Serum replacement with albumin-associated lipids prevents excess aggregation and enhances growth of induced pluripotent stem cells in suspension culture

2016 ◽  
Vol 32 (4) ◽  
pp. 1009-1016 ◽  
Author(s):  
Ikki Horiguchi ◽  
Yasuyuki Sakai
Keyword(s):  
Stem Cells ◽  
Suspension Culture ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Serum Replacement ◽  
Induced Pluripotent

283 GENERATION AND CHARACTERIZATION OF BOVINE INDUCED PLURIPOTENT STEM CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 289
Author(s):  
O. J. Koo ◽  
H. S. Kwon ◽  
D. K. Kwon ◽  
K. S. Kang ◽  
B. C. Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Transgenic Animals ◽  
Mouse Embryonic Fibroblast ◽  
Embryoid Bodies ◽  
Differentiation Potential ◽  
Serum Replacement ◽  
Induced Pluripotent ◽  
Large Animals

Stem cells in large animals are an excellent model for cell therapy research and fine resources for producing transgenic animals. However, there are only few reports of stem cells in large animals because of technical differences between species. In this report, we successfully generate bovine induced pluripotent stem cells (iPSC) using 4 human reprogramming factors (Oct4, Sox2, Klf4, and c-myc) under control of PiggyBac transposition vector. Fibroblasts derived from bovine fetuses were transfected using FugeneHD agent. After 21 days, colony-shaped structures on the culture plates were mechanically detached and then seeded on a mouse embryonic fibroblast (MEF) feeder layer pretreated with mitomycin C. The culture medium was DMEM/F12 supplemented with 20% serum replacement, 5 ng mL–1 basic fibroblast growth factor (bFGF), 0.1 mM β-mercaptoethanol, 1% NEAA, and 1% penicillin-streptomycin antibiotics. The iPSC colonies showed alkaline phosphatase activity and expressed several pluripotency markers (Oct4, Sox2, SSEA1, and SSEA4). To confirm differentiation potential, the iPSC were cultured as embryoid bodies and then plated again. βIII-tubulin (ectoderm) and GFAP or α-SMA (mesoderm) were well expressed on the attached cells. The results revealed that the bovine fibroblasts were well inducted to iPSC that had potential of multilineage differentiation. We hope this technology contributes to improving transgenic cattle production. This study was financially supported by IPET (grant # 109023-05-3-CG000, 111078-03-1-CG000) and the BK21 program for Veterinary Science.

Suspension Culture of Undifferentiated Human Embryonic and Induced Pluripotent Stem Cells

2010 ◽  
Vol 6 (2) ◽  
pp. 248-259 ◽  
Author(s):  
Michal Amit ◽  
Judith Chebath ◽  
Victoria Margulets ◽  
Ilana Laevsky ◽  
Yael Miropolsky ◽  
...  
Keyword(s):  
Stem Cells ◽  
Suspension Culture ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Induced Pluripotent

scholarly journals Generation of functional cardiomyocytes from rat embryonic and induced pluripotent stem cells using feeder-free expansion and differentiation in suspension culture

PLoS ONE ◽  
2018 ◽  
Vol 13 (3) ◽  
pp. e0192652 ◽  
Author(s):  
Julia Dahlmann ◽  
George Awad ◽  
Carsten Dolny ◽  
Sönke Weinert ◽  
Karin Richter ◽  
...  
Keyword(s):  
Stem Cells ◽  
Suspension Culture ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Free Expansion ◽  
Induced Pluripotent

scholarly journals Generation of Human iMelanocytes from Induced Pluripotent Stem Cells through a Suspension Culture System

2020 ◽  
Vol 1 (1) ◽  
pp. 100004
Author(s):  
Li-Ping Liu ◽  
Ning-Ning Guo ◽  
Yu-Mei Li ◽  
Yun-Wen Zheng
Keyword(s):  
Stem Cells ◽  
Suspension Culture ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Culture System ◽  
Induced Pluripotent

scholarly journals Establishment of Bovine-Induced Pluripotent Stem Cells

2021 ◽  
Vol 22 (19) ◽  
pp. 10489
Author(s):  
Yue Su ◽  
Ling Wang ◽  
Zhiqiang Fan ◽  
Ying Liu ◽  
Jiaqi Zhu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Normal Karyotype ◽  
Colony Morphology ◽  
Significant Advance ◽  
Serum Replacement ◽  
Induced Pluripotent ◽  
Knockout Serum Replacement

Pluripotent stem cells (PSCs) have been successfully developed in many species. However, the establishment of bovine-induced pluripotent stem cells (biPSCs) has been challenging. Here we report the generation of biPSCs from bovine mesenchymal stem cells (bMSCs) by overexpression of lysine-specific demethylase 4A (KDM4A) and the other reprogramming factors OCT4, SOX2, KLF4, cMYC, LIN28, and NANOG (KdOSKMLN). These biPSCs exhibited silenced transgene expression at passage 10, and had prolonged self-renewal capacity for over 70 passages. The biPSCs have flat, primed-like PSC colony morphology in combined media of knockout serum replacement (KSR) and mTeSR, but switched to dome-shaped, naïve-like PSC colony morphology in mTeSR medium and 2i/LIF with single cell colonization capacity. These cells have comparable proliferation rate to the reported primed- or naïve-state human PSCs, with three-germ layer differentiation capacity and normal karyotype. Transcriptome analysis revealed a high similarity of biPSCs to reported bovine embryonic stem cells (ESCs) and embryos. The naïve-like biPSCs can be incorporated into mouse embryos, with the extended capacity of integration into extra-embryonic tissues. Finally, at least 24.5% cloning efficiency could be obtained in nuclear transfer (NT) experiment using late passage biPSCs as nuclear donors. Our report represents a significant advance in the establishment of bovine PSCs.

