ABSTRACT
Objective: To investigate the effect of all-trans retinoic acid (ATRA) and vascular endothelial growth factor (VEGF) on the osteogenic differentiation of mouse embryonic fibroblasts (MEFs). Methods: The fetal mice in the uterus of NIH pregnant mice (pregnancy 12-15 days) were collected, and the heads and hearts etc. were removed. Then MEFs were separated from the rest tissues of the fetal mice and cultured by trypsin digestion and adherent culture. HEK-293 cells were used to obtain recombinant adenovirus-red fluorescent protein (Ad-RFP) and Ad-VEGF by repeatedly freezing and thawing. Alkaline phosphatase (ALP) staining and quantitative detection were used to detect the changes of ALP activity in MEFs applied with ATRA or VEGF alone or combined use of ATRA and VEGF on the 3rd and 5th days. The cultured 3rd to 4th generation MEFs were divided into groups A, B, C, and D, and were cultured with DMSO plus Ad-RFP, ATRA, Ad-VEGF, ATRA plus Ad-VEGF, respectively. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect the mRNA expressions of osteogenic markers including ALP, collagen type Ⅰ, osteopontin (OPN), osteocalcin (OCN), and angiogenic markers including VEGF, angiopoietin 1 (ANGPT1), and endomucin (EMCN) on the 3rd and 7th days. Immunohistochemical staining was used to detect the protein expressions of OPN and VEGF on the 3rd, 5th, and 7th days in each group. Alizarin red staining was used to detect calcium salt deposition levels in each group at 14 and 21 days after osteogenic induction. Fifteen athymic female nude mice aged 4 to 6 weeks were randomly divided into 3 groups and 5 mice in each group. Then MEFs treated with ATRA, Ad-VEGF, and ATRA plus Ad-VEGF were injected subcutaneously into the dorsal and ventral sides, respectively. X-ray observation, gross observation, and histological staining (Masson, HE, and Safranin O-fast green stainings) were performed at 5 weeks after implantation to observe the ectopic bone formation in nude mice in each group. Results: MEFs were successfully isolated and cultured. The acquired Ad-RFP and Ad-VEGF were successfully transfected into MEFs with approximately 50% and 20% transfection rates. ALP activity tests showed that ATRA or Ad-VEGF could enhance ALP activity in MEFs ( P<0.05), and ATRA had a stronger effect than Ad-VEGF; and the combined use of ATRA and Ad-VEGF significantly enhanced the ALP activity in MEFs ( P<0.05). qRT-PCR test showed that the combined use of ATRA and Ad-VEGF also increased the relative mRNA expressions of early-stage osteogenesis-related markers ALP, OPN, and collagen type I ( P<0.05); the relative mRNA expressions of angiogenesis-related markers VEGF, EMCN, and ANGPT1 increased at 7 days ( P<0.05). Immunohistochemical staining showed that ATRA combined with Ad-VEGF not only enhanced OPN protein expression, but also increased VEGF protein expression on 7th day. Alizarin red staining showed that the application of ATRA or Ad-VEGF induced weak calcium salt deposition, and the combined use of ATRA and Ad-VEGF significantly enhanced the effect of calcium salt deposition in MEFs. The results of implantation experiments in nude mice showed that X-ray films observation revealed obvious bone mass in the ATRA plus Ad-VEGF group, and the bone was larger than that in other groups. Histological staining showed a large amount of collagen and mature bone trabeculae, bone matrix formation, and gray-green collagen bone tissue, indicating that the combined use of ATRA and Ad-VEGF significantly enhanced the osteogenic effect of MEFs in vivo. Conclusion: The combined use of ATRA and VEGF can induce the osteogenic differentiation of MEFs.
ABSTRACT
Objective To explore the effects of hypoxia on the growth,mitochondria distribution and function of mouse embryonic fibroblasts(MEFs).Methods MEFs were sub-cultured in the hypoxia group containing 5％ oxygen and normal oxygen group containing 20％ oxygen,every 24 hours,living MEFs were counted by using trypan blue staining.Mito-Tracker Green was used to stain mitochondria,then cells were observed by using laser confocal microscope.The ATP kit was used to detect ATP synthesis.Results During the logarithmic phase,the numbers of living cells in the hypoxia group were higher than those in the normal oxygen group,the differences were statistically significant (P＜0.05).The percentages of perinuclear mitochondrial in the hypoxia group were higher than those in the normal oxygen group,the differences were statistically significant (P＜0.05).Meanwhile,the significant difference was found in the ATP level between the two groups (P＜0.05).Conclusion The distribution of mitochondria in MEFs and energy synthesis are influenced by the hypoxic culture condition,which could be better for promoting cell growth compared with normal oxygen culture condition.
ABSTRACT
Objective To isolate and cultivate mouse hepatic progenitor cells (mHPCs) from E14.5 mouse fetal liver in vitro and induce mHPCs differentiation into cholangiocytes.Methods Isolation of mHPCs from mouse fetal liver was based on the cell surface antigen delta-like protein 1/preadipocyte factor 1 (Dlk/Pref-1) by a fluorescence-activated cell sorter (FACS).Then mHPCs isolated were co-cultured with/without mouse embryonic fibroblasts (MEFs) by using Transwell.The cell antigen alpha-fetoprotein (AFP), albumin (ALB) and cytokeratin19 (CK19) expression in freshly isolated DLK1+cells or co-cultured for 4 days and 6 days were observed with immunocytochemical method.Results When co-cultured with MEFs, the division and proliferation were observed in most of DLK1+ cells and grape-like aggregation was formed.Cells began to adhere to growth and began to become spindle-shaped on 4th day.The DLK1+cells isolated freshly by FACS were expressed AFP and low levels of ALB but not expressed CK19.But, these cells expressed CK 19 and weak expression of ALB on 4th day.In addition, the expression of CK19 increased and the expression of ALB almost not detected on 6th day.Conclusions Most of DLK1+ cells, isolated from E14.5 fetal livers by FACS, are proved to be mHPCs.Furthermore, these cells can proliferate quickly and differentiate into cholangiocytes by co-culture with MEFs.
