Mitogen activated protein kinase signaling pathways participate in the active principle region of Buyang Huanwu decoction-induced differentiation of bone marrow mesenchymal stem cells

Our preliminary studies confirmed that an active principle region of Buyang Huanwu decoction, comprising alkaloid, polysaccharide, aglycon, glucoside and volatile oil, can induce bone marrow mesenchymal stem cell differentiation into neurons. Mitogen-activated protein kinase signaling was identified as one of the key pathways underlying this differentiation process. The present study shows phosphorylated extracellular signal-regulated protein kinase and phosphorylated p38 protein expression was increased after differentiation. Cellular signaling pathway blocking agents, PD98059 and SB203580, inhibited extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways respectively, mRNA and protein expression of the neuronal marker, neuron specific enolase, and neural stem cell marker, nestin, were decreased in bone marrow mesenchymal stem cells after treatment with the active principle region of Buyang Huanwu decoction. Experimental findings indicate that, extracellular signal-regulated protein kinase and p38 in mitogen-activated protein kinase signaling pathways participate in bone marrow mesenchymal stem cell differentiation into neuron-like cells, induced by the active principle region of Buyang Huanwu decoction.

Insight of a novel functional gene related to differentiation of neural stem cells

A novel fimctional gene P12 was isolated from neural stem cells cultured in differentiation medium. The fully length cDNA of P12 gene was cloned and sequenced. Result showed that it contains an open reading frame encoding a protein of 291 amino acids. Further, this gene was transferred into neural stem cell. Functional analysis suggests that the expression of p12 protein is closely correlated with differentiation of neural dendrite configuration. In addition, to obtain encoding protein, P12 sequence was also expressed in Pichia pastoris yeast.

Neural cell injury microenvironment induces neural differentiation of human umbilical cord mesenchymal stem cells

This study aimed to investigate the neural differentiation of human umbilical cord mesenchymal stem cells （hUCMSCs） under the induction of injured neural cells. After in vitro isolation and culture, passage 5 hUCMSCs were used for experimentation, hUCMSCs were co-cultured with normal or AI31.4o-injured PC12 cells, PC12 cell supernatant or PC12 cell lysate in a Transwell co-culture system. Western blot analysis and flow cytometry results showed that choline acetyltransferase and microtubule-associated protein 2, a specific marker for neural cells, were expressed in hUCMSCs under various culture conditions, and highest expression was observed in the hUCMSCs co-cultured with injured PC12 cells. Choline acetyltransferase and microtubule-associated protein 2 were not expressed in hUCMSCs cultured alone （no treatment）. Cell Counting Kit-8 assay results showed that hUCMSCs under co-culture conditions promoted the proliferation of injured PC12 cells. These findings suggest that the microenvironment during neural tissue injury can effectively induce neural cell differentiation of hUCMSCs. These differentiated hUCMSCs likely accelerate the repair of injured neural ceils.

Expression of Embryonic Stem Cell Marker Oct-4 and Its Prognostic Significance in Rectal Adenocarcinoma

Objective： Recent evidence suggests that Oct-4 is highly expressed in several cancers, and its expression contributes to tumor growth. In this study, we investigated the level of Oct-4 expression in rectal adenocarcinoma, and evaluated the prognostic significance of Oct-4 expression in these cases. Methods： The immunohistochemical expression of Oct-4 was evaluated in 52 formalin-fixed paraffin-embedded postoperative rectal adenocarcinoma tissue samples. The impact of the immunoreactivity of Oct-4 in regard to clinical outcome was determined by Kaplan-Meier and log-rank. Results： The expression level of Oct-4 ranged from 0 to 18.5%. There was no significant association between Oct-4 expression and gender （P=0.772）, age （P=0.123）, clinical stage （P=0.391）, and histological grade （P=0.056）. The 3-year local recurrence-free rates with negative and positive expression of Oct-4 were 83.5% and 75.0%, respectively （P=0.583）. The 3-year metastasis-free rates with negative and positive expression of Oct-4 were 88.6% and 61.9%, respectively （P=0.035）. The 3-year overall survival rates with negative and positive expression of Oct-4 were 77.9% and 49.0%, respectively （P=0.037）. Conclusion： The results suggest that embryonic stem cell marker Oct-4 expression may have prognostic significance in patients with rectal adenocarcinoma. However, to confirm this more and larger studies are required.