219 Invitro culture environment influences the ability to generate porcine primordial germ cell-like from induced pluripotent stem cells

2020 ◽  
Vol 32 (2) ◽  
pp. 237
Author(s):  
N. Pieri ◽  
R. Botigelli ◽  
A. de Souza ◽  
K. Recchia ◽  
R. de Castro ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Growth Factor ◽  
Germ Cell ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Culture Condition ◽  
Primordial Germ Cell ◽  
Serum Replacement ◽  
Induced Pluripotent

The ability to generate primordial germ cell-like (PGCLs) from induced pluripotent stem cells (iPSCs) in swine could greatly contribute to regenerative medicine. Herein, we aimed to generate porcine PGCLs (ipPGCLs) from iPSCs derived from different culture systems. Porcine (p)iPSCs from fibroblasts of stillborn animals (n=3) were transduced with lentiviral vectors containing murine OCT4, SOX2, c-MYC, and KLF4 cDNAs and maintained in iPSC medium on mouse embryonic fibroblasts (MEFs). The cells were divided into three groups: (1) supplemented with 10ngmL−1 basic fibroblast growth factor (bFGF) and murine leukemia inhibitory factor (LIF), (2) only bFGF, or (3) only LIF. The piPSC colonies were generated and characterised for pluripotency. To induce piPSCs into ipPGCLs, three or more cell lines from each culture condition (after passage 20) were differentiated into epiblast stem cell-like cells (EpiLCs) by culture with 20ngmL−1 Activin A, 12ngmL−1 bFGF, and 1% knockout serum replacement (KSR) for 2 days. Then, cells were further induced to differentiate by nonadherent culture and supplementation with 500ngmL−1 bone morphogenetic protein (BMP)4, 500ngmL−1 BMP8a, LIF, 100ngmL−1 stem cell factor (SCF), and 50ngmL−1 epidermal growth factor for 4 days. The ipPGCLs were characterised by cell morphology and detection of germ cell markers by immunofluorescence and gene expression. Statistical analysis was determined by one-way ANOVA (Prism Software). Co-location quantification was determined using the plugin Colocalization Threshold in Image J software (National Institutes of Health). On average, the efficiency rate of iPSC generation was 71% for the iPSCs-bFGF group, 17% for the LIF group, and 85% for the bFGF+LIF group. All iPSCs colonies were positive for alkaline phosphatase and OCT4, SOX2, NANOG, TRA1-60, TRA1-81, SSEA1, and SSEA4 by immunofluorescence. Embryoid body assay revealed that the piPSCs were able to differentiate into three germ layers. The culture condition did not influence the expression of OCT4, NANOG, and KLF4 based on qRT-PCR, however; SOX2 was upregulated in the LIF group (P<0.05). The ipPGCLs generated showed a round morphology. Analysis of endogenous pluripotent genes OCT4, SOX2, and NANOG throughout differentiation (fibroblasts, iPSCs, EpiLCs, and PGCLs) revealed a mild upregulation in ipPGCLs, while OCT4 was slightly downregulated in ipPGCLs from iPSCs-LIF group. PRDM14 and STELLA were not observed in ipPGCLs, although BLIMP1 was present; DAZL and VASA were mildly upregulated. The STELLA, VASA, OCT4, and SOX2 proteins were detected in ipPGCLs, and DAZL was detected only in ipPGCLs from the iPSCs-FGF group. Protein co-localization analysis showed that ipPGCLs from the iPSCs-FGF group were 100% OCT4+STELLA-positive, 55% positive for DAZL+SOX2, and 66% positive for VASA+NANOG; for the LIF group: 99.3% were OCT4+STELLA positive, DAZL was not detected, 95.2% were positive for SOX2 and 85.6% for VASA+NANOG. In the bFGF+LIF group, 95.8% were positive for OCT4+STELLA, DAZL and SOX2 were not observed, and 70% were positive for VASA+NANOG. Exogenous reprogramming factors were still expressed and did not differ between groups. These results indicate that, under our conditions, the iPSCs-FGF group may represent the best culture condition for induction into ipPGCLs. Financial support for this study was provided by FAPESP (2015/25564-0 and 2015/26818-5).

Author response for "Protection of human induced pluripotent stem cells against shear stress in suspension culture by Bingham plastic fluid"

2020 ◽  
Author(s):  
Ikki Horiguchi ◽  
Fuad Gandhi Torizal ◽  
Hotaka Nagate ◽  
Haruka Inose ◽  
Kousuke Inamura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Shear Stress ◽  
Suspension Culture ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Author Response ◽  
Bingham Plastic ◽  
Plastic Fluid ◽  
Induced Pluripotent
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