Combined radiation-wound injury (CRWI) is characterized by blood vessel damage and pro-inflammatory cytokine deficiency. Studies have identified that the direct application of leptin plays a significant role in angiogenesis and inflammation. We established a sustained and stable leptin expression system to study the mechanism. A lentivirus method was employed to explore the angiogenic potential and peripheral inflammation of irradiated human umbilical vein endothelial cells (HUVECs). Leptin was transfected into human placenta-derived mesenchymal stem cells (HPMSCs) with lentiviral vectors. HUVECs were irradiated by X-ray at a single dose of 20 Gy. Transwell migration assay was performed to assess the migration of irradiated HUVECs. Based on the Transwell systems, co-culture systems of HPMSCs and irradiated HUVECs were established. Cell proliferation was measured by cell counting kit-8 (CCK-8) assay. The secretion of pro-inflammatory cytokines (human granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-1α, IL-6, and IL-8) was detected by enzyme-linked immunosorbent assay (ELISA). The expression of pro-angiogenic factors (vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)) mRNA was detected by real-time quantitative polymerase chain reaction (RT-qPCR) assay. Relevant molecules of the nuclear factor-κB (NF-κB) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways were detected by western blot assay. Results showed that leptin-modified HPMSCs (HPMSCs/ leptin) exhibited better cell proliferation, migration, and angiogenic potential (expressed more VEGF and bFGF). In both the single HPMSCs/leptin and the co-culture systems of HPMSCs/leptin and irradiated HUVECs, the increased secretion of pro-inflammatory cytokines (human GM-CSF, IL-1α, and IL-6) was associated with the interaction of the NF-κB and JAK/STAT signaling pathways. We conclude that HPMSCs/leptin could promote angiogenic potential and peripheral inflammation of HUVECs after X-ray radiation.
Cell Proliferation ; Cells, Cultured ; Cytokines/biosynthesis* ; Female ; Human Umbilical Vein Endothelial Cells/radiation effects* ; Humans ; Inflammation/etiology* ; Leptin/pharmacology* ; Mesenchymal Stem Cells/physiology* ; Neovascularization, Physiologic/physiology* ; Placenta/cytology* ; Pregnancy ; STAT3 Transcription Factor/genetics* ; Transcription Factor RelA/genetics* ; X-Rays

The technique of stem cells or hepatocytes transplantation has recently improved in order to bridge the time before whole-organ liver transplantation. In the present study, unfractionated bone marrow stem cells (BMSCs) were harvested from the tibial and femoral marrow compartments of male mice, which were cultured in Dulbecco's modified Eagle's medium (DMEM) with and without hepatocyte growth factor (HGF), and then transplanted into Schistosoma mansoni-infected female mice on their 8th week post-infection. Mice were sacrificed monthly until the third month of bone marrow transplantation, serum was collected, and albumin concentration, ALT, AST, and alkaline phosphatase (ALP) activities were assayed. On the other hand, immunohistopathological and immunohistochemical changes of granuloma size and number, collagen content, and cells expressing OV-6 were detected for identification of liver fibrosis. BMSCs were shown to differentiate into hepatocyte-like cells. Serum ALT, AST, and ALP were markedly reduced in the group of mice treated with BMSCs than in the untreated control group. Also, granuloma showed a marked decrease in size and number as compared to the BMSCs untreated group. Collagen content showed marked decrease after the third month of treatment with BMSCs. On the other hand, the expression of OV-6 increased detecting the presence of newly formed hepatocytes after BMSCs treatment. BMSCs with or without HGF infusion significantly enhanced hepatic regeneration in S. mansoni-induced fibrotic liver model and have pathologic and immunohistopathologic therapeutic effects. Also, this new therapeutic trend could generate new hepatocytes to improve the overall liver functions.
Alanine Transaminase/blood ; Alkaline Phosphatase/blood ; Animals ; Antigens, Differentiation/biosynthesis ; Aspartate Aminotransferases/blood ; Bone Marrow Cells/cytology ; *Bone Marrow Transplantation ; Cell Differentiation ; Cell- and Tissue-Based Therapy ; Cells, Cultured ; Collagen/metabolism ; Female ; Granuloma/parasitology/pathology ; Hepatocyte Growth Factor/pharmacology ; Hepatocytes/*cytology ; Liver/parasitology/pathology ; Liver Cirrhosis/parasitology/pathology/*therapy ; Male ; Mice ; Mice, Inbred BALB C ; Schistosoma mansoni/pathogenicity ; Schistosomiasis mansoni/mortality/*therapy ; *Stem Cell Transplantation ; Stem Cells/cytology

