scholarly journals Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

10.3791/3854 ◽  
2012 ◽  
Author(s):  
Justyna Jozefczuk ◽  
Katharina Drews ◽  
James Adjaye
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Mouse Embryonic Fibroblast ◽  
Fibroblast Cells ◽  
Induced Pluripotent ◽  
Embryonic Fibroblast

scholarly journals Generation of induced pluripotent stem cells from bovine embryonic fibroblast cells

Cell Research ◽  
2011 ◽  
Vol 21 (10) ◽  
pp. 1509-1512 ◽  
Author(s):  
Xiaoping Han ◽  
Jianyong Han ◽  
Fangrong Ding ◽  
Suying Cao ◽  
Seong Soo Lim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Fibroblast Cells ◽  
Induced Pluripotent ◽  
Embryonic Fibroblast

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.

199 GERM-CELL-LIKE CELLS GENERATION FROM GOAT INDUCED PLURIPOTENT STEM CELLS

2014 ◽  
Vol 26 (1) ◽  
pp. 214
Author(s):  
D. K. Singhal ◽  
H. N. Malik ◽  
R. Singhal ◽  
S. Saugandhika ◽  
A. Dubey ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Germ Cell ◽  
Induced Pluripotent Stem Cells ◽  
Adult Female ◽  
Germ Cells ◽  
Pluripotent Stem Cells ◽  
Fibroblast Cells ◽  
Rt Pcr ◽  
Induced Pluripotent

Primordial germ cells (PGCs) generated from embryonic stem (ES) cells in different species may be an alternative approach to dealing with the worldwide problem of increasing female infertility. Reprogramming of fibroblasts into induced pluripotent stem cells has been achieved by overexpression of different transcription factors. Here, we report the generation of female goat germ cells from goat induced pluripotent stems cells (giPSC). Goat induced pluripotent stem cells (giPSC) were produced by transduction of adult female goat fibroblast cells with Oct4, Sox2, and Nanog lentiviral particles and further sub-cultured on fibroblast feeder layers. GiPSC were characterised by different methods. These iPSC were found to express alkaline phosphatase, SSEA1, SSEA4, Tra-1–81, and Tra-1–60 surface markers. However, SSEA3 was not observed in giPSC. GiPSC also expressed Oct4, Nanog, and Sox2. Along with Oct4, Nanog, and Sox2, the expression of different transcription factors such as Cdx1, Dapp5, Dax1, Ecat, Eras, Fgf4, Gata6, Lin28, Rex1, and Utf1 was confirmed by RT-PCR. GiPSC were in vitro differentiated and three germ layers were characterised by immunostaining of Gata4 for endoderm, α-Actinin for mesoderm, and β-III tubulin for ectoderm and RT-PCR analysis of GATA4, α-Actinin and BMP4. IPSCs were directed differentiated into germ cells using retinoic acid and bone morphogenetic protein 4 without the inactivation of exogenous factors as these are also required for germ cells development. Differentiated germ cells were characterised by immunostaining against VASA and Dazl proteins. RT–PCR assay was performed for Dazl, Nanog, Nanos1, PUM8, SCP3, Stella, and VASA genes expression. Quantitative PCR was also performed for detection of VASA and Dazl expression during the course of germ cell differentiation. Flow-cytometric analysis of differentiated germ cells was confirmed the presence of germ cells in population of differentiated giPSC. Oocytes/ova-like structures, which were comparable to natural goat oocytes, were observed under scanning electron microscope (SEM). Cumulus–oocyte complex like structure was observed, which was further used for SEM. The study concluded that adult female goat fibroblast cells can be reprogrammed into induced pluripotent stem cells using ectopic expression of Oct4, Nanog, and Sox2 genes and the germ-cells-like cells generated from reprogrammed giPSC could be differentiated into goat oocytes/ova-like structure which have immense applications in human and animal reproduction.

