Migration deficits of the neural crest caused by CXADR triplication in a human Down syndrome stem cell model.

Down syndrome (DS) is the most common chromosomal abnormality in live-born infants and is caused by trisomy of chromosome 21. Most individuals with DS display craniofacial dysmorphology, including reduced sizes of the skull, maxilla, and mandible. However, the underlying pathogenesis remains largely unknown. Since the craniofacial skeleton is mainly formed by the neural crest, whether neural crest developmental defects are involved in the craniofacial anomalies of individuals with DS needs to be investigated. Here, we successfully derived DS-specific human induced pluripotent stem cells (hiPSCs) using a Sendai virus vector. When DS-hiPSCs were induced to differentiate into the neural crest, we found that trisomy 21 (T21) did not influence cell proliferation or apoptosis. However, the migratory ability of differentiated cells was significantly compromised, thus resulting in a substantially lower number of postmigratory cranial neural crest stem cells (NCSCs) in the DS group than in the control group. We further discovered that the migration defects could be partially attributed to the triplication of the coxsackievirus and adenovirus receptor gene (CXADR; an adhesion protein) in the DS group cells, since knockdown of CXADR substantially recovered the cell migratory ability and generation of postmigratory NCSCs in the DS group. Thus, the migratory deficits of neural crest cells may be an underlying cause of craniofacial dysmorphology in individuals with DS, which may suggest potential targets for therapeutic intervention to ameliorate craniofacial or other neural crest-related anomalies in DS.

Characterization and Therapeutic Application of Mesenchymal Stem Cells with Neuromesodermal Origin from Human Pluripotent Stem Cells.

Rationale: Mesenchymal stem cells (MSC) hold great promise in the treatment of various diseases including autoimmune diseases, inflammatory diseases, etc., due to their pleiotropic properties. However, largely incongruent data were obtained from different MSC-based clinical trials, which may be partially due to functional heterogeneity among MSC. Here, we attempt to derive homogeneous mesenchymal stem cells with neuromesodermal origin from human pluripotent stem cells (hPSC) and evaluate their functional properties. Methods: Growth factors and/or small molecules were used for the differentiation of human pluripotent stem cells (hPSC) into neuromesodermal progenitors (NMP), which were then cultured in animal component-free and serum-free induction medium for the derivation and long-term expansion of MSC. The resulted NMP-MSC were detailed characterized by analyzing their surface marker expression, proliferation, migration, multipotency, immunomodulatory activity and global gene expression profile. Moreover, the in vivo therapeutic potential of NMP-MSC was detected in a mouse model of contact hypersensitivity (CHS). Results: We demonstrate that NMP-MSC express posterior HOX genes and exhibit characteristics similar to those of bone marrow MSC (BMSC), and NMP-MSC derived from different hPSC lines show high level of similarity in global gene expression profiles. More importantly, NMP-MSC display much stronger immunomodulatory activity than BMSC in vitro and in vivo, as revealed by decreased inflammatory cell infiltration and diminished production of pro-inflammatory cytokines in inflamed tissue of CHS models. Conclusion: Our results identify NMP as a new source of MSC and suggest that functional and homogeneous NMP-MSC could serve as a candidate for MSC-based therapies.

Enhanced generation of human induced pluripotent stem cells by ectopic expression of Connexin 45.

Somatic cells can be successfully reprogrammed into pluripotent stem cells by the ectopic expression of defined transcriptional factors. However, improved efficiency and better understanding the molecular mechanism underlying reprogramming are still required. In the present study, a scrape loading/dye transfer assay showed that human induced pluripotent stem cells (hiPSCs) contained functional gap junctions partially contributed by Connexin 45 (CX45). We then found CX45 was expressed in human embryonic stem cells (hESCs) and human dermal fibroblasts (hDFs) derived hiPSCs. Then we showed that CX45 was dramatically upregulated during the reprogramming process. Most importantly, the ectopic expression of CX45 significantly enhanced the reprogramming efficiency together with the Yamanaka factors (OCT4, SOX2, KLF4, cMYC - OSKM), whereas knockdown of endogenous CX45 expression significantly blocked cellular reprogramming and reduced the efficiency. Our further study demonstrated that CX45 overexpression or knockdown modulated the cell proliferation rate which was associated with the reprogramming efficiency. In conclusion, our data highlighted the critical role of CX45 in reprogramming and may increase the cell division rate and result in an accelerated kinetics of iPSCs production.

Controlling the strontium-doping in calcium phosphate microcapsules through yeast-regulated biomimetic mineralization.

