Chitosan Sulfate Research Articles

Свойства водной дисперсии комплекса хитотозана и хондроитина сульфата, полученных из гидробионтов

Свойства водной дисперсии комплекса хитотозана и хондроитина сульфата, полученных из гидробионтов

Sulfated polyelectrolyte complex nanoparticles structured nanoflitration membrane for dye desalination

Polysaccharide nanofiltration (NF) membrane with traditional modification normally suffers from poor water permeability as a result of its tight packing of polymeric chains. In this work, a novel membrane building block, polyelectrolyte complex (PEC) nanoparticles (NPs) armed with adjustable content of sulfated groups was developed using chitosan and dextran sulfate sodium. The sulfated polyelectrolyte complex membranes (SPECMs) were first prepared by solution-casting and glutaraldehyde crosslinking process, and their structural characteristics and surface properties were systematically investigated. Intrinsic aggregation structure combined with numerous sulfate groups attenuates packing density of polymeric chains and promotes hydrophilicity, endowing SPECMs with high flux and perm-selectivity. SPECMs achieved a water permeability of 6.71Lm−2h−1bar−1, which was ∼2.3 times higher than pristine sulfated chitosan membrane. The selectivity for NaCl/Na2SO4 separation and NaCl/methyl blue dye separation were as high as 13.1 and 850.0, respectively. Moreover, SPECMs featured efficient dye antifouling property with low flux decline ratio (6.8%) and high recovery ratio (96.8%), which was primarily attributed to its hydrophilicity and smooth surface. The prominent perm-selectivity associated with antifouling property suitably position SPECMs for practical small organic molecule/inorganic salt mixture separation in a long-term process.

Surface interactions of gold nanorods and polysaccharides: From clusters to individual nanoparticles

Gold nanorods (AuNRs) are suitable for constructing self-assembled structures for the development of biosensing devices and are usually obtained in the presence of cetyltrimethylammonium bromide (CTAB). Here, a sulfated chitosan (ChiS) and gum arabic (GA) were employed to encapsulate CTAB/AuNRs with the purpose of studying the interactions of the polysaccharides with CTAB, which is cytotoxic and is responsible for the instability of nanoparticles in buffer solutions. The presence of a variety of functional groups such as the sulfate groups in ChiS and the carboxylic groups in GA, led to efficient interactions with CTAB/AuNRs as evidenced through UV–vis and FTIR spectroscopies. Electron microscopies (HR-SEM and TEM) revealed that nanoparticle clusters were formed in the GA-AuNRs sample, whereas individual AuNRs, surrounded by a dense layer of polysaccharides, were observed in the ChiS-AuNRs sample. Therefore, the presented work contributes to the understanding of the driving forces that control the surface interactions of the studied materials, providing useful information in the building-up of gold self-assembled nanostructures.

Tripolyphosphate-responsive release property of monoolein cubic phase containing sodium dodecyl sulfate and oligo chitosan

ABSTRACTTripolyphosphate (TPP)-responsive MO cubic phase was prepared by immobilizing oligo chitosan in the water channel through its electrostatic attraction with sodium dodecyl sulfate (SDS). The phase transition temperature (PTT) increased with increasing the content of SDS. The PTT of cubic phase whose SDS content was 0%, 0.21%, 0.42%, 0.84%, and 1.68%, determined by polarized microscopy, was about 69.5°C, 72°C, 75°C, 80.5°C, and 95°C, respectively. The PTT did not markedly deviate from that determined by differential scanning calorimetry. The release degree for 72 h of dye (i.e., amaranth and methylene blue) was dependent on the pH value of release medium (pH 3.0 and pH 7.0). Moreover, the release degree significantly increased when the TPP concentration in the release medium increased to 0.4% (w/v). Oligo chitosan was electrostatically complexed with TPP and the complexation took place extensively at the oligo chitosan/TTP mass ratio of 1:0.125 and 1:0.25 and at the oligo chitosan concentration of 1.6% (w/v), evidenced by optical spectroscopy and scanning electron microscopy. It was thought that the complexation was responsible for the TPP concentration-dependent release.

