolateral membrane colocalization of CLC2-like proteins and Na+-K+-ATPase α-subunits. We conclude that active K+ absorption in rat distal colon involves electroneutral basolateral K+ exit, which may reflect IK and CLC2 channels operating in parallel.NEW & NOTEWORTHY This study demonstrates that during active electroneutral K+ absorption in rat distal colon, K+ exit across the basolateral membrane mainly reflects intermediate conductance K+ channels operating in conjunction with chloride channel 2, with a smaller, but significant, contribution from K+-Cl- cotransporter-1 (KCC1) activity.

Polysulfone membranes coated with polymerized 3,4-dihydroxy-l-phenylalanine are a versatile and cost-effective synthetic substrate for defined long-term cultures of human pluripotent stem cells.

Clinical and industrial applications of human pluripotent stem cells (hPSC) require large amounts of cells that have been expanded under defined conditions. Labor-intensive techniques and ill-defined or expensive compounds and substrates are not applicable. Here we describe a chemically defined synthetic substrate consisting of polysulfone (PSF) membranes coated with polymerized 3,4-dihydroxy-l-phenylalanine (DOPA). DOPA/PSF is inexpensive and can be easily produced at various shapes and sizes. DOPA/PSF supports long-term self-renewal of undifferentiated human embryonic (hESC) and human induced pluripotent stem cells (hiPSC) under defined conditions. Pluripotency is maintained for at least 10 passages. Adhesion of hPSC to DOPA/PSF is mainly mediated by a specific integrin heterodimer. Proliferation and gene expression patterns on DOPA/PSF and control substrates are comparable. Labor-intensive cultivation methods and use of serum or coating with proteins are not required. Together, these features make DOPA/PSF attractive for applications where large-scale expansion of human pluripotent stem cells under defined conditions is essential.

Human embryonic stem cells differentiate into functional renal proximal tubular-like cells.

Renal cells are used in basic research, disease models, tissue engineering, drug screening, and in vitro toxicology. In order to provide a reliable source of human renal cells, we developed a protocol for the differentiation of human embryonic stem cells into renal epithelial cells. The differentiated stem cells expressed markers characteristic of renal proximal tubular cells and their precursors, whereas markers of other renal cell types were not expressed or expressed at low levels. Marker expression patterns of these differentiated stem cells and in vitro cultivated primary human renal proximal tubular cells were comparable. The differentiated stem cells showed morphological and functional characteristics of renal proximal tubular cells, and generated tubular structures in vitro and in vivo. In addition, the differentiated stem cells contributed in organ cultures for the formation of simple epithelia in the kidney cortex. Bioreactor experiments showed that these cells retained their functional characteristics under conditions as applied in bioartificial kidneys. Thus, our results show that human embryonic stem cells can differentiate into renal proximal tubular-like cells. Our approach would provide a source for human renal proximal tubular cells that are not affected by problems associated with immortalized cell lines or primary cells.

Role of lineage-specific matrix in stem cell chondrogenesis.

Cartilage repair in clinics is a challenge owing to the limited regenerative capacities of cartilage. Synovium-derived stem cells (SDSCs) are suggested as tissue-specific stem cells for chondrogenesis. In this study, we hypothesize that decellularized extracellular matrix (dECM) deposited by SDSCs could provide a superior tissue-specific matrix microenvironment for optimal rejuvenation of adult SDSCs for cartilage regeneration. dECMs were deposited by adult stem cells with varying chondrogenic capacities; SDSCs (strong) (SECM), adipose-derived stem cells (weak) (AECM) and dermal fibroblasts (weak) (DECM), and urine-derived stem cells (none) (UECM). Plastic flasks (Plastic) were used as a control substrate. Human SDSCs were expanded on the above substrates for one passage and examined for chondrogenic capacities. We found that each dECM consisted of unique matrix proteins and exhibited varied stiffnesses, which affected cell morphology and elasticity. Human SDSCs grown on dECMs displayed a significant increase in cell proliferation and unique surface phenotypes. Under induction media, dECM expanded cells yielded pellets with a dramatically increased number of chondrogenic markers. Interestingly, SECM expanded cells had less potential for hypertrophy compared to those grown on other dECMs, indicating that a tissue-specific matrix might provide a superior microenvironment for stem cell chondrogenic differentiation.