Yeast cells have controllable biosorption on metallic ions during metabolism. However, few studies were dedicated to using yeast-regulated biomimetic mineralization process to control the strontium-doped positions in calcium phosphate microcapsules. In this study, the yeast cells were allowed to pre-adsorb strontium ions metabolically and then served as sacrificing template for the precipitation and calcination of mineral shell. The pre-adsorption enabled the microorganism to enrich of strontium ions into the inner part of the microcapsules, which ensured a slow-release profile of the trace element from the microcapsule. The co-culture with human marrow stromal cells showed that gene expressions of alkaline phosphatase and Collagen-I were promoted. The promotion of osteogenic differentiation was further confirmed in the 3D culture of cell-material complexes. The strategy using living microorganism as 'smart doping apparatus' to control incorporation of trace element into calcium phosphate paved a pathway to new functional materials for hard tissue regeneration.

Biological performance of functionalized biomedical polymers for potential applications as intraocular lens.

To study the biological performance of surface-modified biomedical polymer materials, a model of the functional mechanism of nonspecific adsorption resistance was constructed. Cell behavior on the surface and in vivo transplantation features of intraocular lens (IOL) materials, such as hydrophobic acrylic ester and polymethyl methacrylate (PMMA), were investigated. The results of cell adhesion and proliferation studies showed that the addition of hirudin can significantly resist epithelial cell adhesion, better than the pure amination process, and thereby inhibit excessive proliferation on the surface. Experiments on the eyes of rabbits indicated that the IOL surfaces with hirudin modification reduced the incidence of cell aggregation and inflammation. Combined with a study of protein-resistant layer construction with recombinant hirudin on the material surface, the mechanism of surface functionalization was determined. The biological performance indicated that nonspecific adsorption is greatly decreased due to the existence of amphiphilic ions or hydration layers, which lead to stability and long-term resistance to nonspecific adsorption. These results offer a theoretical basis for the use of traditional biomedical polymer materials in long-term clinical applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1961-1967, 2016.

Evaluation of insulin medium or chondrogenic medium on proliferation and chondrogenesis of ATDC5 cells.

BACKGROUND: The ATDC5 cell line is regarded as an excellent cell model for chondrogenesis. In most studies with ATDC5 cells, insulin medium (IM) was used to induce chondrogenesis while chondrogenic medium (CM), which was usually applied in chondrogenesis of mesenchymal stem cells (MSCs), was rarely used for ATDC5 cells. This study was mainly designed to investigate the effect of IM, CM, and growth medium (GM) on chondrogenesis of ATDC5 cells. METHODS: ATDC5 cells were, respectively, cultured in IM, CM, and GM for a certain time. Then the proliferation and the chondrogenesis progress of cells in these groups were analyzed. RESULTS: Compared with CM and GM, IM promoted the proliferation of cells significantly. CM was effective for enhancement of cartilage specific markers, while IM induced the cells to express endochondral ossification related genes. Although GAG deposition per cell in CM group was significantly higher than that in IM and GM groups, the total GAG contents in IM group were the most. CONCLUSION: This study demonstrated that CM focused on induction of chondrogenic differentiation while IM was in favor of promoting proliferation and expression of endochondral ossification related genes. Combinational use of these two media would be more beneficial to bone/cartilage repair.

Phosphatidylserine enhances osteogenic differentiation in human mesenchymal stem cells via ERK signal pathways.

Phosphatidylserine (PS) has been demonstrated to promote bone mineralization. It has also been used in bone repairing biomaterials as a functional molecule. However, the effect of PS on mesenchymal stem cells (MSCs) is not clear. In this study, we determined the effect of PS on the osteogenic differentiation of human MSCs (hMSCs) cultured in growth or osteogenic differentiation medium and the role of the ERK1/2 signaling pathway on PS activity. Cytotoxicity of PS was measured by MTT assay in growth medium for 5 days. Cell osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity analysis, Alizarin Red S staining and real-time PCR assay. Western blotting and ERK blocking assay were used to examine the role of ERK1/2 signaling pathway on PS activity. The results showed no cytotoxicity for the doses of PS administered. For 21 days, 50-100 µM PS increased ALP expression and mineralization of hMSCs. The expression of the osteogenic gene marker, ALP, osteocalcin (OC), and RUNX2 was enhanced by 50 µM PS treatment at day 14. Phospho-ERK was activated by 50 µM PS at 30 min and 1h in growth medium. In osteogenic medium, 50 µM PS extended phospho-ERK activation by osteogenic induction medium from 30 min to 8 h. U0126, an ERK inhibitor, suppressed the ALP expression induced by PS. Our data indicate that the ERK signal is potentially a mediator in the process of osteogenic differentiation of hMSCs induced by PS. PS, as a functional molecule, has high potential for use in bone repairing biomaterials and bone tissue engineering.

Importance of suitable reference gene selection for quantitative RT-PCR during ATDC5 cells chondrocyte differentiation.