Chitosan sulfate: engineering a biomimetic growth factor delivery system

Chitosan sulfate: engineering a biomimetic growth factor delivery system

Novel chitosan-based nanobiohybrid membranes for wound dressing applications

Montmorillonite nanolayers were treated with chitosan sulfate, as a functional biocompatibilizer, and then exfoliated in chitosan matrix to design nanohybrid membranes for potential wound dressing applications with enhanced physical, mechanical and antibacterial properties.

Balancing osteoblast/osteoclast ratio in vitro by means of chitosan scaffolds either surficial sulfated or modified by hemocyanines and calcium phosphate phases. A co-culture study.

Event Abstract Back to Event Balancing osteoblast/osteoclast ratio in vitro by means of chitosan scaffolds either surficial sulfated or modified by hemocyanines and calcium phosphate phases. A co-culture study. Christiane Heinemann1, Jana Farack1, Susanne Hoehne2, Ronny Bruenler3, Simy Weil4, Dilibaier Aibibu3, Amir Sagi4 and Thomas Hanke1 1 Technische Universitaet Dresden, IfWW - Max-Bergmann-Center of Biomaterials, Germany 2 Leibniz Association, Institute of Polymer Research Dresden, Germany 3 Technische Universitaet Dresden, Institute of Textile Machinery and High Performance Material Technology, Germany 4 Ben Gurion University of the Negev, Department of Life Sciences and the National Institute for Biotechnology in the Negev, Israel Introduction: Bone tissue engineering is still a field of interest. Due to a variety of material properties the natural polysaccharide chitosan is an excellent candidate for a scaffold material[1]. Recently, development focuses on modification for improvement of the impact on the healing process of bone. Main point of application is the ratio of osteoblasts and osteoclasts in the bone multicellular unit. The present study reports on differently sulfated chitosan scaffolds and scaffolds modified by hemocyanines, collagen and calcium phosphate phases. Materials and Methods: (1) Solid-phase sulf­ation was achieved by treating embroidered chitosan scaffolds with sulfur trioxide dimethylformamide complex (DMF/SO3) or chlorethan sulfonic acid (CLESA) or sulphur trioxide pyridine complex (Pyr/SO3)[2]. (2) Net shape nonwoven-(NSN)-chitosan scaffolds were functionalized by collagen coating[3]. The microfibrous scaffolds were coated with bovine type I collagen by dipping the scaffolds into a solution of 2 mg/ml collagen dissolved in 0.1M TRIS buffer solution with pH 7.4. Then the scaffolds were freeze-dried and subsequently chemically cross-linked. (3) For the macroporous scaffolds homogeneous clear solutions of 1 % chitosan in acetic acid were prepared. Chitosan solution was transferred to 48-well plates and freeze-dried. After neutralization with NaOH and several washing steps the scaffolds were lyophilized again. To get mineralized chitosan scaffolds brushite or hydroxyapatite were added to the same weight of chitosan, while it was dissolved acetic acid. Hemocyanin modification was carried out by dip-coating. (4) Single culture experiments with hMSC/ Osteoblasts[4] and Monocytes/ Osteoclasts[1] as well as co-culture experiments[5] were carried out as described in our cited previous studies. Results and Discussion: (1) All sulfation methods resulted in enhanced proliferation and enhanced osteogenic differentiation. During cultivation, Pyr/SO3 scaffolds turned out to provide the best conditions for both. On the contrary, ostoclastogenesis was most effective for CLESA-sulfation. That is a key-result because the use of differently sulfated scaffolds could be a tool to adjust the ratio of bone building and bone resorbing activity. (2) Collagen coating of the NSN scaffolds enhances the adhesion and proliferation of hMSC and their differention to osteoblasts, as well. So, the bone building activity could be supported by NSN chitosan collagen-hybrid scaffolds. (3) Modification of chitosan scaffolds by hemocyanines enhances attachment and proliferation of hMSC/osteoblasts as well as the bioactivity of the scaffold material. Co-culture experiments on special hemocyanine-calcium phosphate-chitosan compositions show the enhancement of osteogenic hMSC differentiation in comparison with the osteoclastogenesis. Conclusion: The in vitro-findings give reason to expect that all three approaches are applicable to the manipulation of the ratio of bone resorption and bone formation during remodeling. Deutsche Forschungsgemeinschaft Grants HA5284/2-1 and HA 5284/4-2; Bundesministerium für Forschung und Technologie Grant 13GW0025

