High efficient serum free differentiation of endothelial cells from human iPS cells without sorting
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PROVIDER: PRJNA824455 | ENA |
REPOSITORIES: ENA
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Project description:Introduction: Endothelial cells (ECs) form the inner lining of all blood vessels of the body, play important roles in vascular tone regulation, hormone secretion, anticoagulation, regulation of blood cell adhesion and immune cell extravasation. Limitless ECs sources are required to further in-vitro investigations of ECs’ physiology and pathophysiology as well as for tissue engineering approaches. Ideally, the differentiation protocol avoids animal derived components such as fetal serum and yields ECs at efficiencies that make further sorting obsolete for most applications. Method: hiPSC are cultured under serum free conditions, and induced into mesodermal progenitor cells via stimulation of Wnt signaling for 24 hours. Mesodermal progenitor cells are further differentiated into ECs by utilizing a combination of human vascular endothelial growth factor A165 (VEGF), basic fibroblast growth factor (bFGF), 8-Bromoadenosine 3′,5′-cyclic monophosphate sodium salt monohydrate (8Bro), and melatonin (Mel) for 48 hours. Result: This combination generates hiPSC derived ECs (hiPSC-ECs) at a fraction of 90.9 ± 1.5% and is easily transferable from the two dimensional (2D) mono layer into three dimensional (3D) scalable bioreactor suspension cultures. hiPSC-ECs are positive for CD31, VE-Cadherin, von-Willebrand-factor, and CD34. Furthermore, the majority of hiPSC-ECs express the vascular endothelial marker CD184 (CXCR4). Conclusion: The differentiation method presented here generates hiPSC-ECs in only 6 days, without addition of animal sera and at an efficiency eliminating the need for further enrichment strategies, hence providing a scalable source of hiPSC-ECs.
2022-04-08 | GSE200399 | GEO
Project description:IntroductionEndothelial cells (ECs) form the inner lining of all blood vessels of the body play important roles in vascular tone regulation, hormone secretion, anticoagulation, regulation of blood cell adhesion and immune cell extravasation. Limitless ECs sources are required to further in vitro investigations of ECs' physiology and pathophysiology as well as for tissue engineering approaches. Ideally, the differentiation protocol avoids animal-derived components such as fetal serum and yields ECs at efficiencies that make further sorting obsolete for most applications.MethodHuman induced pluripotent stem cells (hiPSCs) are cultured under serum-free conditions and induced into mesodermal progenitor cells via stimulation of Wnt signaling for 24 h. Mesodermal progenitor cells are further differentiated into ECs by utilizing a combination of human vascular endothelial growth factor A165 (VEGF), basic fibroblast growth factor (bFGF), 8-Bromoadenosine 3',5'-cyclic monophosphate sodium salt monohydrate (8Bro) and melatonin (Mel) for 48 h.ResultThis combination generates hiPSC derived ECs (hiPSC-ECs) at a fraction of 90.9 ± 1.5% and is easily transferable from the two-dimensional (2D) monolayer into three-dimensional (3D) scalable bioreactor suspension cultures. hiPSC-ECs are positive for CD31, VE-Cadherin, von Willebrand factor and CD34. Furthermore, the majority of hiPSC-ECs express the vascular endothelial marker CD184 (CXCR4).ConclusionThe differentiation method presented here generates hiPSC-ECs in only 6 days, without addition of animal sera and at high efficiency, hence providing a scalable source of hiPSC-ECs.
| S-EPMC9188069 | biostudies-literature
Project description:The ectopic expression of a defined set of transcription factors, Oct4, Klf4, Sox2, and c-Myc, reprograms mouse embryonic fibroblasts (MEFs) and adult tail-tip fibroblasts (TTFs) into embryonic stem (ES)-like cells called induced pluripotent stem (iPS) cells. iPS cells have been generated from a variety of somatic cells, including embryonic and adult dermal fibroblasts, epithelial cells of the liver and stomach, pancreatic b cells, mature B lymphocytes, and adult neural stem cells (NSCs). While these studies demonstrated that most somatic cells are capable of acquiring pluripotency with minimal gene transduction, the poor efficiency of cell reprogramming and the uneven quality of iPS cells are still important problems. In particular, the choice of cell type most suitable for inducing high-quality iPS cells remains unclear. Here, we generated iPS cells from PDGFRa+ Sca-1+ (PaS) cells that represent highly enriched adult mouse mesenchymal stem cells (MSCs) and PDGFRa- Sca-1- osteo-progenitor (OP) cells. When we compared the induction efficiency and quality of individual iPS clones, MSCs had a higher reprogramming efficiency compared with OP cells and TTFs. The iPS cells induced from MSCs by Oct3/4, Sox2, and Klf4 appeared to be the closest equivalent to ES cells by DNA microarray gene profile and germline-transmission efficiency. We conclude that prospectively isolated MSCs are a promising candidate for the efficient and stable generation of high-quality iPS cells.