Real-time quantitative reverse transcription-polymerase chain reaction (qPCR) is an efficient and accurate method to detect and compare patterns of gene expression. The reliability of qPCR is highly dependent on the selection of appropriate reference genes used for normalization. By analyzing 16 potential candidates of reference genes (GAPDH, Actb, 18 s, PGK1, Hprt, Tbp, Rpl5, B2M, Gusb, Ppia, UBC, Sdha, Eef1a1, H2afz, Tkt and Ldha) through geNorm, we identified Ppia, Tbp, Hprt and Eef1a1 as the most stable reference genes while UBC, B2M, Gusb as the least stable ones during the chondrocyte differentiation of ATDC5 cells. Considering the low expression of Eef1a1 and Tbp would cause divergent results for they failed to provide accurate normalization for RNA extraction and reverse transcription efficiency, we recommended the use of Ppia and Hprt as the most suitable genes to normalize qPCR. In addition, although GAPDH, Actb and 18 s were usually adopted in most of studies using ATDC5 cells, they were found unstable and then were not ideal reference genes for qPCR assay in ATDC5 cells chondrocyte differentiation. Also, we further confirmed that the Ppia and Hprt worked well during chondrocyte differentiation of mouse mesenchymal cells.

Surface modification on polyethylene terephthalate films with 2-methacryloyloxyethyl phosphorylcholine.

In this study, the surface of polyethylene terephthalate (PET) was modified to improve the protein and cell adhesion behavior with low temperature ammonia plasma treatment followed by 2-methacryloyloxyethyl phosphorylcholine (MPC) grafting. The x-ray photoelectron spectroscopy (XPS) results showed that the -COO(-), -N-C=O and -P-O-H groups were successfully incorporated onto the sample surface after MPC grafting. Furthermore, formation of new bonds, -N= and N-H on the sample surface grafted with MPC was recorded by Fourier transform infrared spectroscopy (FTIR). A large number of spherical particles at submicron to nanometer scale were also observed on the surface by atomic force microscopy (AFM). The cell adhesion experiments on PET film surfaces were evaluated and the highly hydrophilic surfaces could not promote cell adhesion and spreading. All results achieved in this study have clearly indicated that the method combining low temperature ammonia plasma treatment and MPC grafting is an effective way of producing a suitably hydrophilic PET surface with the capability of weakening the protein adsorption greatly.

In vitro generation of osteochondral differentiation of human marrow mesenchymal stem cells in novel collagen-hydroxyapatite layered scaffolds.

Integrated, layered osteochondral (OC) composite materials and/or engineered OC grafts are considered as promising strategies for the treatment of OC damage. A novel biomimetic collagen-hydroxyapatite (COL-HA) OC scaffold with different integrated layers has been generated by freeze-drying. The capacity of the upper COL layer and the lower COL/HA layer to promote the growth and differentiation of human mesenchymal stem cells (hMSCs) into chondrocytes and osteoblasts respectively was evaluated. Cell viability and proliferation on COL and COL/HA scaffolds were assessed by the MTT test. The chondrogenic differentiation of hMSCs on both scaffolds was evaluated by glucosaminoglycan (GAG) quantification, alcian blue staining, type II collagen immunocytochemistry assay and real-time polymerase chain reaction in chondrogenic medium for 21 days. Osteogenic differentiation was evaluated by alkaline phosphatase activity assay, type I collagen immunocytochemistry staining, alizarin S staining and mRNA expression of osteogenic gene for 14 days in osteogenic medium. The results indicated that hMSCs on both COL and COL/HA scaffolds were viable and able to proliferate over time. The COL layer was more efficient in inducing hMSC chondrogenic differentiation than the COL/HA layer, while the COL/HA layer possessed the superiority on promoting hMSC osteogenic induction over either COL layer or pure HA. In conclusion, the layered OC composite materials can effectively promote cartilage and bone tissue generation in vitro and are potentially usable for OC tissue engineering.

[Mutational research on the role of lysine 21 in the Pichia stipitis xylose reductase].

The xylose reductase of Pichia stipitis is one of the most important enzymes. It can be used to build up recombinant Saccharomyces cerevisiae strain for utilizing xylose and producing ethanol. Intercellular redox imbalance caused by NADPH preference over NADH for Pichia stipitis xylose reductase (PsXR) has been considered to be one of the main factors for poor ethanol productivity. Some key amino acids of PsXR, which affect the activity or coenzyme preference, were investigated in our previous study. In this study, Lys21 were rational designed for site-directed mutagenesis to alter coenzyme specificity of PsXR from NADPH and NADH into single NADH. The wild gene and mutagenesis genes were ligated into pET28b, and were transferred into E.coli BL21(DE3). After induced by IPTG, the xylose reductases were purified. Purified mutants K21A (Lys21-->Ala), K21R(Lys21-->Arg) were characterized by steady-state kinetic analysis. The results showed that the coenzyme dependence of K21A was completely reversed to NADH.