Anti-trombogenic property of sulfated chitosan films

Event Abstract Back to Event Anti-trombogenic property of sulfated chitosan films Anaftalia F. Moraes1, Niédja F. Vasconcelos1, Lorena Mara A. Silva2, Kirley M. Canuto2, Débora C. Maia1 and Rodrigo S. Vieira1 1 Universidade Federal do Ceará, Brazil 2 Embrapa - Brazilian Company Agricultural Reaserch, Brazil Introduction: Biomaterials used in medical devices in contact with blood can induce thrombus formation and inflammation. These two processes are interdependent and involve the coagulation cascade and complement system activation, with the consequent platelets and leukocytes adhesion and activation. The hemocompatibility of these devices can be improved by using natural polymers, since they have properties as biocompatibility, non-toxicity, biodegradability and antimicrobial potential. Chitosan is apromising biomaterial studied today, however, has thrombogenic characteristics in its natural form. This biopolymer is easy chemically modified due to its OH- and NH3+ groups, which are highly reactive. Thus, this study aims to analyze the anti-thrombogenic property of chitosan films chemically modified by sulfation reaction. Materials and Methods: Natural chitosan (QN) and sulfated (QS) films were produced and characterized by elemental analysis, ATR-FTIR, H-NMR and SEM/EDS. Platelet adhesion assay was performed with whole blood in vitro to test the hemocompatibility and the results were quantified by fluorescence microscope. Results and Discussion: The sulfur amount in the QS was 12% and its degree of substitution (DS) was 1.37, both obtained by elemental analysis. The introduction of sulfated group in the chitosan structure was confirmed by FTIR-ATR, with the appearance of a characteristic band of this group at 1215 cm-1 (S=O) and 806 cm-1 (C-S-O). The morphology of the films was observed using SEM/EDS, showing a smooth and homogeneous surface, with sulfur species homogeneously distributed on the modified surface. The degree of deacetylation (DD) of the films was measured H-NMR, which was found to natural chitosan DD of 77% and 58% formodified film. The DD reduction for modified chitosan is very important for applications involving blood contact, since the protonated amino groups from raw chitosan can interact with charged blood proteins and contributed to thrombus formation and inflammation. In the platelet adhesion assay was observed a reduction in the adherence of blood components on sulfated film compared with the QN film (Figure 1). From the image area (1,045393 x 10-4 mm2) was recorded a number of platelets in the QN of 1,15 x 10-8 mm2, as that for the QS was 0,3 x 10-9 mm2. Conclusions: The chemical modification of chitosan by sulfonation reaction indicated an improvement in the hemocompatibility potential for biomedical applications. CAPES; Embrapa; Central Analítica UFC

Structural characterization and in vitro biomedical activities of sulfated chitosan from Sepia pharaonis

A low molecular weight sulfated chitosan (SP-LMWSC) was isolated from the cuttlebone of Sepia pharaonis. Elemental analysis established the presence of C, H and N. The sulfation of SP-LMWSC was confirmed by the presence of characteristic peaks in FT-IR and FT-Raman spectra. The thermal properties of SP-LMWSC were studied by thermogravimetric analysis and differential scanning calorimetry. Electrolytic conductivity of SP-LMWSC was measured by cyclic voltammetry and the molecular weight was determined by MALDI–TOF/MS. The molecular structure and sulfation sites of SP-LMWSC were unambiguously confirmed using 1H, 13C, 2D COSY and 2D HSQC NMR spectroscopy. SP-LMWSC exhibited increased anticoagulant activity in avian blood by delaying coagulation parameters and displayed cytostatic activity by inhibiting the migration of avian leucocytes. SP-LMWSC demonstrated avian antiviral activity by binding to Newcastle disease virus receptors at a low titer value of 1/64. These findings suggested that SP-LMWSC isolated from an industrial discard holds immense potentials as carbohydrate based pharmaceuticals in future.

