Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Immacolata Belviso; Anna Maria Sacco; Veronica Romano; Fabrizio Schonauer; Daria Nurzynska; Stefania Montagnani; Franca Di Meglio; Clotilde Castaldo

doi:10.3791/60629

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripo...

JoVE Journal
Bioengineering

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JoVE Journal Bioengineering

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

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10:52 min

January 19, 2020

DOI: 10.3791/60629-v

Immacolata Belviso*¹, Anna Maria Sacco*¹, Veronica Romano¹, Fabrizio Schonauer¹, Daria Nurzynska¹, Stefania Montagnani¹, Franca Di Meglio¹, Clotilde Castaldo¹

¹Department of Public Health,University of Naples Federico II

Overview

This protocol outlines the generation of autologous human-induced pluripotent stem cells (iPSCs) from human dermal fibroblasts. It utilizes non-modified RNAs and immune evasion factors to enhance reprogramming efficiency without viral methods.

Key Study Components

Area of Science

Cell Reprogramming
Stem Cell Biology
Regenerative Medicine

Background

iPSCs are crucial for regenerative medicine and disease modeling.
Traditional methods often involve viral integration, which can pose risks.
This protocol aims to provide a safer, non-viral approach.
Understanding the reprogramming process is essential for effective application.

Purpose of Study

To develop a protocol for generating iPSCs from dermal fibroblasts.
To facilitate research in regenerative medicine and disease modeling.
To provide a method accessible to researchers with limited experience.

Methods Used

Isolation of human dermal fibroblasts from skin punch biopsies.
Application of non-modified RNAs for reprogramming.
Incorporation of immune evasion factors to reduce cellular defenses.
Visual demonstrations to aid in the protocol execution.

Main Results

Successful generation of xeno-free iPSC colonies.
Protocol allows for efficient reprogramming with minimal contamination risks.
Autologous iPSCs can be utilized for various applications.
Guidance provided to avoid common experimental mistakes.

Conclusions

The protocol offers a reliable method for generating iPSCs.
It supports advancements in regenerative medicine and disease research.
Accessible to researchers with varying levels of experience.

Frequently Asked Questions

What are induced pluripotent stem cells (iPSCs)?

iPSCs are stem cells that can be generated directly from adult cells and have the ability to differentiate into any cell type.

Why is a non-viral method preferred for reprogramming?

Non-viral methods reduce the risk of insertional mutagenesis and other complications associated with viral vectors.

What is the significance of using autologous cells?

Autologous cells minimize the risk of immune rejection and enhance the compatibility of the stem cells for therapeutic applications.

How can iPSCs be used in disease modeling?

iPSCs can be differentiated into specific cell types to study disease mechanisms and test potential therapies in a laboratory setting.

What precautions should be taken during the reprogramming process?

It is essential to prevent microbial contamination and ensure the integrity of RNA used in the reprogramming process.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

The following protocol provides instructions to generate autologous human-induced pluripotent stem cells through the fast and efficient reprogramming of human dermal fibroblasts isolated from a skin punch biopsy. The procedures can be carried out by researchers with little or no experience in cell reprogramming and with proper adjustment, allows the product of xeno-free induced pluripotent stem cells. Autologous IPS cells might be used for regenerative medicine applications and for modeling diseases like genetic disorders or cancer to investigate on underlying molecular mechanisms and develop more specific therapies.

The workload is high. It is extremely helpful to study the protocol prior to carefully plan the experiment, including the steps during which reagents and materials should be prepared. The visual demonstration of the procedure helps the researches with little or no experience perform the cell isolation and the reprogramming, avoiding common mistakes related to possible microbial and RNA contamination.

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