PEI-FeONanoparticles for Human Amniotic Epithelial Cells Labeling / 中国医学科学院学报

Objective To label human amniotic epithelial cells(hAECs) by using PEI-FeOnanoparticles. Methods The PEI-FeOnanoparticles were characterized by using transmission electron microscopy and dynamic light scattering. The primary cultured hAECs were labeled with the nanoparticles,and the labeling efficiency was evaluated by Prussian blue staining. The cell survival rate and viability were tested by using placenta blue staining and CCK-8 assay,respectively. Results The PEI-FeOnanoparticles were compact spheres with an average particle size of 13 nm,a hydrodynamic radius of 17.56 nm,and a zeta potential of+34.5 mV. The labeling efficiency of the nanoparticles on hAECs reached 91% when the concentrations were greater than 20 μg/ml. When the concentrations of nanoparticles were at 50 μg/ml(t=16.37,P<0.0001;t=10.39,P<0.0001) and 100 μg/ml(t=29.89,P<0.0001;t=16.86,P<0.0001),the cell survival rates and cell viabilities were significantly reduced versus controls. Conclusion The PEI-FeOnanoparticles can be used for labeling hAECs without obvious cytotoxicity at its working concentration.

Effect of epidermal growth factor on migration of human amniotic mesenchymal stem cells / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the mechanism via which the epidermal growth factor (EGF) affects the migration of human amnion-derived mesenchymal stem cells (hAMSCs).</p><p><b>METHODS</b>In vitro cultured hAMSCs were divided into control (untreated), EGF group, inhibitor AG1478 + EGF group, inhibitor LY294002 + EGF group, and inhibitor U0126 + EGF group. The migration ability of hAMSCs in each group was measured using Transwell chamber. The expressions of phosphorylated EGFR (P-EGFR), phosphorylated AKT (P-AKT), and phosphorylated ERK1/2 (P-ERK1/2) as well as the expressions of metalloproteinase (MMP) -2 and MMP-9 were detected using Western blot analysis. The differentially expressed genes in the culture solutions in EGF groups and control group were analyzed with RNA-Seq technique.</p><p><b>RESULTS</b>Cells in EGF group had significantly stronger migration ability than in control group (P = 0.0361), inhibitor AG1478 + EGF group (P = 0.0113), inhibitor LY294002 + EGF group (P = 0.0169), and inhibitor U0126 + EGF group (P = 0.0293). EGF increased the phosphorylation levels of EGFR, AKT and ERK, and increased the expression of MMP-2. However, the increased expressions of P-AKT and P-ERK could be suppressed by AG1478 and LY294002. As shown by GO functional enrichment analysis and KEGG pathway analysis, EGF increased the transcription of genes, which were mainly involved in transcriptional regulation, protein modification, and apoptosis inhibition. Genes that were involved in the MARK pathway included DUSP5, IL1B, DUSP6, NGF, and HSPA2.</p><p><b>CONCLUSION</b>EGF-induced migration of hAMSCs may be mediated by the signaling pathways of PI3K and ERK, which needs MMP-2 expression and the co-expression of genes involved in transcriptional regulation, protein modification, and apoptosis inhibition.</p>

Effect of the human amniotic membrane loaded with human amniotic mesenchymal stem cells on the skin wounds of SD rats / 中国医学科学院学报

<p><b>OBJECTIVE</b>To observe the effect of the human amniotic membrane (HAM) loaded with human amniotic mesenchymal stem cells (hAMSCs) on the skin wounds of SD rats.</p><p><b>METHODS</b>The amniotic epithelial cells were removed by trypsin digestion, hAMSCs were loaded onto HAM and then covered on rats' skin defects. The wound healing was observed by HE staining and immunohistochemistry, and the results were compared with the amniotic membrane group and blank control group.</p><p><b>RESULTS</b>The average wound healing time was (18.3 +/- 0.9) d in the HAM load with hAMSCs group, which was significantly faster than those in the blank control group [(26.4 +/- 0.7) d, P < 0.01] and the amniotic membrane group [(21.5 +/- 1.2) d, P < 0.05]. After 11 d and 14 d, the wound healing rates in the HAM load with hAMSCs group were (81.5 +/- 7.2)% and (94.3 +/- 3.6)%, respectively, which were significantly higher than those in the blank control group [(48.5 +/- 3.2)% and (74.3 +/- 4.3 )%] and the amniotic membrane group [(68.5 +/- 4.5)% and (86.8 +/- 4.8)%] (all P < 0.01). Skin biopsy/HE staining confirmed that the quality of wound healing in the HAM load with hAMSCs group was significantly better than in the amniotic membrane group and the blank control group. Immunohistochemical staining showed that the number of CK19-positive epidermal stem cells in the HAM load with hAMSCs group (48.2 +/- 3.2) was significantly larger than those in the amniotic membrane group (37.7 +/- 3.1) (P < 0.05) and the blank control group (29.6 +/- 2.4) (P < 0.01). Furthermore, the vascular endothelial growth factor expression (64.5 +/- 4.5) in the HAM load with hAMSCs group was also significantly higher than those in the amniotic membrane group (52.6 +/- 3.8) (P < 0.05) and the blank control group (40.7 +/- 3.1) (P < 0.01).</p><p><b>CONCLUSION</b>HAM loaded with hAMSCs may promote the repair of skin wounds by promoting the regeneration of epidermal stem cells and capillaries.</p>