Extent of shielding by counterions determines the bactericidal activity of N,N,N-trimethyl chitosan salts

In this study, we show that the bactericidal activity of quaternized chitosans (TMCs) with sulfate, acetate, and halide counterions against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) correlates with the “availability” of N-quaternized groups [−+N(CH3)3] in the TMCs backbones. N,N,N-trimethyl chitosan sulfate (TMCS) and N,N,N-trimethyl chitosan acetate (TMCAc) displayed the highest activities, probably due to their delocalized π system. Among TMCs with halide counterions, activity was higher for N,N,N-trimethyl chitosan chloride (TMCCl), whereas N,N,N-trimethyl chitosan iodide (TMCI) and N,N,N-trimethyl chitosan bromide (TMCBr) exhibited lower, similar values to each other. This is consistent with the shielding of −+N(CH3)3 groups inferred from chemical shifts for halide counterions in 1HNMR spectra. We also demonstrate that TMCs with distinct bactericidal activities can be classified according to their vibrational spectra using principal component analysis. Taken together, these physicochemical characterization approaches represent a predictive tool for the bactericidal activity of chitosan derivatives.

Effect of peroxide depolymerization of chitosan on properties of chitosan sulfate particles produced from this substance

Effect of the oxidative destruction of chitosan on the rate at which a dispersed phase is formed in its dilute solutions in the presence of sulfate ions and on the composition, size and ζ-potential of submicrometer chitosan sulfate particles being formed was studied. It was found that the particle size steadily decreases as the molecular mass of chitosan becomes smaller, and the sedimentation stability of aqueous dispersions increases in the absence of surfactants. The \(\nu _{SO_4 } :\nu _{NH_2 }\) molar ratio in chitosan sulfate particles is independent of the molecular mass of chitosan and varies within the range 0.45–0.46. A pH-dependence of the sign of the ζ-potential with isoelectric point at pH 5.0 was found for particles based on destructed chitosan.

EX VITRO ADAPTATION OF REGENERATED STRAWBERRY (MOSCOW DELICACY VARIETY)

Adaptation of regenerated plants of strawberry (varietу Moscow Delicaсy) towards conditions in vitro were performed on a hydroponic installation "minivan 0.35" and calcined river sand. Plants were adapted to hydroponic installation characterized by intensive development of shoots and leaves, which are 1.5-2 times higher than those figures have regenerated, adapted in the sand. Thus the root system formed in hydroponic conditions had roots of the second order. Before planting in the ground regenerated plants were treated with 0.1% sodium sulfate, chitosan and ultrasound. Ultrasonic irradiation was carried out using the apparatus of "Wave" with a frequency mechanical vibrations of 22±1,65 kHz. Spraying the leaves of 0.1% sodium sulfate chitosan virtually had no impact on plant growth and development. Soaking the root system in the same solution also significantly increased the number of whiskers (1.75 pcs.) and leaves (0.35 pcs.), the length of the whiskers (1.86 cm) and significantly increased the height of the outlet 1.79 cm. Treatment of plants with ultrasound for 10 minutes slightly increased the amount of whiskers (1.54 pcs.) and length (1.26 cm), and significantly increases the height of the slots (1.79 cm). Designed experiments aimed to assess the ability of reproduction of plants of strawberry produced in the traditional way and in vitro studies have shown that plants propagated in vitro formed 6,43±0,99 whiskers per socket, while the plants produced by conventional method had no whiskers.

Monosaccharide compositions of sulfated chitosans obtained by analysis of nitrous acid degraded and pyrazolone-labeled products

Chemically sulfated chitosans are important biomaterials. However, a reliable analytical method for quality control over such compounds is still lacking. In this study, we prepared four different kinds of selectively sulfated chitosans and developed a novel method to analyze their monosaccharide compositions by HPLC. In this method, nitrous acid was used to generate 2, 5-hydro mannose (M), 3-O-sulfated M (M3), 6-O-sulfated M (M6), and 3, 6-O-disulfated M (M9) from the sulfated chitosans. PMP, that is 1-phenyl-3-methyl-5-pyrazolone with a UV absorbance at 245nm, was used to label all the Ms quantitatively. The monosaccharide compositions for each sulfated chitosan were obtained by C18 HPLC separation and online UV detection of all PMP-labeled Ms. The identities of all Ms were confirmed by MS analysis with the help of standard Ms generated from a heparin pentasaccharide and chitosan. The overall results indicated that the newly developed method had advantages over 13C NMR in defining the monosaccharide compositions of sulfated chitosans and was useful for quality control purpose.

