Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

Jesse J. Hubbard; Spencer K. Sullivan; Jason A. Mills; Brian J. Hayes; Beverly J. Torok-Storb; Aravind Ramakrishnan

doi:10.3791/52009

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

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Biology

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Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

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08:14 min

October 28, 2014

DOI: 10.3791/52009-v

Jesse J. Hubbard¹, Spencer K. Sullivan², Jason A. Mills³, Brian J. Hayes¹, Beverly J. Torok-Storb¹, Aravind Ramakrishnan¹

¹Clinical Research Division,Fred Hutchinson Cancer Research Center, ²Division of Hematology,The Children's Hospital of Philadelphia, ³Department of Pathology,The Children's Hospital of Philadelphia

Overview

This article presents a protocol for generating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood. The method utilizes an episome-based reprogramming strategy combined with histone deacetylase inhibitors to enhance reprogramming efficiency.

Key Study Components

Area of Science

Stem Cell Biology
Cell Reprogramming
Regenerative Medicine

Background

Induced pluripotent stem cells (iPSCs) are valuable for research and therapeutic applications.
Traditional methods of generating iPSCs often involve integrating vectors, which can pose risks of insertional mutagenesis.
This protocol aims to provide a safer alternative by using non-integrating episomal plasmids.
Peripheral blood is an accessible source for iPSC generation.

Purpose of Study

To develop a reliable method for generating iPSCs from peripheral blood.
To eliminate the risk of genomic integration associated with viral methods.
To enhance the efficiency of reprogramming using defined growth factors and inhibitors.

Methods Used

Isolation of peripheral blood mononuclear cells.
Culturing cells in defined growth factors to yield erythrocyte progenitors.
Introduction of non-integrating episomal plasmids via nucleofection.
Plating nucleofected cells on irradiated mouse embryonic fibroblasts for reprogramming.

Main Results

Successful generation of iPSC colonies identified by morphological characteristics.
Positive alkaline phosphatase activity in reprogrammed cells.
Expression of pluripotency markers confirmed through immunochemistry.
Endogenous pluripotency genes were expressed in the reprogrammed cells.

Conclusions

This protocol provides a consistent method for generating integration-free iPSCs.
The use of peripheral blood as a starting material is advantageous for clinical applications.
The approach minimizes risks associated with viral-based reprogramming techniques.

Frequently Asked Questions

What are induced pluripotent stem cells (iPSCs)?

iPSCs are stem cells that are generated from adult cells and can differentiate into various cell types.

Why is it important to use integration-free methods for generating iPSCs?

Integration-free methods reduce the risk of insertional mutagenesis, making them safer for therapeutic applications.

What is the role of histone deacetylase inhibitors in this protocol?

Histone deacetylase inhibitors enhance the reprogramming efficiency of somatic cells into iPSCs.

How are the reprogrammed iPSCs identified?

Reprogrammed iPSCs are identified based on their morphology, alkaline phosphatase activity, and expression of pluripotency markers.

Can this method be applied to other types of cells?

While this protocol focuses on peripheral blood, similar methods can potentially be adapted for other cell types.

What are the advantages of using peripheral blood for iPSC generation?

Peripheral blood is a non-invasive and easily accessible source for obtaining cells for reprogramming.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

The overall goal of the following experiment is to generate integration free induced pluripotent stem cells from a small amount of peripheral blood. This is achieved by culturing isolated peripheral blood mononuclear cells in defined growth factors to yield an erythrocyte progenitor population that is very responsive to reprogramming. As a second step non integrating episomal plasmids carrying reprogramming genes are introduced into the erythroblasts by nucleo affection to initiate the reprogramming process.

Next, the nucle affected cells are plated onto a layer of irradiated mouse embryonic fibroblasts to allow for continued reprogramming ultimately successfully. Reprogrammed induced pluripotent stem cell colonies can be identified based on their morphological characteristics, alkaline phosphatase activity, positive immunochemistry for pluripotency markers and expression of endogenous pluripotency genes. The main advantage of this technique over existing methods like viral based reprogramming, is that this protocol ensures consistent generation of integration free IPSC Demonstrating the procedure today will be Jesse, myself, and Gretchen Johnson.

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IPS Cell Generation Episomes HDAC Inhibitors Peripheral Blood Integration-free Nonintegrating Reprogramming Vectors Erythroblasts Nucleofection EBNA1 Mouse Embryonic Fibroblasts Epigenetic Remodeling