Episomal iPSC Generation from Fibroblasts

1×10⁵ fibroblasts (cultured without antibiotics for 24 hours) were electroporated with 0.5 µg iPSC episomal vector mixture(17 (link)) using a 10 µl kit neon transfection system (Thermo Fisher MPK1096) (1300V, 20ms, 2 pulses), and plated into one well of a Matrigel-coated 6-well plate. Cells were cultured in E8 media(18 (link)) plus 0.1µM hydrocortisone until confluency was ~20% (typically 5–10 days), at which point cells were cultured in E7 media (E8 without TGF-β) until PSC colonies were visible (typically 25–30 days post-electroporation). Individual colonies were picked and transferred to Matrigel-coated 24-well dishes and expanded and maintained in E8. For TSC2-shRNA transduced fibroblasts: cultures were transferred to E8 + hydrocortisone 48 hours following electroporation.

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Julian L.M., Delaney S.P., Wang Y., Goldberg A.A., Doré C., Yockell-Lelièvre J., Tam R.Y., Giannikou K., McMurray F., Shoichet M.S., Harper M.E., Henske E.P., Kwiatkowski D.J., Darling T.N., Moss J., Kristof A.S, & Stanford W.L. (2017). Human pluripotent stem cell-derived TSC2-haploinsufficient smooth muscle cells recapitulate features of Lymphangioleiomyomatosis. Cancer research, 77(20), 5491-5502.

Publication 2017

MPK1096

Antibiotics Cells Cultured media Dishes Electroporation Episomal Fibroblasts Hydrocortisone Ipsc Matrigel Neon Pulses Shrna Tgf β Transfection Tsc2 Vector

Corresponding Organization : Ottawa Hospital

Other organizations : University of Ottawa, McGill University Health Centre, Brigham and Women's Hospital, Harvard University, Uniformed Services University of the Health Sciences, National Heart Lung and Blood Institute

Top 5 similar protocols

Variable analysis

independent variables

Electroporation with 0.5 µg iPSC episomal vector mixture
Transduction with TSC2-shRNA

dependent variables

Generation of iPSC colonies

control variables

Number of fibroblasts (1×10^5)
Culture duration (5-10 days until ~20% confluency, 25-30 days post-electroporation)
Culture media (E8, E7)
Hydrocortisone treatment
Matrigel-coated culture plates

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!