Sensitization of Cancer Cells via Non-Viral Delivery of Apoptosis Inducing Proteins Using a Cationic Bolaamphiphile.

Cationic bolaamphiphile polymers had been previously studied as efficient delivery system for the delivery of proteins with relatively low toxicity. Here, the authors investigate the use of a protein delivery system based on a cationic bolaamphiphile to sensitize cancer cells toward apoptosis-inducing drugs as a novel approach for cancer therapy. The authors demonstrates the efficacy of the system by two strategies. The first strategy involves delivery of a survivin antibody to inhibit survivin activity. Sensitization of MCF-7 cells to doxorubicin is observed by survivin inhibition by antibodies. The IC50 of doxorubicin is reduced ≈2.5-fold after delivery of survivin antibodies to breast cancer cells and induction of apoptosis is shown by Western blotting with apoptosis specific antibodies. In a second approach, functional wild type p53 is delivered into p53-null liver cancer (Hep3B) cells, sensitizing the cells toward the p53 pathway drug, Nutlin. Nutlin reduced the viability of Hep3B cells by ≈42% at 15 µM concentration, demonstrating the effectiveness of p53 delivery. The expression of p21, a downstream target of p53 further confirmed the functional status of the delivered protein. In conclusion. The successful delivery of apoptosis inducing proteins and sensitization of cancer cells via cationic bolaamphiphile polymer represents a promising system for cancer therapeutics.

Biological assemblies provide novel templates for the synthesis of hierarchical structures and facilitate cell adhesion.

Mechanical mismatch and the lack of interactions between implants and the natural tissue environment are the major drawbacks in bone tissue engineering. Biomaterials mimicking the self-assembly process and the composition of the bone matrix should provide new route for fabricating biomaterials possessing novel osteoconductive and osteoinductive properties for bone repair. In the present study, we employ bio-inspired strategies to design de novo self-assembled chimeric protein hydrogels comprising leucine zipper motifs flanked by dentin matrix protein 1 domain, which was characterized as a mineralization nucleator. Results showed that this chimeric protein could function as a hydroxyapatite nucleator in pseudo-physiological buffer with the formation of highly oriented apatites similar to biogenic bone mineral. It could also function as an inductive substrate for osteoblast adhesion, promote cell surface integrin presentation and clustering, and modulate the formation of focal contacts. Such biomimetic "bottom-up" construction with dual osteoconductive and osteoinductive properties should open new avenues for bone tissue engineering.Mechanical mismatch and the lack of interactions between implants and the natural tissue environment are the major drawbacks in bone tissue engineering. Biomaterials mimicking the self-assembly process and the composition of the bone matrix should provide new route for fabricating biomaterials possessing novel osteoconductive and osteoinductive properties for bone repair. In the present study, we employ bio-inspired strategies to design de novo self-assembled chimeric protein hydrogels comprising leucine zipper motifs flanked by dentin matrix protein 1 domain, which was characterized as a mineralization nucleator. Results showed that this chimeric protein could function as a hydroxyapatite nucleator in pseudo-physiological buffer with the formation of highly oriented apatites similar to biogenic bone mineral. It could also function as an inductive substrate for osteoblast adhesion, promote cell surface integrin presentation and clustering, and modulate the formation of focal contacts. Such biomimetic "bottom-up" construction with dual osteoconductive and osteoinductive properties should open new avenues for bone tissue engineering.

Lineage-specific exosomes could override extracellular matrix mediated human mesenchymal stem cell differentiation.

