Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Viviana Meraviglia; Alessandra Zanon; Alexandros A. Lavdas; Christine Schwienbacher; Rosamaria Silipigni; Marina Di Segni; Huei-Sheng Vincent Chen; Peter P. Pramstaller; Andrew A. Hicks; Alessandra Rossini

doi:10.3791/52885

Generazione di pluripotenti indotte cellule staminali da congelata Buffy cappotti con non-integra...

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JoVE Journal Developmental Biology

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Generazione di pluripotenti indotte cellule staminali da congelata Buffy cappotti con non-integrazione episomiale Plasmidi

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10:52 min

June 5, 2015

DOI: 10.3791/52885-v

Viviana Meraviglia*¹, Alessandra Zanon*¹, Alexandros A. Lavdas¹, Christine Schwienbacher¹, Rosamaria Silipigni², Marina Di Segni², Huei-Sheng Vincent Chen³, Peter P. Pramstaller¹, Andrew A. Hicks¹, Alessandra Rossini¹

¹Center for Biomedicine,European Academy Bozen/Bolzano (EURAC), ²Laboratory of Medical Genetics,Fondazione IRCCS Ca´ Granda, Ospedale Maggiore Policlinico, ³Del E. Webb Center for Neuroscience, Aging & Stem Cell Research,Sanford-Burnham Medical Research Institute

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Overview

This article presents a protocol for generating induced pluripotent stem cells (iPSCs) from human peripheral blood mononuclear cells (PBMNCs) derived from frozen buffy coats. The method is cost-effective and utilizes a virus-free approach for reprogramming.

Key Study Components

Area of Science

Stem Cell Biology
Cell Reprogramming
Clinical Applications

Background

Induced pluripotent stem cells (iPSCs) are valuable for patient-specific tissue generation.
PBMNCs can be isolated from frozen buffy coats for reprogramming.
Traditional methods often involve viral vectors, which this protocol aims to avoid.
Using non-integrating episomal plasmids enhances safety for clinical applications.

Purpose of Study

To develop a reliable method for generating iPSCs from PBMNCs.
To provide a detailed protocol that is accessible and cost-effective.
To demonstrate the efficiency of reprogramming through immunofluorescence and gene expression analysis.

Methods Used

Isolation of PBMNCs from buffy coats via centrifugation.
Expansion of PBMNCs in a specific blood culture medium.
Transfection of PBMNCs with episomal plasmids using electroporation.
Plating transfected cells onto mouse embryonic fibroblast feeder cells.

Main Results

Successful reprogramming of PBMNCs into iPSCs was achieved.
iPSCs displayed typical human morphology.
Immunofluorescence confirmed the expression of pluripotency markers.
Gene expression analysis validated the reprogramming efficiency.

Conclusions

The protocol provides a safe and effective method for generating iPSCs.
This approach can facilitate patient-specific research and therapeutic applications.
Future studies may explore the clinical implications of these iPSCs.

Frequently Asked Questions

What are induced pluripotent stem cells?

Induced pluripotent stem cells (iPSCs) are cells that have been genetically reprogrammed to an embryonic stem cell-like state.

Why use PBMNCs for iPSC generation?

PBMNCs are easily accessible and can be obtained from patients, allowing for personalized medicine approaches.

What is the advantage of using a virus-free protocol?

A virus-free protocol reduces the risk of insertional mutagenesis and other complications associated with viral vectors.

How are PBMNCs isolated from buffy coats?

PBMNCs are isolated through centrifugation of whole blood without a density gradient.

What methods confirm the successful reprogramming of PBMNCs?

Immunofluorescence and gene expression analysis are used to confirm the successful reprogramming of PBMNCs into iPSCs.

Cellule staminali pluripotenti indotte (iPSCs) rappresentano una fonte di tessuti paziente-specifici per le applicazioni cliniche e di ricerca di base. Qui vi presentiamo un protocollo dettagliato per riprogrammare le cellule umane mononucleari di sangue periferico (PBMNCs) ottenute da buffy coats congelati in iPSCs-virali gratuito utilizzando non integrano plasmidi episomiale.

L'obiettivo generale di questa procedura è quello di generare cellule staminali pluripotenti indotte da cellule mononucleate di sangue periferico umano ottenute da buffy coat congelati utilizzando un protocollo economico e privo di virus. Ciò si ottiene in primo luogo mediante l'isolamento di PB MNC da Buffy coat dopo la centrifugazione del sangue intero senza gradiente di densità, seguita dall'espansione in uno specifico mezzo di coltura del sangue. Il secondo passo consiste nel trasfettare le MNC PB di Buffy coat con plasmidi episomiali mediante elettroporazione.

Le MNC PB trasfettate vengono quindi piastrate su cellule alimentatrici di fibroblasti embrionali di topo fresche. Il passaggio finale consiste nel prelevare manualmente le cellule staminali pluripotenti indotte dalla coltura e trasferirle su nuove piastre cellulari di alimentazione per l'espansione. In definitiva, l'efficiente riprogrammazione delle MNC PB in cellule staminali pluripotenti indotte con una tipica morfologia umana può essere dimostrata mediante immunofluorescenza e misurazione dell'espressione genica.

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