Cell reprogramming: control key genes to obtain needed cells / 中国医学科学院学报

Cell reprogramming is a progress in which the memory of a mature cell is erased and then the cell develops novel phenotype and function; ultimately, the fate of the cell changes. Cell reprogramming usually occurs at genes expression levels that no genomic DNA sequence change will be involved. By changing the programs of the genetic expressions of cells in terms of space and time, cell reprogramming alters the differentiation of cells and thus produces the required cells. Further research on cells reprogramming will elucidate the mechanisms that govern the cell development, and thus provides more information of the sources of seed cells used for regeneration medicine. More cells differentiated from many terminally differentiated cells will be obtained, which is extremely important for the understanding of molecular differentiation and for the development of cell replacement therapy. This article summarizes the classification, influencing factors, approaches and latest advances of cells reprogramming.

Relationship between neuronal restricted silencing factor and induced differentiation from rat mesenchymal stem cells to neurons / 中国医学科学院学报

<p><b>OBJECTIVE</b>To analyze the change of the neuronal restricted silencing factor (NRSF) gene as well as the NRSF regulation genes in beta-mercaptoethanol induction of the marrow mesenchymal stem cells (MSCs) to neurons, and to discuss the function of NRSF in neural induction of the MSCs and the mechanism of the differentiation from MSCs to neurons.</p><p><b>METHOD</b>We used beta-mercaptoethanol, serum-free DMEM, and dimethyl sulfoxide to induce rat MSCs to differentiate to neurons, and then analyzed the changes of the expressions of NRSF gene and NRSF-regulated genes through real-time PCR.</p><p><b>RESULTS</b>The rat MSCs were successfully induced to differentiate into neuron-like cells. The induced neuron marker, neuron-specific enolase, was positive. Real-time PCR showed that the expression of NRSF gene remarkably declined. The expressions of neurotrophic tyrosine kinase receptor, type 3, synaptosomal-associated protein 25, L1 cell adhesion molecular,neuronal pentraxin receptor in the NRSF-regulated genes also increased at varied extents.</p><p><b>CONCLUSIONS</b>The differentiation from MSCs to neurons is relevant with the decline of NRSF expression and the increase of the expressions of NRSF-regulated genes. The NRSF may be the key gene during the differentiation from MSCs to neurons.</p>

Construction and identification of the lentiviral vector of repressor element-1/neuron-restrictive silencer element double-stranded RNA / 中国医学科学院学报

<p><b>OBJECTIVE</b>To construct a lentiviral vector of repressor element-1/neuron-restrictive silencer element (RE-1/NRSE) double-stranded RNA (dsRNA).</p><p><b>METHODS</b>The RE-1/NRSE cDNA containing both sense and antisense oligo DNA fragments of the targeting sequence was synthesized and cloned into the pGC-LV vector. The obtained lentiviral vector containing RE-1/NRSE dsRNA was confirmed by PCR and sequencing. A total of 293T cells were cotransfected with lentiviral vector of L-smNRSE/RE-1, pHelper 1.0, and pHelper 2.0. The titer of virus was measured based on the expression level of green fluorescent protein. The transfection efficiency of green fluorescent protein into rat mesenchymal stem cells was calculated.</p><p><b>RESULTS</b>PCR and DNA sequencing demonstrated that the constructed lentivirus vector of L-smNRSE/RE-1 produced RE-1/NRSE dsRNA.The titer of the concentrated virus was 4x108 TU/m1. The virus was stably transfected into rat mesenchymal stem cells, and the infection efficiency reached 100% when the multiplicity of infection was 80.</p><p><b>CONCLUSION</b>The lentivirus vector of RE-1/NRSE dsRNA is successfully constructed.</p>