Lineage specification is an essential process in stem cell fate, tissue homeostasis and development. Microenvironmental cues provide direct and selective extrinsic signals to regulate lineage specification of stem cells. Microenvironmental milieu consists of two essential components, one being extracellular matrix (ECM) as the substratum, while the other being cell secreted exosomes and growth factors. ECM of differentiated cells modulates phenotypic expression of stem cells, while their exosomes contain phenotype specific instructive factors (miRNA, RNA and proteins) that control stem cell differentiation. This study demonstrates that osteoblasts-derived (Os-Exo) and adipocytes-derived (Ad-Exo) exosomes contain instructive factors that regulate the lineage specification of human mesenchymal stem cells (hMSCs). Analyses of exosomes revealed the presence of transcription factors in the form of RNA and protein for osteoblasts (RUNX2 and OSX) and adipocytes (C/EBPα and PPARγ). In addition, several miRNAs reported to have osteogenic and adipogenic differentiation potentials are also identified in these exosomes. Kinetic and differentiation analyses indicate that both osteoblast and adipocyte exosomes augment ECM-mediated differentiation of hMSCs into the respective lineage. The combination of osteoblast/adipocyte ECM and exosomes turned-on the lineage specific gene expressions at earlier time points of differentiation compared to the respective ECM or exosomes administered individually. Interestingly, the hMSCs differentiated on osteoblast ECM with adipogenic exosomes showed expression of adipogenic lineage genes, while hMSCs differentiated on adipocyte ECM with osteoblast exosomes showed osteogenic lineage genes. Based on these observations, we conclude that exosomes might override the ECM mediated instructive signals during lineage specification of hMSC.

Biological markers for evaluation of root resorption.

BACKGROUND: External apical root resorption is a pathologic consequence of orthodontic tooth movement. Cementum and dentin are removed from the root surface while active force is present. OBJECTIVE: The aim of this study was to identify and quantify extracellular matrix proteins, dentin matrix protein 1 (DMP1), dentin phosphophoryn (PP), and dentin sialoprotein (DSP) in the gingival crevicular fluid (GCF) of subjects undergoing orthodontic treatment. METHODS: Subjects with mild (less than 2mm) and severe (more than 2mm) root resorption during orthodontic treatment were identified by radiographs. A control group of subjects with neither signs of root loss nor undergoing orthodontic treatment was also identified. GCF was collected from the upper incisors by using filter paper strips (Periopaper). The absorbed GCF was eluted and the proteins were separated by SDS-PAGE analysis and stained. Western blot and ELISA were also performed. One-way ANOVA and Scheffé test were used for statistical analysis. RESULTS: SDS-PAGE analysis identified proteins at 77, 66, 55, 50 and 26kDa. Immunoblotting did not show any differential expression pattern between control and study groups. ELISA results revealed a significant difference in the concentrations of DMP1, PP and DSP between control and root resorption groups. Concentration of PP and DSP in severe root resorption group was also statistically higher than in mild root resorption group. CONCLUSION: DSP and PP could be suitable biological markers for monitoring root resorption during orthodontic treatment, since a significant difference in the level of these dentin specific proteins is detected in all groups.

Engineering Concepts in Stem Cell Research.

The field of regenerative medicine integrates advancements made in stem cells, molecular biology, engineering, and clinical methodologies. Stem cells serve as a fundamental ingredient for therapeutic application in regenerative medicine. Apart from stem cells, engineering concepts have equally contributed to the success of stem cell based applications in improving human health. The purpose of various engineering methodologies is to develop regenerative and preventive medicine to combat various diseases and deformities. Explosion of stem cell discoveries and their implementation in clinical setting warrants new engineering concepts and new biomaterials. Biomaterials, microfluidics, and nanotechnology are the major engineering concepts used for the implementation of stem cells in regenerative medicine. Many of these engineering technologies target the specific niche of the cell for better functional capability. Controlling the niche is the key for various developmental activities leading to organogenesis and tissue homeostasis. Biomimetic understanding not only helped to improve the design of the matrices or scaffolds by incorporating suitable biological and physical components, but also ultimately aided adoption of designs that helped these materials/devices have better function. Adoption of engineering concepts in stem cell research improved overall achievement, however, several important issues such as long-term effects with respect to systems biology needs to be addressed. Here, in this review the authors will highlight some interesting breakthroughs in stem cell biology that use engineering methodologies.