We established methods to isolate human amniotic fluid-derived progenitor cells (hAFPCs), and analyze the ability of hAFPCs to secrete human coagulation factor IX (hFIX) after gene modification. The hAFPCs were manually isolated by selection for attachment to gelatin coated culture dish. hFIX cDNA was transfected into hAPFCs by using a lentiviral vector. The hFIX protein concentration and activity produced from hAFPCs were determined by enzyme-linked immunosorbent assay (ELISA) and clotting assay. The isolated spindle-shaped cells showed fibroblastoid morphology after three culture passages. The doubling time in culture was 39.05 hours. Immunocytochemistry staining of the fibroblast-like cells from amniotic fluid detected expression of stem cell markers such as SSEA4 and TRA1-60. Quantitative PCR analysis demonstrated the expression of NANOG, OCT4 and SOX2 mRNAs. Transfected hAFPCs could produce and secrete hFIX into the culture medium. The observed concentration of secreted hFIX was 20.37% +/- 2.77% two days after passage, with clotting activity of 16.42% +/- 1.78%. The amount of hFIX:Ag reached a plateau of 50.35% +/- 5.42%, with clotting activity 45.34% +/- 4.67%. In conclusion, this study established method to isolate and culture amniotic fluid progenitor cells. Transfected hAFPCs can produce hFIX at stable levels in vitro, and clotting activity increases with higher hFIX concentration. Genetically engineered hAFPC are a potential method for prenatal treatment of hemophilia B.
Amniotic Fluid ; cytology ; Blood Coagulation ; Cell Culture Techniques ; Cell Separation ; methods ; DNA, Complementary ; Factor IX ; biosynthesis ; Genetic Engineering ; Genetic Vectors ; Humans ; Stem Cells ; cytology ; metabolism ; Transfection

The anti-melanogenesis effect of glyceollins was examined by melanin synthesis, tyrosinase activity assay in zebrafish embryos and in B16F10 melanoma cells. When developing zebrafish embryos were treated with glyceollins, pigmentation of the embryos, melanin synthesis and tyrosinase activity were all decreased compared with control zebrafish embryos. In situ expression of a pigment cell-specific gene, Sox10, was dramatically decreased by glyceollin treatment in the neural tubes of the trunk region of the embryos. Stem cell factor (SCF)/c-kit signaling pathways as well as expression of microphthalmia-associated transcription factor (MITF) were determined by western blot analysis. Glyceollins inhibited melanin synthesis, as well as the expression and activity of tyrosinase induced by SCF, in a dose-dependent manner in B16F10 melanoma cells. Pretreatment of B16F10 cells with glyceollins dose-dependently inhibited SCF-induced c-kit and Akt phosphorylation. Glyceollins significantly impaired the expression and activity of MITF. An additional inhibitory function of glyceollins was to effectively downregulate intracellular cyclic AMP levels stimulated by SCF in B16F10 cells. Glyceollins have a depigmentation/whitening activity in vitro and in vivo, and that this effect may be due to the inhibition of SCF-induced c-kit and tyrosinase activity through the blockade of downstream signaling pathway.
Animals ; Embryo, Nonmammalian/drug effects ; Melanins/*biosynthesis ; Melanoma, Experimental/metabolism/pathology ; Mice ; Monophenol Monooxygenase/metabolism ; Phosphorylation/drug effects ; Pigmentation/drug effects ; Proto-Oncogene Proteins c-kit/*metabolism ; Pterocarpans/chemistry/*pharmacology ; SOXE Transcription Factors/metabolism ; Sesquiterpenes/chemistry/*pharmacology ; Signal Transduction/*drug effects ; Soybeans/*chemistry ; Stem Cell Factor/*pharmacology ; Zebrafish/embryology/metabolism