186 Improving Success Rate of Establishment and Maintenance of Porcine Induced Pluripotent Stem Cells by Investigation of Colony Morphology

2018 ◽  
Vol 30 (1) ◽  
pp. 233
Author(s):  
P. Setthawong ◽  
P. Phakdeedindan ◽  
M. Techakumphu ◽  
T. Tharasanit
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Induced Pluripotent Stem Cells ◽  
Success Rate ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Ips Cells ◽  
Fibroblast Cells ◽  
Induced Pluripotent

Induced pluripotent stem cells (iPS cells) are generated by reprogramming of somatic cells using ectopic introduction of 4 transcription factors, including OCT4, SOX2, KLF4, and c-MYC (OSKM). Fibroblast cells are the most commonly used as a primary cell source for iPS cells because they are easy to harvest and culture. However, reprogramming efficiency of porcine fibroblasts is poor (~0.1%). During reprogramming process, mixed populations of primary colonies become the major obstacle in iPS establishment. In this study, we characterised 2 different colony morphologies at colony pick-up (compact and loose iPS morphology). We hypothesised that compact colonies will proceed to long-term culture and pluripotency. The fibroblast cells were isolated from tails of piglets and transfected with retroviral vectors expressing OSKM. The primary colonies were counted on Day 7 after gene transduction. The iPS-like colonies were divided into compact (n = 10) and loose (n = 10) morphology at colony pick-up. The characteristics of iPS-like cell lines were analysed by morphology, alkaline phosphatase staining, G-banding karyotype, immunofluorescence staining (OCT4), pluripotent gene expression (RT-PCR), and embryoid body formation. A total of 1,697 iPS-like colonies (2.34%) were observed. The compact colonies contained with tightly packed cells with distinct border between iPS colony and feeder cells, while colonies with irregular shape and border were classified as loose colonies. These 2 types of iPS-like colonies expressed alkaline phosphatase and OCT4. A total 5 of 10 (50%) compact morphology cell lines could be maintained in the undifferentiated state for more than 50 passages. But only 3 of 10 (30%) loose morphology cell lines demonstrated pluripotent characteristics. G-Banding karyotype analysis revealed normal chromosome number (n = 38). All of the cell lines also expressed endogenous pluripotent genes, including OSKM and NANOG and formed three-dimensional aggregating masses. In this study, we found that the cell lines from compact morphology could be maintained for longer than those of the loose morphology. A high rate of differentiation of loose iPS colony may also indicate that this type of colony has different pluripotency signals or incomplete reprogramming compared with compact colony. In conclusion, selection of compact colony morphology at colony pick-up is simple and reflects long-term propagation of porcine iPS cell lines. This information is important for improving the success rate of establishment and maintenance of porcine iPS cells.

scholarly journals Generation of induced pluripotent stem cells from human β-thalassemia fibroblast cells

Cell Research ◽  
2009 ◽  
Vol 19 (9) ◽  
pp. 1120-1123 ◽  
Author(s):  
Yixuan Wang ◽  
Yonghua Jiang ◽  
Sheng Liu ◽  
Xiaofang Sun ◽  
Shaorong Gao
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Fibroblast Cells ◽  
Induced Pluripotent

scholarly journals Reprogramming with Small Molecules instead of Exogenous Transcription Factors

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Tongxiang Lin ◽  
Shouhai Wu
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Small Molecules ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Mouse Embryonic Fibroblast ◽  
Patient Specific ◽  
Full Potential ◽  
Ipsc Generation ◽  
Induced Pluripotent

Induced pluripotent stem cells (iPSCs) could be employed in the creation of patient-specific stem cells, which could subsequently be used in various basic and clinical applications. However, current iPSC methodologies present significant hidden risks with respect to genetic mutations and abnormal expression which are a barrier in realizing the full potential of iPSCs. A chemical approach is thought to be a promising strategy for safety and efficiency of iPSC generation. Many small molecules have been identified that can be used in place of exogenous transcription factors and significantly improve iPSC reprogramming efficiency and quality. Recent studies have shown that the use of small molecules results in the generation of chemically induced pluripotent stem cells from mouse embryonic fibroblast cells. These studies might lead to new areas of stem cell research and medical applications, not only human iPSC by chemicals alone, but also safe generation of somatic stem cells for cell based clinical trials and other researches. In this paper, we have reviewed the recent advances in small molecule approaches for the generation of iPSCs.

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