Characterization of Chlorhexidine-Loaded Calcium-Hydroxide Microparticles as a Potential Dental Pulp-Capping Material.

This study explores the delivery of novel calcium hydroxide [Ca(OH)2] microparticles loaded with chlorhexidine (CHX) for potential dental therapeutic and preventive applications. Herein, we introduce a new approach for drug-delivery to deep dentin-surfaces in the form of drug-loaded microparticles. Unloaded Ca(OH)2 [Ca(OH)2/Blank] and CHX-loaded/Ca(OH)2 microparticles were fabricated by aqueous chemical-precipitation technique. The synthesized-microparticles were characterized in vitro for determination of surface-morphology, crystalline-features and thermal-properties examined by energy-dispersive X-ray scanning and transmission electron-microscopy (EDX-SEM/TEM), Fourier-transform infrared-spectroscopy (FTIR), X-ray diffraction (XRD), thermogravimetric analysis (TGA) and differential scanning-calorimetry (DSC). Time-related pH changes, initial antibacterial/biofilm-abilities and cytotoxicity of CHX-loaded/Ca(OH)2 microparticles were evaluated. Microparticles were delivered to dentin-surfaces with subsequent SEM examination of treated dentin-substrates. The in vitro and ex vivo CHX-release profiles were characterized. Ca(OH)2/Blank were hexagonal-shaped with highest z-average diameter whereas CHX-inclusion evidenced micro-metric spheres with distinguishable surface "rounded deposits" and a negative-shift in diameter. CHX:Ca(OH)2/50 mg exhibited maximum encapsulation-efficiency with good antibacterial and cytocompatible properties. SEM examination revealed an intact layer of microparticles on exposed dentin-surfaces with retention of spherical shape and smooth texture. Microparticles loaded on dentin-surfaces showed prolonged release of CHX indicating substantial retention on dentin-substrates. This study validated the inherent-applicability of this novel drug-delivery approach to dentin-surfaces using micro-metric CHX-loaded/Ca(OH)2 microparticles.

Current Perspective of Stem Cell Therapy in Neurodegenerative and Metabolic Diseases.

Neurodegenerative diseases have been an unsolved riddle for quite a while; to date, there are no proper and effective curative treatments and only palliative and symptomatic treatments are available to treat these illnesses. The absence of therapeutic treatments for neurodegenerative ailments has huge economic hit and strain on the society. Pharmacotherapies and various surgical procedures like deep brain stimulation are being given to the patient, but they are only effective for the symptoms and not for the diseases. This paper reviews the recent studies and development of stem cell therapy for neurodegenerative disorders. Stem cell-based treatment is a promising new way to deal with neurodegenerative diseases. Stem cell transplantation can advance useful recuperation by delivering trophic elements that impel survival and recovery of host neurons in animal models and patients with neurodegenerative maladies. Several mechanisms, for example, substitution of lost cells, cell combination, release of neurotrophic factor, proliferation of endogenous stem cell, and transdifferentiation, may clarify positive remedial results. With the current advancements in the stem cell therapies, a new hope for the cure has come out since they have potential to be a cure for the same. This review compiles stem cell therapy recent conceptions in neurodegenerative and neurometabolic diseases and updates in this field. Graphical Absract ᅟ.

Targeting Warburg Effect in Cancers with PEGylated Glucose.

