PEI-Fe3O4 Nanoparticles for Human Amniotic Epithelial Cells Labeling

Objective To label human amniotic epithelial cells(hAECs) by using PEI-Fe3O4 nanoparticles. Methods The PEI-Fe3O4 nanoparticles 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-Fe3O4 nanoparticles 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-Fe3O4 nanoparticles can be used for labeling hAECs without obvious cytotoxicity at its working concentration.

Repression of COUP-TFI Improves Bone Marrow-Derived Mesenchymal Stem Cell Differentiation into Insulin-Producing Cells.

Identifying molecular mechanisms that regulate insulin expression in bone marrow-derived mesenchymal stem cells (bmMSCs) can provide clues on how to stimulate the differentiation of bmMSCs into insulin-producing cells (IPCs), which can be used as a therapeutic approach against type 1 diabetes (T1D). As repression factors may inhibit differentiation, the efficiency of this process is insufficient for cell transplantation. In this study, we used the mouse insulin 2 (Ins2) promoter sequence and performed a DNA affinity precipitation assay combined with liquid chromatography-mass spectrometry to identify the transcription factor, chicken ovalbumin upstream promoter transcriptional factor I (COUP-TFI). Functionally, bmMSCs were reprogrammed into IPCs via COUP-TFI suppression and MafA overexpression. The differentiated cells expressed higher levels of genes specific for islet endocrine cells, and they released C-peptide and insulin in response to glucose stimulation. Transplantation of IPCs into streptozotocin-induced diabetic mice caused a reduction in hyperglycemia. Mechanistically, COUP-TFI bound to the DR1 (direct repeats with 1 spacer) element in the Ins2 promoter, thereby negatively regulating promoter activity. Taken together, the data provide a novel mechanism by which COUP-TFI acts as a negative regulator in the Ins2 promoter. The differentiation of bmMSCs into IPCs could be improved by knockdown of COUP-TFI, which may provide a novel stem cell-based therapy for T1D.

In vitro reprogramming of rat bmMSCs into pancreatic endocrine-like cells.

Islet transplantation provides curative treatments to patients with type 1 diabetes, but donor shortage restricts the broad use of this therapy. Thus, generation of alternative transplantable cell sources is intensively investigated worldwide. We previously showed that bone marrow-derived mesenchymal stem cells (bmMSCs) can be reprogrammed to pancreatic-like cells through simultaneously forced suppression of Rest/Nrsf (repressor element-1 silencing transcription factor/neuronal restrictive silencing factor) and Shh (sonic hedgehog) and activation of Pdx1 (pancreas and duodenal transcription factor 1). We here aimed to reprogram bmMSCs further along the developmental pathway towards the islet lineages by improving our previous strategy and by overexpression of Ngn3 (neurogenin 3) and NeuroD1 (neurogenic differentiation 1), critical regulators of the development of endocrine pancreas. We showed that compared to the previous protocol, the overexpression of only Pdx1 and Ngn3 reprogrammed bmMSCs into cells with more characteristics of islet endocrine lineages verified with bioinformatic analyses of our RNA-Seq datasets. These analyses indicated 2325 differentially expressed genes including those involved in the pancreas and islet development. We validated with qRT-PCR analysis selective genes identified from the RNA-Seq datasets. Thus, we reprogrammed bmMSCs into islet endocrine-like cells and advanced the endeavor to generate surrogate functional insulin-secreting cells.

MiR-378b Promotes Differentiation of Keratinocytes through NKX3.1.

MicroRNA (miRNA) is a kind of short non-coding RNA, involved in various cellular processes. During keratinocyte differentiation, miRNAs act as important regulators. In this study, we demonstrated by microarray assay that the expression of miR-378b significantly increased during keratinocytes differentiation. Our findings showed that miR-378b could inhibit proliferation, migration and differentiation in keratinocytes. Luciferase reporter assays showed that miR-378b directly target NKX3.1. Silencing of NKX3.1 could coincide with the effects of miR-24 overexpression. In conclusion, our results demonstrate miR-378b promote keratinocytes differentiation by targeting NKX3.1. Manipulation of miR-378b may afford a new strategy to clinic treatment of skin injury and repair.

In vitro reprogramming of rat bone marrow-derived mesenchymal stem cells into insulin-producing cells by genetically manipulating negative and positive regulators.

Islet cell replacement therapy represents the most promising approach for the cure of type 1 diabetes if autoimmunity to ß cells is under control. However, this potential is limited by a shortage of pancreas donors. To address the donor shortage problem, we determined whether bone marrow-derived mesenchymal stem cells (bmMSCs) can be directly reprogrammed to islet lineages by simultaneously forced suppression and over-expression of key regulator genes that play critical roles during pancreas development. Here, we report that rat bmMSCs were converted in vitro into insulin-producing cells by suppressing two-repressor genes repressor element-1 silencing transcription factor/neuronal restrictive silencing factor (Rest/Nrsf) and sonic hedgehog (Shh) and by over-expressing pancreas and duodenal transcription factor 1 (Pdx1). The reprogrammed bmMSCs expressed both genes and proteins specific for islet cells. These converted cells were capable of releasing insulin in a glucose-responsive manner. Our study suggests that bmMSCs may ultimately be reprogrammed to functional insulin-secreting cells.

Quantity and proliferation rate of mesenchymal stem cells in human cord blood during gestation.

UCB (umbilical cord blood) as a resource of MSCs (mesenchymal stem cells) is widely accepted, but the quantity and characteristics of UCB-MSCs from different gestational ages have not been well studied. We have quantified the number of MSCs in UCB at different gestational ages using a multi-colour flowcytometer and compared the cell proliferation rates of these UCB-MSCs. Defining MSCs as CD44+/CD105+/CD34-/CD45 population, their numbers declined in the UCB at the gestational age. Proliferation rates were significantly higher in UCB before term than at full term. Non-full term UCB samples may be a better source of MSCs.

[Effect of epidermal growth factor on migration of human amniotic mesenchymal stem cells].

OBJECTIVE: To explore the mechanism via which the epidermal growth factor (EGF) affects the migration of human amnion-derived mesenchymal stem cells (hAMSCs). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Effect of the human amniotic membrane loaded with human amniotic mesenchymal stem cells on the skin wounds of SD rats].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSION: HAM loaded with hAMSCs may promote the repair of skin wounds by promoting the regeneration of epidermal stem cells and capillaries.

[Cell reprogramming: control key genes to obtain needed cells].

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].

OBJECTIVE: 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. METHOD: 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. RESULTS: 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. CONCLUSIONS: 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.

[Construction and identification of the lentiviral vector of repressor element-1/neuron-restrictive silencer element double-stranded RNA].

OBJECTIVE: To construct a lentiviral vector of repressor element-1/neuron-restrictive silencer element (RE-1/NRSE) double-stranded RNA (dsRNA). METHODS: 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. RESULTS: 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. CONCLUSION: The lentivirus vector of RE-1/NRSE dsRNA is successfully constructed.