Effect of IKVAV Peptide Nanofiber on Proliferation,Adhesion and Differentiation into Neurocytes of Bone Marrow Stromal Cells

This study examined the effect of IKVAV peptide nanofiber on proliferation, adhesion and differentiation into neurocytes of bone marrow stromal cells （BMSCs）. IKVAV Peptide-amphiphile was synthesized and purified. Then, hydrogen chloride was added to the diluted aqueous solutions of PA to induce spontaneous formation of nanofiber in vitro. The resultant samples was observed tmder transmission electron microscope. BMSCs were cultured with IKVAV peptide nanofiber. The effect of IKVAV nanofiber on the proliferation, adhesion and induction differentiation of BMSCs was observed by inverted microscopy, calcein-AM/PI staining, cell counting and immunofluorescence staining. The results demonstrated that IKVAV peptide-amphiphile could self-assemble to form nanofiber gel. BMSCs cultured in combination with IKVAV peptide nanofiber gel grew well and the percentage of live cells was over 90%. IKVAV peptide nanofiber gel exerted no influence on the proliferation of BMSCs and could promote the adhesion of BMSCs and raise the ra- tio of neurons when BMSCs were induced to differentiate into neurocytes. It is concluded that BMSCs could proliferate and adhere well and yield more neurons during when induced to differente into neurocytes on IKVAV peptide nanofiber gel.

The Three-Dimensional Collagen Scaffold Improves the Sternness of Rat Bone Marrow Mesenchymal Stem Cells

Mesenchymal stem cells （MSCs） show the great promise for the treatment of a variety of diseases because of their self-renewal and multipotential abilities. MSCs are generally cultured on two-dimensional （2D） substrate in vitro. There are indications that they may simultaneously lose their sternness and multipotentiality as the result of prolonged 2D culture. In this study, we used three-dimensional （3D） collagen scaffolds as rat MSCs cartier and compared the properties of MSCs on 3D collagen scaffolds with monolayer cultured MSCs. The results demonstrated that collagen scaffolds were suitable for rat MSCs adherence and proliferation. More importantly, compared to MSCs under 2D culture, 3D MSCs significantly maintained higher expression levels of stemness genes （Oct4, Sox2, Rex-1 and Nanog）, yielded high frequencies of colony-forming units-fibroblastic （CFU-F） and showed enhanced osteogenic and adipogenic differentiation efficiency upon induction. Thus, 3D collagen scaffolds may be beneficial for expanding rat MSCs while maintaining the stem cell properties in vitro.

Derivation and characterization of human embryonic stem cell lines from poor quality embryos

Poor quality embryos discarded from in vitro fertilization （IVF） laboratories are good sources for deriving human embryonic stem cell （hESC） lines. In this study, 166 poor quality embryos donated from IVF centers on day 3 were cultured in a blastocyst medium for 2 days, and 32 early blastocysts were further cultured in a blastocyst optimum culture medium for additional 2 days so that the inner cell masses （ICMs） could be identified and isolated easily. The ICMs of 17 blastocysts were isolated by a mechanical method, while those of the other 15 blastocysts were isolated by immunosurgery. All isolated ICMs were inoculated onto a feeder layer for subcultivation. The rates of ICM attachment, primary ICM colony formation and the efficiency of hESC derivation were similar between the ICMs isolated by the two methods （P〉0.05）. As a result, four new hESC lines were established. Three cell lines had normal karyotypes and one had an unbalanced Robertsonian translocation. All cell lines showed normal hESC characteristics and had the differentiation ability. In conclusion, we established a stable and effective method for hESC isolation and culture, and it was confirmed that the mechanical isolation was an effective method to isolate ICMs from poor embryos. These results further indicate that hESC lines can be derived from poor quality embryos discarded by IVF laboratories.