In highly proliferative cancer cells, energy is predominantly produced by a high rate of glycolysis, followed by lactic acid fermentation, despite the availability of oxygen - an observation known as the Warburg effect. As a consequence, cells employing this glycolytic pathway require high uptake of glucose and increased metabolic rates to maintain their proliferation. It has been hypothesized that by blocking glucose uptake using modified glucose molecules, apoptosis in the cancer cells can be induced. In this study, it has been showed that several poly(ethylene glycol) (PEG)-modified glucose compounds could reduce cell proliferation in various cancer cell lines by a phenomenon that blocked the availability of the glucose transporters and reduced AKT1 (serine/threonine-specific protein kinase) activation. Xenograft cancer models that are intravenously administered with glucose-conjugated branched PEG (GBrP) daily for 14 d show little tumor development, as compared to the control group without GBrP treatment. The toxicological effects and the pharmacokinetics of the PEGylated glucose are studied in rodents. The PEGylated glucose exerts no systemic toxicity at 40 mg kg(-1) dosage. However, doses above 80 mg kg(-1) show dose-dependent toxicity in all the organs analyzed. The present results suggest PEGylated glucose as a promising "metabolic therapy" approach for the treatment of cancer.

Functional Kidney Bioengineering with Pluripotent Stem-Cell-Derived Renal Progenitor Cells and Decellularized Kidney Scaffolds.

Recent advances in developmental biology and stem cell technology have led to the engineering of functional organs in a dish. However, the limited size of these organoids and absence of a large circulatory system poses limits to its clinical translation. To overcome these issues, decellularized whole kidney scaffolds with native microstructure and extracellular matrix (ECM) are employed for kidney bioengineering, using human-induced pluripotent-stem-cell-derived renal progenitor cells and endothelial cells. To demonstrate ECM-guided cellular assembly, the present work is focused on generating the functional unit of the kidney, the glomerulus. In the repopulated organ, the presence of endothelial cells broadly upregulates the expression level of genes related to renal development. When the cellularized native scaffolds are implanted in SCID mice, glomeruli assembly can be achieved by co-culture of the renal progenitors and endothelial cells. These individual glomerular units are shown to be functional in the context of the whole organ using a simulated bio-reactor set-up with urea and creatinine excretion and albumin reabsorption. Our results indicate that the repopulation of decellularized native kidney using clinically relevant, expandable patient-specific renal progenitors and endothelial cells may be a viable approach for the generation of a functional whole kidney.

Alginate Microfiber System for Expansion and Direct Differentiation of Human Embryonic Stem Cells.

Pluripotent human embryonic stem cells (hESCs) are a potential renewable cell source for regenerative medicine and drug testing. To obtain adequate cell numbers for these applications, there is a need to develop scalable cell culture platforms to propagate hESCs. In this study, we encapsulated hESCs in calcium alginate microfibers as single cells, for expansion and differentiation under chemically defined conditions. hESCs were suspended in 1% (w/v) alginate solution at high cell density (>10(7) cells/mL) and extruded at 5 m/min into a low calcium concentration bath (10 mM) for gelation. Mild citrate buffer (2.5 mM), which did not affect hESCs viability, was used to release the cells from the calcium alginate hydrogel. Encapsulation as single cells was critical, as this allowed the hESCs to grow in the form of relatively small and uniform aggregates. This alginate microfiber system allowed for expansion of an hESC line, HUES7, for up to five passages while maintaining pluripotency. Immunohistochemistry, polymerase chain reaction, and other analyses showed that passage 5 (P5) HUES7 cells expressed proteins and genes characteristic of pluripotent stem cells, possessed normal karyotype, and were able to form representative tissues of the three embryonic germ layers in vitro and in vivo. Encapsulated HUES7 cells at P5 could also be induced to directly differentiate into liver-like cells. Collectively, our experiments show that the alginate microfiber system can be used as a three-dimensional cell culture platform for long-term expansion and differentiation of hESCs under defined conditions.

Identification of differentially expressed cDNA transcripts from a rat odontoblast cell line.

