I-Ming Chu

Compare the effects of chondrogenesis by culture of human mesenchymal stem cells with various type of the chondroitin sulfate C

Chondroitin sulfate C (CSC) is a kind of glycosaminoglycans (GAGs) with molecular weights of 10,000 to 50,000 Da and a high charge density. GAGs are major components in extracellular matrix (ECM), which play important role in the regulation of cell proliferation, migration, and differentiation. In this study, we studied the effects of chondroitin sulfate C (CSC) on the differentiation of human mesenchymal stem cells (MSCs) toward the chondrocyte lineage. The MSCs were either cultured on type II collagen (COL II) scaffolds with high molecular weight CSC addition in the medium (free CSC) or with free oligosaccharide CSC. Special attention was given to the effects of MSCs cultured on CSC cross-linked type II scaffolds (cross-linked CSC). According to the analysis of histology stain, gene expression, and ECM secretion, our results showed that MSCs cultured with free CSC, free oligosaccharides CSC, and on the cross-linked CSC scaffolds all would be induced into chondrocytes. Moreover, free oligosaccharide CSC present in the microenvironment could significantly up-regulate MSC chondrogenesis gene expression and stimulate cartilage ECM accumulation more than free CSC with high molecular weight after 3-week induction. Importantly, cross-linked CSC had the most excellent effects on the MSC chondrogenesis. Thus, we believed that cross-linked CSC in the scaffold would play the similar roles with free oligosaccharide CSC in the medium. Cross-linked CSC would be a potential candidate for cartilage repair in the cell therapy and tissue engineering.

Evaluating osteochondral defect repair potential of autologous rabbit bone marrow cells on type II collagen scaffold

The feasibility of using genipin cross-linked type II collagen scaffold with rabbit bone marrow mesenchymal stem cells (RBMSCs) to repair cartilage defect was herein studied. Induction of RBMSCs into chondrocytic phenotype on type II collagen scaffold in vitro was conducted using TGF-β 3 containing medium. After 3-weeks of induction, chondrocytic behavior, including marker genes expression and specific extracellular matrix (ECM) secretion, was observed. In the in vivo evaluation experiment, the scaffolds containing RBMSCs without prior induction were autologous implanted into the articular cartilage defects made by subchondral drilling. The repairing ability was evaluated. After 2xa0months, chondrocyte-like cells with lacuna structure and corresponding ECM were found in the repaired sites without apparent inflammation. After 24xa0weeks, we could easily find cartilage structure the same with normal cartilage in the repair site. In conclusion, it was shown that the scaffolds in combination of in vivo conditions can induce RBMSCs into chondrocytes in repaired area and would be a possible method for articular cartilage repair in clinic and cartilage tissue engineering.

Characterization and transplantation of induced megakaryocytes from hematopoietic stem cells for rapid platelet recovery by a two-step serum-free procedure

OBJECTIVEnA complete process for mass generation of megakaryocytes from hematopoietic stem cells under serum-free conditions has great clinical potential for rapid platelet reconstruction in thrombocytopenia patients. We have previously reported on the generation of an optimized serum-free medium (serum-free hematopoietic stem cell medium) for ex vivo expansion of CD34(+) cells. Here, we further generated large amounts of functional megakaryocytes from serum-free expanded CD34(+) cells under a complete and optimal serum-free condition for complying with clinical regulations.nnnMATERIALS AND METHODSnSerum substitutes and cytokines were screened and optimized for their concentration for megakaryocyte generation by systemically methods. Serum-free induced megakaryocytes were characterized by surface antigens, gene expression, ex vivo megakaryocyte activation ability, and ability of megakaryocyte and platelet recovery in nonobese diabetic/severe combined immunodeficient mice.nnnRESULTSnThe optimal serum-free megakaryocyte induction medium was Iscoves modified Dulbeccos medium containing serum substitutes (i.e., human serum albumin, human insulin, and human transferrin) and a cytokine cocktail (i.e., thrombopoietin, stem cell factor, Fms-like tyrosine kinase 3 ligand, interleukin-3, interleukin-6, interleukin-9, and granulocyte-macrophage colony-stimulating factor). After induction, induced megakaryocytes expressed CD41a and CD61 surface antigens, nuclear factor erythroid-derived 2 and GATA-1 transcription factors and megakaryocyte activation ability. Importantly, transplantation of induced megakaryocytes could accelerate megakaryocyte and platelet recovery in irradiated nonobese diabetic/severe combined immunodeficient mice.nnnCONCLUSIONnIn conclusion, we have developed a serum-free megakaryocyte induction medium, and the combination of serum-free megakaryocyte and serum-free hematopoietic stem cell media can generate a large amount of functional megakaryocytes efficiently. Our method represents a promising source of megakaryocytes and platelets for future cell therapy.

