Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Feb;74(1-2):157-65.

Establishment and optimal culture conditions of microrna-induced pluripotent stem cells generated from HEK293 cells via transfection of microrna-302s expression vector

Naoshi Koide  1 , Kaori Yasuda, Kenji Kadomatsu, Yoshifumi Takei
Affiliations

Establishment and optimal culture conditions of microrna-induced pluripotent stem cells generated from HEK293 cells via transfection of microrna-302s expression vector

Naoshi Koide et al. Nagoya J Med Sci. 2012 Feb.

Abstract

Induced pluripotent stem cells (iPSCs) have been directly generated from fibroblast cultures though retrovirus- or lentivirus-mediated ectopic overexpression of only a few defined transcriptional factors. This remarkable achievement has greatly enhanced our ability to explore the causes of, and potential cures for, many genetic diseases, and strengthened the promise of regenerative medicine. In fact, to date, many kinds of somatic cells from different tissues have exhibited a capacity for reprogramming toward an embryonic stem cell-like state, but major bottlenecks in iPSC derivation and therapeutic use remain, including low reprogramming efficiencies and the tumorigenesis of the generated iPSC. Here, we successfully generated miR-302s-induced pluripotent stem cells (mirPS cells) from human embryonic kidney (HEK) 293 cells via transfection of the miR-302s expression vector. We also determined the optimal culture conditions to generate mirPS on feeder cells, which included the use of serum-free N2B27 medium. The mirPS cells generated by our improved conditions showed the expression of pluripotent marker genes such as OCT3/4, NANOG, and SOX2 under growth conditions via reverse transcription-PCR, whereas no expression of these genes was observed in HEK293 cells. On the other hand, under differentiation conditions, mirPS cells formed ball-shaped structures (embryoid bodies), and showed the ability to differentiate into three germ layers (ectoderm, mesoderm, and endoderm) in vitro. The results suggested that our generated mirPS cells are actually functional as a cell resource to apply to regenerative medicine, and mirPS cells are suitable materials to clarify the mechanism underlying the reprogramming from somatic cells.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Generation of mirPS cells from HEK293 cells via transfection of the miR-302s expression vector. (A) Flow chart of the generation of mirPS cells. (B) Morphology of HEK293 cells. Scale bar, 250 μm. (C) EGFP expression was detected in the transfected HEK293 cells (white arrow). Scale bar, 50 μm. (D) Expanded EGFP-positive cells exhibited the packed-dome colony formation. Scale bar, 250 μm. (E) Morphology of HEK293 cells transfected with the miR-302s expression vector onto type IV collagen-coated plates without any feeder cells (MEFs). Scale bar, 100 μm. (F) EGFP-positive cells were not able to form the colony without any feeder cells (MEFs). Scale bar, 100 μm.
Fig. 2
Fig. 2
Expression profiles of mirPS cells generated from HEK293 cells. Lane 1, HEK293 cells; and lane 2, mirPS cells. mirPS cells showed the expression of some typical undifferentiated ES cell-marker genes, such as octamer-binding transcription factor 4 (OCT3/4), also known as POU domain, class 5, transcription factor 1 (POU5F1), sex determining region Y (SRY)-box2 (SOX2), NANOG, embryonic cell-specific gene 1 (ESG1), and reduced expression 1 (REX1). The predicted size of each PCR product is shown.
Fig. 3
Fig. 3
In vitro differentiation of mirPS cells through EB formation. (A) Time schedule of the differentiation experiment in vitro. (B) mirPS cells formed EB-like spheroids under a floating culture condition at day 8. Scale bar, 250 µm. (C-E) Images of differentiated cells at day 16. Immunocytochemical analysis of Tuj-1 (C), α-smooth muscle actin (D), and α-fetoprotein (E) was performed. Scale bars, 100 μm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by difined factors. Cell, 2006; 126: 663−676. - PubMed
    1. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda H, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by difined factors. Cell, 2007; 131: 861−872. - PubMed
    1. Feng B, Ng JH, Heng JC, Ng HH. Molecules that promote or enhance reprogramming of somatic cells to induced pluripotent stem cells. Cell Stem Cell, 2009; 4: 301−312. - PubMed
    1. Aoi T, Yae K, Nakagawa M, Ichisaka T, Okita K, Takahashi K, Chiba T, Yamanaka S. Generation of pluripotent stem cells from adult mouse liver and stomach cells. Science, 2008; 321: 699−702. - PubMed
    1. Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka S. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol, 2008; 26: 101−106. - PubMed

Publication types

MeSH terms

LinkOut - more resources