The aim of this study was to investigate the mechanism of deposition of extracellular matrix induced by TGF-β1 in skeletal muscle-derived stem cells (MDSCs). Rat skeletal MDSCs were obtained by using preplate technique, and divided into four groups: group A (control group), group B (treated with TGF-β1, 10 ng/mL), group C (treated with TGF-β1 and anti-connective tissue growth factor (CTGF), both in 10 ng/mL), and group D (treated with anti-CTGF, 10 ng/mL). The expression of CTGF, collagen type-I (COL-I) and collagen type-III (COL-III) in MDSCs was examined by using RT-PCR, Western blot and immunofluorescent stain. It was found that one day after TGF-β1 treatment, the expression of CTGF, COL-I and COL-III was increased dramatically. CTGF expression reached the peak on the day 2, and then decreased rapidly to a level of control group on the day 5. COL-I and COL-III mRNA levels were overexpresed on the day 2 and 3 respectively, while their protein expression levels were up-regulated on the day 2 and reached the peak on the day 7. In group C, anti-CTGF could partly suppress the overexpression of COL-I and COL-II induced by TGF-β1 one day after adding CTGF antibody. It was concluded that TGF-β1 could induce MDSCs to express CTGF, and promote the production of COL-I and COL-III. In contrast, CTGF antibody could partially inhibit the effect of TGF-β1 on the MDSCs by reducing the expression of COL-I and COL-III. Taken together, we demonstrated that TGF-β1-CTGF signaling played a crucial role in MDSCs synthesizing collagen proteins in vitro, which provided theoretical basis for exploring the methods postponing skeletal muscle fibrosis after nerve injury.
Animals ; Cell Differentiation ; drug effects ; physiology ; Cells, Cultured ; Fibrillar Collagens ; biosynthesis ; Male ; Myoblasts, Skeletal ; cytology ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology ; drug effects ; metabolism ; Transforming Growth Factor beta1 ; pharmacology

To investigate the effect of hSCGF-alpha on human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs), we obtained hSCGF-alpha using genetic engineering, hSCGF-alpha gene was amplified from hUCMSCs cDNA using two-step PCR and was inserted into pET-28a(+) plasmid vector. Induced by IPTG at 20 degrees Celsius for 24 h, the fusion protein expressed in E. coli BL21 (DE3) was mainly existing in soluble form. The recombinant hSCGF-a was purified using NI-NTA affinity chromatography and the purity was up to 90%. The colony forming test revealed that combined use hSCGF-alpha and rmGM-CSF (recombinant murine GM-colony stimulating factor, rmGM-CSF) had granulocyte/macrophage (GM) promoting effects on murine bone marrow GM progenitor. In addition, the results indicated that hSCGF-alpha and rhGM-CSF had stimulatory effect on hUCMSCs and their synergetic effect was the strongest.
Cell Proliferation ; drug effects ; Cells, Cultured ; Cloning, Molecular ; Drug Synergism ; Escherichia coli ; genetics ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Stem Cell Factor ; biosynthesis ; genetics ; Umbilical Cord ; cytology

OBJECTIVETo study the effect of astragaloside IV on the expression of cytokines in bone mesenchymal stem cells (MSCs) in rats.

METHODSMSCs were isolated from Wistar rats by the method of adhesive cultiration and clone, and then their biological activities were assessed using indirect immunofluorescence. Proliferation of MSCs stimulated with astragaloside IV was ascertained by the MTT method. Expression of cytokines was ascertained using RT-PCR in MSCs with astragaloside IV stimulation or not.

RESULTSMSCs were effectively isolated and purified in vitro, and had expression of many cytokines except IL-3, such as stem cell factor (SCF), thrombopoietin (TPO), granulocyte macrophage colony stimulating factor (GM-CSF) and transforming growth factor (TGF-beta1). Astragaloside IV stimulation promoted MSCs proliferation, and 200 mg/mL astragaloside IV treatment produced a peak effect 72 hrs after culture. The SCF expression in MSCs stimulated with astragaloside IV increased significantly compared with that in MSCs without astragaloside IV stimulation.

CONCLUSIONSAstragaloside IV may promote MSCs proliferation and increase SCF secretion in vitro.

Animals ; Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Cell Proliferation ; drug effects ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; secretion ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Saponins ; pharmacology ; Stem Cell Factor ; biosynthesis ; genetics ; secretion ; Triterpenes ; pharmacology