Large generation of megakaryocytes from serum-free expanded human CD34+ cells

Ex vivo generation of megakaryocytes from hematopoietic stem cells (HSCs) is crucial to HSC research and has important clinical potential for thrombocytopenia patients to rapid platelet reconstruction. In this study, factorial design and steepest ascent method were used to screen and optimize the effective cytokines (10.2 ng/ml TPO, 4.3 ng/ml IL-3, 15.0 ng/ml SCF, 5.6 ng/ml IL-6, 2.8 ng/ml FL, 2.8 ng/ml IL-9, and 2.8 ng/ml GM-CSF) in megakaryocyte induction medium that facilitate ex vivo megakaryopoiesis from CD34(+) cells. After induction, the maximum fold expansion for accumulated megakaryocytes was almost 5000-fold, and the induced megakaryocytes were characterized by analysis of gene expression, polyploidy and platelet activation ability. Furthermore, the combination of megakaryocyte induction medium and HSC expansion medium can induce and expand a large amount of functional megakaryocytes efficiently, and might be a promising source of megakaryocytes and platelets for cell therapy in the future.

Design of polyanionic nanocarriers based on modified poly (aspartic acid)s for oral administration: synthesis and characterization

This study designed a series of polyanionic nanocarriers based on biodegradable and biocompatible poly (aspartic acid)s for oral administration. First, polysuccinimide (PSI) was synthesized from L-aspartic acid using acid-catalyzed bulk thermal polycondensation and acid-catalyzed thermal polycondensation in a mixture of mesitylene/sulfolane. PSI-C16 was then synthesized by aminolysis with nucleophile, hexadecylamine to react with PSI known as nucleophilic addition. Finally, a series of partially esterified poly (aspartic acid)s was produced by alkaline treatment to afford an amphiphilic polyanion, poly (sodium aspartate-g-hexadecyl aspartate) (Na-PASP-g-C16-PASP). 1HNMR, FTIR, DSC and GPC were utilized to demonstrate and characterize the polymers. The synthesized polyanion could be self-assembled into the nano-scaled micelles and be independent of pH in phosphoric buffer solutions. The hydrodynamic diameter and zeta potential were measured using the dynamic light scattering (DLS) method, and the critical micelle concentration (CMC) was determined using the fluorescence spectrophotometer. The micellar morphologies were examined using transmission electron microscopy (TEM), and atomic force microscopy (AFM) to present the nano-dimensional sphere. The stability of size transition at different pH levels, from strong acid to alkaline, proved that the micelles could stably transport from the stomach to intestinal lumen prior to arriving in the epithelium of the small intestine.

