Federica Pisano —BIO-PROTOCOL

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Massimiliano Gnecchi Department of Cardiothoracic and Vascular Sciences-Coronary Care Unit and Laboratory of Clinical and Experimental Cardiology-Fondazione IRCCS Policlinico San Matteo, Italy
1 protocol

Antoine de Morree Stanford university
36 protocols

Ningfei An University of Chicago
6 protocols

Vivien Coulson-Thomas University of Houston
24 protocols

Reviewer

Federica Pisano

Post-Doc, Department of Cardiothoracic and Vascular Sciences-Coronary Care Unit and Laboratory of Clinical and Experimental Cardiology-Fondazione IRCCS Policlinico San Matteo

Research focus

Stem cell
mesenchymal stromal cells, acute miocardial infarction, regeneration

1 Author merit
Reviewer merit
Editor merit

Education

PhD in Bioengineering and Bioinformatics, University of Pavia, 2014

Lab information

Stem cell lab - molecular medicine department

Publications

Pisano Federica, Mura Manuela, Ciuffreda Maria Chiara, Calabrò Federica, Lanzo Nicola, Gnecchi Massimiliano. Optimized lentiviral transduction of human amniotic mesenchymal stromal cells. Pharmacological Research, [in press].

Manuela Mura, Ashish Mehta, Chrishan J. Ramachandra, Rita Zappatore, Federica Pisano, Maria Chiara Ciuffreda, Vincenzo Barbaccia, Lia Crotti, Peter J. Schwartz, Winston Shim, Massimiliano Gnecchi. The KCNH2-IVS9-28A/G mutation causes aberrant isoform expression and hERG trafficking defect in cardiomyocytes derived from patients affected by Long QT Syndrome type 2. Int J Cardiol. 2017 Aug 1;240:367-371.

Pisano F, Gnecchi M. Transfection of Embryoid Bodies with miRNA Precursors to Differentiate Cells into Cardiomyocytes.Bio-protocol, 2016. bio-protocol.org/e1726.

Gnecchi M, Pisano F, Bariani R. MicroRNA and cardiac regeneration. MicroRNA: basic science. Adv Exp Med Biol. 2015;887:119-41. doi: 10.1007/978-3-319-22380-3_7.

Pisano F, Altomare C, Cervio E, Barile L, Rocchetti M, Ciuffreda M C, Malpasso G, Copes F, Mura M, Danieli P, Viarengo G, Zaza A, Gnecchi M. Combination of miRNA499 and miRNA133 exerts a synergic effect on cardiac differentiation. Stem Cells. 2015 Apr; 33(4):1187-99. doi: 10.1002/stem.1928.

Danieli P, Malpasso G, Ciuffreda MC, Cervio E, Calvillo L, Copes F, Pisano F, Mura M, Kleijn L, de Boer RA, Viarengo G, Rosti V, Spinillo A, Roccio M, Gnecchi M. Conditioned Medium From Human Amniotic Mesenchymal Stromal Cells Limits Infarct Size and Enhances Angiogenesis. Stem Cells Transl Med. 2015 Mar 30. pii: sctm.2014-0253. Stem Cells Translational Medicine 2015;4:1-11.

I. Del Vecchio, A. Zuccotti, F. Pisano, Canneva, S.C. Lenzken, F. Rousset, E. Corsini, S. Govoni, M. Racchi, Functional Mapping of the Promoter Region of the GNB2L1 Human Gene Coding for RACK1 Scaffold Protein. Gene 2009; 430: 17-29.

1 Protocol published

Transfection of Embryoid Bodies with miRNA Precursors to Induce Cardiac Differentiation

Authors: Federica Pisano and Massimiliano Gnecchi, date: 02/05/2016, view: 7099, Q&A: 1

In recent years, the utilization of stem cell therapy to regenerate cardiac tissue has been proposed as a possible strategy to treat cardiac damage (Gnecchi et al., 2012, Aguirre et al., 2013; Sanganalmath and Bolli, 2013). ...