Investigation of Anti-Alga Properties and Anti-Bacteria Effects of Composite Nanofiltration Membranes Based on Chitosan Derivatives

The anti-alga properties and anti-bacteria effects of composite nanofiltration (NF) membranes prepared from sulfated chitosan (SCS) and N, O-carboxymethyl chitosan (NOCC) were investigated in this study. The base membranes, polyacrylonitrile (PAN) and polysulfone (PS) ultrafiltration (UF) membranes, were used to be as the controls. Compared with the controls, the adsorptions of the alga on the composite NF membranes were less severe. It suggested that the SCS and NOCC composite NF membranes have anti-alga and antifouling abilities. The chosen bacteria were escherichia coli, bacillus subtilis, staureus, penicillium chrysogenum, and streptomyces jinyangensis. By comparing the colony diameters of different bacteria on various membranes and the growth of bacteria after different time periods, the qualitative conclusions of the anti-bacterial effects of the membranes were drawn. It suggested that all the investigated membranes have some anti-bacterial effects on the five kinds of bacteria and the anti-bacterial effects are related to the active layer material of the composite NF membrane and the cross-linking agent.

Synthesis of Chitosan-Coated Magnetic Microparticle Using Glutaraldehyde as Crosslinker and PEG as Spacer Arm and Its Application as Adsorbent of Peat Humic Acid

A simple procedure for synthesis of chitosan-coated magnetic microparticle (CMMP) using glutaraldehyde as a cross-linker and polyethylene glycol (PEG) as a spacer arm has been developed. The functionalized microparticle were prepared using an inexpensive, simple, rapid, one-pot process, based on the heating of chitosan, PEG, and ferrous sulfate mixture at high pH. X-ray diffraction results indicated that the surfacemodified Fe3O4 microparticle did not lead to phase change unlike the pure Fe3O4. Magnetic chitosan adsorbent has been evaluated for removal of peat humic acid from its aqueous solution.

Biomimetic microbeads containing a chondroitin sulfate/chitosan polyelectrolyte complex for cell-based cartilage therapy.

Articular cartilage has a limited healing capacity that complicates the treatment of joint injuries and osteoarthritis. Newer repair strategies have focused on the use of cells and biomaterials to promote cartilage regeneration. In the present study, we developed and characterized bioinspired materials designed to mimic the composition of the cartilage extracellular matrix. Chondroitin sulfate (CS) and chitosan (CH) were used to form physically cross-linked macromolecular polyelectrolyte complexes (PEC) without the use of additional crosslinkers. A single-step water-in-oil emulsification process was used to either directly embed mesenchymal stem cells (MSC) in PEC particles created with a various concentrations of CS and CH, or to co-embed MSC with PEC in agarose-based microbeads. Direct embedding of MSC in PEC resulted in high cell viability but irregular and large particles. Co-embedding of PEC particles with MSC in agarose (Ag) resulted in uniform microbeads 80-90 μm in diameter that maintained high cell viability over three weeks in culture. Increased serum content resulted in more uniform PEC distribution within the microbead matrix, and both high and low CS:CH ratios resulted in more homogeneous microbeads than 1:1 formulations. Under chondrogenic conditions, expression of sulfated GAG and collagen type II was increased in 10:1 CS:CH PEC-Ag microbeads compared to pure Ag beads, indicating a chondrogenic influence of the PEC component. Such PEC-Ag microbeads may have utility in the directed differentiation and delivery of progenitor cell populations for cartilage repair.

Enhanced bioactivity of transform growth factor-β1 from sulfated chitosan microspheres for in vitro chondrogenesis of mesenchymal stem cells

Abstract Transform growth factor-β1 (TGF-β1) is an extremely powerful protein to induce the chondrogenesis of mesenchymal stem cells (MSCs) both in vitro and in vivo. However, due to the short-life of TGF-β1, the direct application of TGF-β1 may deteriorate its bioactivity and thereby the repair effect. In this study, uniform sulfated chitosan microspheres (SCMs) with a mean diameter of ∼ 2 μm were fabricated by membrane emulsification as a carrier for TGF-β1. The in vitro release study showed that TGF-β1 could be sustainedly released from the microspheres up to 16 days. Under the protection of SCMs, about 13 % TGF-β1 was preserved even after stored for 14 days. The microspheres cytotoxicity was evaluated by coculture of MSCs with different concentrations SCMs and no obvious deterioration of cell viability was observed when the concentration of SCMs is lower than 2 μg/1.0 × 104 cells. In comparison with the blank group, the addition of TGF-β1 either in free state or loaded in SCMs inhibited the proliferation trend of MSCs. Quantitative analysis of GAGs production and genes expression of COL II and aggrecan by qRT-PCR revealed that enhanced bioactivity of TGF-β1 was obtained in the group of TGF-β1/SCMs, indicating that SCMs could be functioned as a promising carrier of TGF-β1 for the in vitro chondrogenesis of MSCs.