BIOLOGICAL EFFECTS OF OLIGOSACCHARIDE CHONDROITIN SULFATE C ON HUMAN ARTICULAR CHONDROCYTES

Chondroitin sulfate C (CSC) is an important extracellular matrix (ECM) component of native cartilage tissue. Since ECM is considered to play an important role in guiding proper cellular functions, such as proliferation, differentiation, migration, synthesis or degradation of ECM, in specific tissues, we would like to elucidate the effects of CSC on chondrocytes are cultured on type-II collagen (COL II) scaffolds in this study. In particular, we want to investigate if the oligosaccharides of CSC (O-CSC) have much stronger effects on the chondrocytes. In this in vitro study, human articular chondrocytes were cultured on porous scaffolds made of COL II, cross-linked by genipin. Media containing different molecular weights of CSC were used to cultivate the cells. The results were examined mainly from the gene expression profiles of the cultured cells. The expression levels of several genes were examined by real-time PCR. These included genes of COL II, aggrecan, SRY-related high mobility group-box gene 9 (SOX9) and cartilage oligo matrix protein (COMP), alkaine phosphtase (ALP), a disintegrin and metalloproteinases with thrombospondin motifs 4 (ADAMTS-4), a disintegrin and metalloproteinases with thrombospondin motifs 5 (ADAMTS-5), matrix metalloproteinases 3 (MMP-3) and tissue inhibitors of metalloproteinases 3 (TIMP-3). The results suggested that O-CSC is more potent in upregulating genes that promote chondrogenesis and downregulating genes that degrade cartilage ECM. The results suggest low-molecular-weight glycosaminoglycans may have therapeutic values in osteoarthritis treatment and may lead to further understanding of the basic mechanism of the interactions between the chondrocytes and their ECM.

Aurintricarboxylic Acid Exerts Insulin-Like Growth Stimulating Effects on Chinese Hamster Ovary Cells under Serum-Free Conditions

When aurintricarboxylic acid (ATA) was added at a concentration of 30 mg/l in DME/F12 medium to Chinese hamster ovary (CHO) NTHU-108 cells in static six-well plates, some of the cells exhibited adherent growth while others grew in suspension. Beyond the critical concentration of ATA, CHO cells grew in single-cell suspension. In the same serum-free medium, insulin at a level of 0.5 mg/l was found to be the most important protein ingredient promoting cell growth. We used the respective kinase inhibitors to investigate their influence on the cell proliferation induces by ATA and insulin. It is interesting that the inhibition by seven kinase inhibitors of ATA-induced proliferation is similar to that of insulin-induced proliferation. It is possible that ATA mimics insulin and influences the mitogen activated signal transduction to induce the proliferation of CHO cells. Although the actual mechanism of the proliferation of CHO cells by ATA is unclear, ATA supported the long-term proliferation of CHO cells under serum-free conditions and thus could be used as a good substitute for insulin in the formulation of protein-free media.

A process for simultaneously achieving phenol biodegradation and polyhydroxybutyrate accumulation using Cupriavidus taiwanesis 187

Cupriavidus taiwanensis 187 is reportedly efficient in achieving the degradation of phenol and accumulation of polyhydroxybutyrate (PHB). This study attempted to optimize the cultivation conditions and fermentation strategies for phenol degradation and PHA accumulation by C. taiwanensis 187. After the cultivation conditions were optimized, the conditions required for achieving phenol degradation (100%) and PHB accumulation (51xa0mg/L) by C. taiwanensis 187 were identified as 30xa0°C and 200xa0rpm, when the cultivation time was around 7xa0h. The accumulation of PHB was further increased from 72 to 213xa0mg/L by feeding phenol in three rounds into the fermenter along with the exhaustion of dissolved oxygen, which could totally degrade the phenol at around 1500xa0mg/L. Production of PHB by C. taiwanensis 187 was confirmed by GC, 1H-NMR, and 13C-NMR analyses. Each analytical result proved that C. taiwanensis 187 was able to use phenol as the sole carbon source for producing PHB. Finally, these results revealed that the phenol degraded by C. taiwanensis 187 mainly contributed to cell growth rather than PHB accumulation. These results indicated that the strain C. taiwanensis 187 could be used to degrade phenol to obtain usable biological polyesters.

Surface display of synthetic phytochelatins on Saccharomyces cerevisiae for enhanced ethanol production in heavy metal-contaminated substrates

The aim of this work was to study the feasibility of surface displaying synthetic phytochelatin (EC) on Saccharomyces cerevisiae to overcome the inhibitory effect of heavy metals on ethanol production. Via the fusion of a gene encoding EC to an α-agglutinin gene, the engineered S. cerevisiae was able to successfully display EC on its surface. This surface engineered yeast strain exhibited an efficient cadmium adsorption capability and a remarkably enhanced cadmium tolerance. Moreover, its ethanol production efficiency was significantly improved as compared to a control strain in the presence of cadmium. Similar results could also be observed in the presence of other metals, such as nickel, lead and copper. Overall, this method allows simultaneous biorefinery and heavy metal removal when using heavy metal-contaminated biomass as raw materials.