ABSTRACT
AIM:To study the direct reprogramming method of mouse embryonic fibroblasts (MEFs) conver-ted into induced neural stem cells (iNSCs).METHODS:Sox2-infected MEFs were cultured in NSCs culture medium for 10 d.Subsequently , repeated suspension and adherent culture were performed for 3 times for the purification of iNSCs .The iNSCs were cultured in suspension medium .Real-time PCR was used to detect the expression of neural stem cell marker genes and pluripotent marker gene .In vivo, iNSCs were microinjected into the mouse cerebral cortex .Immunofluorescence was performed to detect the expression of neural stem cell , neuron, oligodendrocyte and astrocyte markers in vitro and vivo. RESULTS:A variety of neural stem cell marker gene expression was significantly increased in iNSCs detected by real -time PCR.Immunofluorescence confirmed that iNSCs expressed nestin and differentiated into neurons , oligodendrocytes and as-trocytes in vitro and vivo.CONCLUSION:Sox2 is sufficient to trigger the direct reprogramming from MEFs to iNSCs .iN-SCs have the ability of self-renew and 3 differentiation potentials in vivo and vitro.iNSCs are the suitable seed cells of SCI .
ABSTRACT
Millions of people world over suffer visual disability due to retinal dystrophies which can be age-related or a genetic disorder resulting in gradual degeneration of the retinal pigmented epithelial (RPE) cells and photoreceptors. Therefore, cell replacement therapy offers a great promise in treating such diseases. Since the adult retina does not harbour any stem cells, alternative stem cell sources like the embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer a great promise for generating different cell types of the retina. Here, we report the derivation of four iPSC lines from mouse embryonic fibroblasts (MEFs) using a cocktail of recombinant retroviruses carrying the genes for Oct4, Sox2, Klf4 and cMyc. The iPS clone MEF-4F3 was further characterized for stemness marker expression and stable reprogramming by immunocytochemistry, FACS and RT-PCR analysis. Methylation analysis of the nanog promoter confirmed the reprogrammed epigenetic state. Pluripotency was confirmed by embryoid body (EB) formation and lineage-specific marker expression. Also, upon retinal differentiation, patches of pigmented cells with typical cobble-stone phenotype similar to RPE cells are generated within 6 weeks and they expressed ZO-1 (tight junction protein), RPE65 and bestrophin (mature RPE markers) and showed phagocytic activity by the uptake of fluorescent latex beads.
ABSTRACT
Objective To comparethe reprogramming efficiencies of mouse bone marrow mesenchymal stem cells (BMSCs), mouse bone marrow mononuclear cells (BM-MNCs) and mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPS cells). Methods BMSCs, BM-MNCs and MEFs were infected with lentivirus (LV-efla-mOrt4- IRES-EGFP, LV-efla-mklf4-IRES-EGFP, LV-efla-mK/4-IRES-EGFP and LV-efla-mc-Mcc-IRES-EGFP) at a multiplicity of infection. Reprogramming efficiencies of BMSCs, BM-MNCs and MEFs were compared by counting the number of alkaline phosphatase (AP)-positive clones. Pluripotency of the clones was evaluated by detecting the expression of embryonic stem cells markers and assessing their ability to formembryoid bodies and teratomas. Results iPS cells derived from BMSCs, BM-MNCs and MEFs were all able to grow into clones with clear borders, to express enbryonic stem cell-specific cell surface markers (Nanog, Rex-1 and SSEA-1), and to express characteristic genes of all three germ layers both in vitro and vivo. The AP- positive clones derived from BMSCs were notably less than those from BM-MNCs and MEFs. Conclusion BMSCs, BM-MNCs and MEFs can all reprogram into iPS cells, but the reprogramming efficiency of BMSCs in adherent culture is lower than those of BM-MNCs and MEFs.
ABSTRACT
Objective To isolate and identify embryonic stem (ES) cells of rhesus monkey from mature blastocysts cultured in vitro . Methods Rhesus monkey blastocysts were obtained after in vitro maturation of oocytes, fertilization and maturation of early embryos. After the blastocysts were hatched naturally from the zone pellucida, the inner cell mass (ICM) was dislodged from blastocysts using the sealed end of a finely drawn Pasteur pipette and co cultured with the feeder cells. The ICM, resembling embryonic stem cell colony, was isolated, cultured, and identified. Results A total of 92 normal GV oocytes were obtained from 4 FSH prime rhesus monkeys. Six blastocysts with high quality were selected from 22 oocytes after culture in HECM 10 medium. One of the rhesus monkey ES cell lines, i.e. RS5 cell line, was finally isolated from 3 of the inner cell mass isolated from the 6 blastocysts. The RS5 cells presented a high nucleus/cytoplasm ratio, prominent nucleoli, and flatter colonies with individual and distinct cells. After successive passages for 5 months passage, the RS5 cells remained a normal karyotype, i.e. 42 of chromosomes and alkaline phosphatase (AP) positive, which meant the RS5 ES cell colony remained undifferentiated. After high density culture for a longer time, the ES cell could differentiate into multi types of cells. Conclusion The RS5 cell line has the ability to self renew and potential to differentiate. Thus, RS5 cell line belongs to embryonic stem cells.