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serenity Gao

Max plank institute

Hello, Thanks for the protocol. And I have some questions.
Firstly, I am curious about the exact transfection efficiency about this method since you use the EB to be transfected directly if I am right.
Secondly, is the high transfection efficiency just limited to this cell line or the precursor miRNA. I am wondering how is it for the other ESC lines and the general plasmid?
Thirdly, I am wondering what is the difference between the cardiomyocyte differentiated from this embryonal carcinoma cell line and the normal ESC line? If there is no difference, this cell line is really convenient for cardiac differentiation. And how much percentage of the cardiomyocyte in the EBs differentiaed from this cell line?
It would be greatly appreciated if you can give me some idea about these questions. Thank you very much!

3/18/2016 9:22:18 AM Reply

Federica Pisano

Department of Cardiothoracic and Vascular Sciences-Coronary Care Unit and Laboratory of Clinical and Experimental Cardiology-Fondazione IRCCS Policlinico San Matteo

Firstly, I am curious about the exact transfection efficiency about this method since you use the EB to be transfected directly if I am right.

Yes, you are right. We optimized the standard transfection protocol using EB. To quantify transfection efficiency we performed qPCR on dissociated EB (please, see point 12 of our protocol) and using both a positive control (100% of miRNA expression) and a negative control (P19 naïve cells). RNA was extracted from the isolated cells and RT-PCR and qPCR were performed for miRNA1, miRNA133 and miRNA499. The transfection efficiency ranged 88 to 100%.

Secondly, is the high transfection efficiency just limited to this cell line or the precursor miRNA. I am wondering how is it for the other ESC lines and the general plasmid?

P19 cells are not easy to transfect with liposomes and plasmid. However, the low molecular weight of miRNA precursors (60-80 nucleotides) probably facilitates the transfection efficiency. We initially tried to transfect these cell line with a 10 Kb plasmid but the transfection efficiency was very low. For these reason, we switched to lentivirus to transform P19 cells obtaining high efficiency (please, see Pisano et al., 2015 http://onlinelibrary.wiley.com/doi/10.1002/stem.1928/pdf).
We did not test ESC lines in our studies so we do not have an answer for this specific question.

Thirdly, I am wondering what is the difference between the cardiomyocyte differentiated from this embryonal carcinoma cell line and the normal ESC line? If there is no difference, this cell line is really convenient for cardiac differentiation. And how much percentage of the cardiomyocyte in the EBs differentiaed from this cell line?

As aforementioned, we did not perform the transfection protocol on ESC. Anyway, ESC and P19 behave very similarly and we supposed that the cardiac differentiation efficiency could be similar between these two cell types. Please, refer again to our manuscript (Pisano et al., 2015 http://onlinelibrary.wiley.com/doi/10.1002/stem.1928/pdf).

2016-04-06 00:22:02

4 Protocols reviewed

Implantation of Human Peripheral Corneal Spheres into Cadaveric Human Corneal Tissue

Authors: Jeremy John Mathan, Salim Ismail, Jennifer Jane McGhee and Trevor Sherwin, date: 07/20/2017, view: 5226, Q&A: 0

Stem and progenitor cells isolated from human limbal tissue can be cultured in vitro as spheres. These spheres have potential for use as transplantable elements for the repopulation of corneal tissue (Mathan et al., 2016). Herein ...

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Reversible Cryo-arrests of Living Cells to Pause Molecular Movements for High-resolution Imaging

Authors: Jan Huebinger, Martin E. Masip, Jens Christmann, Frank Wehner and Philippe I. H. Bastiaens, date: 04/20/2017, view: 5609, Q&A: 0

Fluorescence live-cell imaging by single molecule localization microscopy (SMLM) or fluorescence lifetime imaging microscopy (FLIM) in principle allows for the spatio-temporal observation of molecular patterns in individual, living cells. However, ...

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