2010-08-21 | GSE23717 | GEO
Project description:A major difficulty of producing induced pluripotent stem cells (iPSCs) has been the low efficiency of reprogramming differentiated cells into pluripotent cells. We previously showed that 5% of mouse embryonic fibroblasts (MEFs) were reprogrammed into iPSCs when they were transduced with a fusion gene composed of Oct4 and the transactivation domain of MyoD (called M(3)O), along with Sox2, Klf4 and c-Myc (SKM). In addition, M(3)O facilitated chromatin remodeling of pluripotency genes in the majority of transduced MEFs, including cells that did not become iPSCs. These observations suggested the possibility that more than 5% of cells had acquired the ability to become iPSCs given more favorable culture conditions. Here, we raised the efficiency of making mouse iPSCs with M(3)O-SKM to 26% by culturing transduced cells at low density in serum-free culture medium. In contrast, the efficiency increased from 0.1% to only 2% with the combination of wild-type Oct4 and SKM (OSKM) under the same culture condition. For human iPSCs, M(3)O-SKM achieved 7% efficiency under a similar serum-free culture condition, in comparison to 1% efficiency with OSKM. This study highlights the power of combining the transactivation domain of MyoD with a favorable culture environment.
| S-EPMC3316619 | biostudies-literature
Project description:The generation of induced pluripotent stem (iPS) cells is a powerful tool in regenerative medicine, and advances in nanotechnology clearly have great potential to enhance stem cell research. Here, we introduce a liposomal magnetofection (LMF) method for iPS cell generation. Efficient conditions for generating virus-free iPS cells from mouse embryonic fibroblast (MEF) cells were determined through the use of different concentrations of CombiMag nanoparticle-DNA(pCX-OKS-2A and pCX-cMyc)-lipoplexes and either one or two cycles of the LMF procedure. The cells were prepared in a short reprogramming time period (? 8 days, 0.032-0.040%). Among the seven LMF-iPS cell lines examined, two were confirmed to be integration-free, and an integration-free LMF-iPS cell line was produced under the least toxic conditions (single LMF cycle with a half-dose of plasmid). This cell line also displayed in vitro/in vivo pluripotency, including teratoma formation and chimeric mouse production. In addition, the safety of CombiMag-DNA lipoplexes for the transfection of MEF cells was confirmed through lactate dehydrogenase activity assay and transmission electron microscopy. These results demonstrated that the LMF method is simple, effective, and safe. LMF may represent a superior technique for the generation of virus-free or integration-free iPS cell lines that could lead to enhanced stem cell therapy in the future.
| S-EPMC3458059 | biostudies-literature
Project description:Sorting nitrifying microorganism with targeted cell sorting
| PRJEB81149 | ENA
Project description:Using magneto-nanofection, iPS cells were efficiently generated by the transient expression of iPS genes in MEF (mouse embryonic fibroblast), suggesting that the non-viral magneto-nanofection method can be used for the efficient generation of iPS cells.
2024-02-29 | GSE21357 | GEO
Project description:Transcriptional profiling of CHO-K1 cells comparing to in-house serum-free and suspension adapted CHO-K1 cells in the exponential phase. Goal was to determine the effects of serum on CHO-K1 cells.
2016-12-24 | GSE92885 | GEO
Project description:Reprogramming process can bring in genetic and epigenetic abnormalities, and these abnormalities can endow effect on gene expression of obtained iPSCs. We used Microarray to detect obvious gene expression differences between iPS cells ,which were induced using one episomal plasmid from C57BL/6 mouse embryonic fibroblasts, and one normal ES cell line from C57BL/6 inbred strain mice. iPS cells and control ES cells were selected in early passage for RNA extraction and hybridization on Affymetrix microarrays. To reduce the effect of in vitro culture, we chose iPS cells and ES cells in passage 7 to 10 for analysis.
2015-09-14 | E-GEOD-61403 | biostudies-arrayexpress
Project description:Human artificial chromosomes (HACs) have unique characteristics as gene-delivery vector, e.g., episomal transmission and transfer of multiple, large transgenes. Here, we demonstrate the advantages of HAC vectors for reprogramming mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Two HAC vectors (iHAC1 and iHAC2) were constructed. Both carried four reprogramming factors, and iHAC2 also encoded a p53-knockdown construct (see Kazuki et al 2011). The iHAC1 partially reprogrammed MEFs, and the iHAC2 efficiently reprogrammed MEFs. Global gene expression showed that the iHACs, unlike other vectors, generated relatively uniform iPS cells. We established iHAC-free iPS cells by isolating cells that spontaneously lost the iHAC2. Analyses of pluripotent markers, teratomas, and chimeras confirmed that these iHAC-free iPS cells were pluripotent. Moreover, iHAC-free iPS cells with a re-introduced HAC encoding Herpes Simplex Virus Thymidine Kinase were eliminated by Ganciclovir treatment; therefore, the HAC safeguard system functioned in iPS cells. Thus, the HAC vector could generate uniform, integration-free iPS cells with a safeguard system.
2011-10-16 | GSE29441 | GEO