Parathyroid hormone-related protein (PTHrP) is synthesized by diverse tissues, and its processing produces several fragments, each with apparently distinct autocrine and paracrine bioactivities. In bone, PTHrP appears to modulate bone formation in part through promoting osteoblast differentiation. The putative effect of PTH-like and PTH-unrelated fragments of PTHrP on human mesenchymal stem cell (MSCs) is not well known. Human MSCs were treated with PTHrP (1-36) or PTHrP (107-139) or both (each at 10 nM) in osteogenic or adipogenic medium, from the start or after 6 days of exposure to the corresponding medium, and the expression of several osteoblastogenic and adipogenic markers was analyzed. PTHrP (1-36) inhibited adipogenesis in MSCs and favoured the expression of osteogenic early markers. The opposite was observed with treatment of MSCs with PTHrP (107-139). Moreover, inhibition of the adipogenic differentiation by PTHrP (1-36) prevailed in the presence of PTHrP (107-139). The PTH/PTHrP type 1 receptor (PTH1R) gene expression was maximum in the earlier and later stages of osteogenesis and adipogenesis, respectively. While PTHrP (107-139) did not modify the PTH1R overexpression during adipogenesis, PTHrP (1-36) did inhibit it; an effect which was partially affected by PTHrP (7-34), a PTH1R antagonist, at 1 microM. These findings demonstrate that both PTHrP domains can exert varying effects on human MSCs differentiation. PTHrP (107-139) showed a tendency to favor adipogenesis, while PTHrP (1-36) induced a mild osteogenic effect in these cells, and inhibited their adipocytic commitment. This further supports the potential anabolic action of the latter peptide in humans.
Adipogenesis/drug effects ; Alkaline Phosphatase/biosynthesis/genetics ; Antigens, Differentiation/biosynthesis/genetics ; Bone Marrow/pathology ; Cell Differentiation/drug effects ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit/biosynthesis/genetics ; Culture Media ; Gene Expression Regulation ; Humans ; Lipoprotein Lipase/biosynthesis/genetics ; Mesenchymal Stem Cells/*drug effects/metabolism/pathology ; Osteoblasts/drug effects/*metabolism/pathology ; Osteogenesis/drug effects ; PPAR gamma/biosynthesis/genetics ; Parathyroid Hormone/*pharmacology ; Peptide Fragments/*pharmacology ; Receptor, Parathyroid Hormone, Type 1/antagonists & inhibitors

OBJECTIVE: To generate and identify the induced pluripotent stem cells (iPSCs) from human embryonic fibroblast cells (hEFs) by introducing 4 defined factors. METHODS: We introduced 4 factors (Oct4, Sox2, Nanog,and Lin28) into hEFs by lentivirus infection. The iPSCs generated from this method were analyzed in many aspects, including surface antigens, gene expression, and telomerase activity, differentiation ability in vivo and in vitro and the patterns of short tandem repeat (STR). RESULTS: The human iPSCs generated from this study were positive for alkaline phosphatase (AKP) staining, expressed hESCs-specific surface antigens, and exhibited high telomerase activity. They also possessed the ability to differentiate into 3-germ layers both in vivo and in vitro. STR analysis indicated that the human iPSCs were derived from the donor material. CONCLUSION The iPSCs lines can be obtained from human embryonic fibroblast by introducing 4 factors. Such human iPSCs should be useful in the study of epigenetic reprogramming and pluripotency maintenance.
Cell Differentiation ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; Fibroblasts ; cytology ; Homeodomain Proteins ; biosynthesis ; genetics ; Humans ; Induced Pluripotent Stem Cells ; cytology ; Lentivirus ; genetics ; metabolism ; Nanog Homeobox Protein ; Octamer Transcription Factor-3 ; biosynthesis ; genetics ; RNA-Binding Proteins ; biosynthesis ; genetics ; SOXB1 Transcription Factors ; biosynthesis ; genetics ; Transfection

Sox9 gene was cloned from immortalized precartilaginous stem cells and its eukaryotic expression vector constructed in order to explore the possibility of bone marrow-derived stromal cells differentiation into precartilaginous stem cells induced by Sox9. A full-length fragment of Sox9 was obtained by RT-PCR, inserted into pGEM-T Easy clone vector, and ligated with pEGFP-IRES2 expression vector by double digestion after sequencing. The compound plasmid was transfected into born marrow-derived stromal cells by Lipofectamine 2000, and the transfection efficacy and the expression of Sox9 and FGFR-3 were observed. Flow cytometry was used to identify the cell phenotype, and MTT was employed to assay proliferative viability of cells. Sequencing, restrictive endonuclease identification and RT-PCR confirmed that the expansion of Sox9 and construction of Sox9 expression vector were successful. After transfection of the recombinant vector into bone marrow-derived stromal cells, the expression of Sox9 and FGFR-3 was detected, and proliferative viability was not different from that of precartilaginous stem cells. It was concluded that Sox9 gene eukaryotic expression vector was successfully constructed, and the transfected bone marrow-derived stromal cells differentiated into the precartilaginous stem cells.
Base Sequence ; Bone Marrow Cells/*cytology ; Cartilage/*cytology ; Cell Differentiation/genetics ; Cells, Cultured ; Cloning, Molecular ; Genetic Vectors/genetics ; Molecular Sequence Data ; Receptor, Fibroblast Growth Factor, Type 3/metabolism ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; SOX9 Transcription Factor/biosynthesis ; SOX9 Transcription Factor/*genetics ; Stem Cells/*cytology ; Stromal Cells/*cytology ; Transfection