Odontoblasts and osteoblasts are two among the myriads of cell types present in the craniofacial complex. Both have a common ectomesenchymal origin and secrete macromolecules that are necessary for the formation of dentin and alveolar bone via matrix-mediated mechanisms. The mineralized matrices of bone and dentin differ in morphology and function but several mineral associated proteins, formerly thought to be tissue specific, have been found to be common in both tissues. To decipher the complex molecular mechanisms involved in mineralized dentin formation, the suppressive subtraction hybridization (SSH) approach has been used to identify the genes expressed by polarized odontoblasts. Employing SSH, 187 cDNA clones were identified from the subtracted cDNA library. Many of these genes have not been previously reported to be expressed by terminally differentiated odontoblasts. Genes were classified into seven groups based on the predicted function of the encoded proteins: extracellular matrix; cytoskeletal components, molecules involved in adhesion and cell-cell interaction; metabolic enzymes, transporters, ion channels; protein processing, protein transport and protein folding molecules; nuclear proteins (transcription factors, DNA processing enzymes); signaling molecules and genes of yet unknown function. Northern blot and in situ hybridization analysis performed for five putative novel genes and one new isoform of amelogenin revealed differential expression levels in the osteoblasts, ameloblasts and the odontoblasts of the developing rat molars. Some of the known genes isolated from this enriched pool were the cleavage products of dentin sialophosphoprotein (DSPP) namely, phosphophoryn (PP) and dentin sialoprotein (DSP). Interestingly amelogenin, ameloblastin and enamelin were also expressed in the odontoblasts during dentin formation.

Enzyme-mediated hyaluronic acid-tyramine hydrogels for the propagation of human embryonic stem cells in 3D.

The propagation of human embryonic stem cells (hESCs) in three-dimensional (3D) scaffolds facilitates the cell expansion process and supplies pluripotent cells of high quality for broad-spectrum applications in regenerative medicine. Herein, we report an enzyme-mediated hyaluronic acid-tyramine (HA-Tyr) hydrogel that encapsulated and propagated hESCs in 3D. HA-Tyr hydrogels were formed by crosslinking the tyramine moieties with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). By changing the HRP and H2O2 concentration, we prepared HA-Tyr hydrogels of different mechanical strength and studied the self-renewal properties of hESCs in these scaffolds. We observed that both the chemical composition and mechanical strength of substrates were important factors affecting cell proliferation and pluripotency. The HA-Tyr hydrogel with a compressive modulus of ∼350Pa supported the proliferation of hESCs at the pluripotent state in both mTeSR1 medium and mouse embryonic fibroblast (MEF)-conditioned medium. Immunohistochemical analyses revealed that hESCs proliferated well and formed spheroid structures in 3D, without undergoing apoptosis. The hESCs cultured in HA-Tyr hydrogels showed high expression of CD44 and pluripotency markers. These cells exhibited the capability to form cell derivatives of all three embryonic germ layers in vitro and in vivo. In addition, the genetic integrity of the hESCs was unaffected in the 3D cultivation system. STATEMENT OF SIGNIFICANCE: The scope of this study is to provide a stable 3D cultivation system for the expansion of human embryonic stem cells (hESCs) towards clinical applications. We report an enzyme mediated hyaluronic acid-tyramine (HA-Tyr) hydrogel that encapsulated and propagated hESCs in 3D. Unlike other HA-based photo-crosslinked hydrogel systems reported, we investigated the effects of mechanical strength of hydrogels on the self-renewal properties of hESCs in 3D. Then, we characterized hESCs cultured in hydrogels with lower mechanical strength that best supported the self-renewal of hESCs. Hence, we demonstrated a reliable approach for the controlled propagation of hESCs in 3D. We believe that such an approach would facilitate the development of stem cell-based therapy towards clinical applications.

Differential expression patterns of the dentin matrix proteins during mineralized tissue formation.