Sulfated chitosan oligosaccharides suppress LPS-induced NO production via JNK and NF-κB inactivation.

Various biological effects have been reported for sulfated chitosan oligosaccharides, but the molecular mechanisms of action of their anti-inflammatory effects are still unknown. This study aimed to evaluate the anti-inflammatory effects of sulfated chitosan oligosaccharides and to elucidate the possible mechanisms of action. The results showed that pretreated low molecular weight sulfated chitosan oligosaccharides inhibited the production of nitric oxide (NO) and inflammatory cytokines such as IL-6 and TNF-α in lipopolysaccharide (LPS)-activated RAW264.7 cells. The sulfated chitosan oligosaccharides also suppressed inducible nitric oxide synthase (iNOS), phosphorylation of JNK and translocation of p65, a subunit of NF-κB, into the nucleus by inhibiting degradation of IκB-α. Our investigation suggests sulfated chitosan oligosaccharides inhibit IL-6/TNF-α in LPS-induced macrophages, regulated by mitogen-activated protein kinases (MAPKs) pathways dependent on NF-κB activation.

Characterization and anti-tumor effects of chondroitin sulfate–chitosan nanoparticles delivery system

We prepared chondroitin sulfate (ChS)–chitosan (CS) nanoparticles (NPs) as a delivery carrier, and doxorubicin (Dox) was used as a model drug. The physicochemical properties and biological activities of the Dox–ChS–CS NPs including the release profile, cell cytotoxicity, cellular internalization, and in vivo anti-tumor effects were evaluated. The ChS–CS NPs and Dox–ChS–CS NPs had a mean size of 262.0 ± 15.0 and 369.4 ± 77.4 nm, and a zeta potential of 30.2 ± 0.9 and 20.6 ± 3.1 mV, respectively. In vitro release tests showed that the 50 % release time for the Dox–ChS–CS NPs was 20 h. Two hepatoma cell models, HepG2 and HuH6, were used for evaluating the cytotoxicity and cell uptake efficiency of the Dox–ChS–CS NPs. A significant difference was observed between doxorubicin solution and the Dox–ChS–CS NPs in the cellular uptake within 60 min (p < 0.01). For the in vivo human xenograft-nude mouse model, the Dox–ChS–CS NPs were more effective with less body weight loss and anti-tumor growth suppression in comparison with the Dox solution. The prepared Dox–ChS–CS NPs offer a new effective targeting nanoparticle delivery system platform for anti-tumor therapy.

6-O-Sulfated Chitosan Promoting the Neural Differentiation of Mouse Embryonic Stem Cells

Embryonic stem cells (ESCs) can be induced to differentiate into nerve cells, endowing them with potential applications in the treatment of neurological diseases and neural repair. In this work, we report for the first time that sulfated chitosan can promote the neural differentiation of ESCs. As a type of sulfated glycosaminoglycan analog, sulfated chitosan with well-defined sulfation sites and a controlled degree of sulfation (DS) were prepared through simple procedures and the influence of sulfated glycosaminoglycan on neural differentiation of ESCs was investigated. Compared with other sulfation sites, 6-O-sulfated chitosan showed the most optimal effects. By monitoring the expression level of neural differentiation markers using immunofluorescence staining and PCR, it was found that neural differentiation was better enhanced by increasing the DS of 6-O-sulfated chitosan. However, increasing the DS by introducing another sulfation site in addition to the 6-O site to chitosan did not promote neural differentiation as much as 6-O-sulfated chitosan, indicating that compared with DS, the sulfation site is more important. Additionally, the optimal concentration and incubation time of 6-O-sulfated chitosan were investigated. Together, our results indicate that the sulfate site and the molecular structure in a sulfated polysaccharide are very important for inducing the differentiation of ESCs. Our findings may help to highlight the role of sulfated polysaccharide in inducing the neural differentiation of ESCs.