Sequential and reciprocal interactions between the oral ectoderm and neural crest-derived mesenchyme are responsible for tooth development. During dentin formation, there are three components that are necessary for proper mineralization, namely, collagen which forms a scaffold, noncollagenous proteins that can specifically bind to the collagen template and function as a mineral nucleator and crystalline calcium phosphate deposited in an ordered manner. It is well established that noncollagenous proteins play an important role during mineralized tissue formation. Here we demonstrate by in situ hybridization techniques that the noncollagenous dentin matrix proteins 1, 2 (DMP1, 2) and dentin sialoprotein (DSP) have characteristic temporal and spatial expression patterns within odontogenic tissues during dentin mineralization. DMP1, DMP2 and DSP mRNA are expressed in the odontoblasts at specific and overlapping time points and are thus presumably used for different functions during dentin formation. In developing rat incisors and molars, high levels of expression of DMP2 mRNA were seen in polarized odontoblasts and preameloblasts, while DSP mRNA was expressed at significantly lower levels and was expressed by highly differentiated odontoblasts. However, their expression was continuously maintained during the mineralization of the organic matrix. In the adult rats, DMP2 and DSP mRNA was also detected in the osteoblasts. The expression of DMP1 mRNA was found to coincide with the start of the mineral nucleation process and gradually decreased during the maturation of the mineralized matrix during odontogenesis. In this study, we have also correlated the expression of these proteins relative to the presence of type I collagen and calcium phosphate crystals. Thus, the temporal and spatial differences between DMP1, DMP2 and DSP might implicate a direct demonstration of the functional difference between these three genes during calcified tissue formation.

Induced pluripotent stem cell-derived hepatocytes and endothelial cells in multi-component hydrogel fibers for liver tissue engineering.

Liver tissue engineering requires a suitable cell source, methodologies to assemble the cells within their niche microenvironments in a spatially defined manner, and vascularization of the construct in vivo for maintenance of hepatocyte viability and function. Recently, we have developed methods of encapsulating cells within separate domains in multi-component hydrogel fibers and methods of assembling fibers to form 3D-patterned tissue constructs. In the present work, we have combined these approaches to encapsulate hepatocytes and endothelial cells within their specific niches, and to assemble them into endothelialized liver tissue constructs. The hepatocytes and endothelial cells were obtained in parallel by differentiating human recombinant protein-induced human pluripotent stem cells, resulting in a construct which contained genetically identical endothelial and parenchymal elements. We were able to demonstrate that the presence of endothelial cells in the scaffold significantly improved hepatocyte function in vitro and facilitated vascularization of the scaffold when implanted in a mouse partial hepatectomy model. The in vivo studies further asserted that integration of the scaffold with host vasculature had occurred, as demonstrated by the presence of human albumin in the mouse serum.

Therapeutic effect of a multi-targeted imidazolium compound in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed lethal cancers in the world. We previously showed two imidazolium salts (IBN-1 and IBN-9) with a moderate efficacy for HCC. Here we report a more potent imidazolium compound IBN-65 (1-benzyl-2-phenyl-3-(4-isopropyl)-benzyl-imidazolium chloride) and the associated mechanisms of action in a mouse model of HCC. The IC50 of this compound in various liver cancer cell lines was around 5 µm. IBN-65 dose-dependently arrested cell cycle at G1 phase and was associated with the down-regulation of the cyclin-dependent kinase-4, -6, cyclin D1, and cyclin E. In addition, IBN-65 induced apoptosis by down-regulating Survivin, Bcl-2 and up-regulating Bax, leading to sequential activation of Caspase-3, Caspase-9 and the cleavage of poly(ADP-ribose) polymerase (PARP). Dysregulation of the epidermal growth factor receptor (EGFR) signaling network has been frequently reported in HCC. We found that IBN-65 displayed a profound inhibitory effect on the EGFR/Raf/MEK/ERK signaling at the phosphorylation level. In Huh7 or Hep3B cells, pretreatment with IBN-65 attenuated EGF-induced phosphorylation of both EGFR and the downstream p44/42 MAPK. A siRNA knockdown of EGFR also proved that IBN-65 induced apoptosis mostly through inhibiting downstream EGFR pathway signaling, much less at the receptor level. Infrequent administration of IBN-65 (i.p., 5 mg/kg once weekly for four weeks) to mice bearing the Huh7 cells significantly reduced the tumor volume by 65% without affecting the body weight. Critically, many of the anti-tumor signaling features observed in the HCC cell lines were recaptured in the xenografted tissues. Thus, the metal-free imidazolium compound IBN-65 could be a potential candidate towards therapeutic development for HCC.