Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

Sandra Meyer; Philipp W&#246;rsd&#246;rfer; Katharina G&#252;nther; Marc Thier; Frank Edenhofer

doi:10.3791/52831

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenit...

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Neuroscience

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JoVE Journal Neuroscience

Derivation of Adult Human Fibroblasts and their Direct Conversion into Expandable Neural Progenitor Cells

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13:58 min

July 29, 2015

DOI: 10.3791/52831-v

Sandra Meyer^1,2, Philipp Wörsdörfer¹, Katharina Günther¹, Marc Thier^2,3, Frank Edenhofer^1,2

¹Institute of Anatomy and Cell Biology,University of Würzburg, ²Institute of Reconstructive Neurobiology,University of Bonn, ³German Cancer Research Center, Heidelberg

Overview

This study presents a method for directly converting adult human fibroblasts into induced neural progenitor cells (INPCs) for therapeutic applications. The approach aims to streamline the traditional Yamanaka IPS method, facilitating the generation of patient-specific neural cells.

Key Study Components

Area of Science

Neuroscience
Stem Cell Biology
Regenerative Medicine

Background

Induced pluripotent stem cells (iPSCs) hold promise for autologous transplants.
Current methods involve lengthy re-differentiation processes.
Direct conversion techniques may enhance clinical applicability.
Fibroblasts can be reprogrammed to generate neural progenitor cells.

Purpose of Study

To develop a faster method for generating INPCs from skin biopsies.
To evaluate the potential of directly converted IPCs for biomedical applications.
To demonstrate the differentiation capability of these cells into neuronal and glial lineages.

Methods Used

Performing a punch biopsy to obtain primary fibroblast cells.
Infecting cultured fibroblasts with reprogramming factor-encoding viruses.
Visual validation and manual selection of converted INPC colonies.
Monoclonal expansion of IPCs in neuro induction medium.

Main Results

Successfully generated INPCs from patient-derived fibroblasts.
Demonstrated differentiation into both neuronal and glial cells.
Provided a virtually unlimited source of cells for research and therapy.
Showed advantages over traditional iPSC methods in terms of efficiency.

Conclusions

The direct conversion method is a promising alternative to classical iPSC derivation.
This approach may enhance the feasibility of patient-specific therapies.
Further research is needed to optimize and validate this technique.

Frequently Asked Questions

What are induced neural progenitor cells?

Induced neural progenitor cells (INPCs) are cells derived from fibroblasts that have the potential to differentiate into various neural lineages.

How does the direct conversion method differ from traditional iPSC methods?

The direct conversion method bypasses the pluripotent state, allowing for a more efficient generation of neural progenitor cells directly from fibroblasts.

What are the potential applications of INPCs?

INPCs can be used for regenerative medicine, modeling neurological diseases, and drug testing.

What is the significance of using patient-derived cells?

Using patient-derived cells ensures that the generated cells are autologous, reducing the risk of immune rejection in therapeutic applications.

What techniques are used to validate the conversion of fibroblasts to INPCs?

Visual validation and immunofluorescence microscopy are employed to confirm the successful conversion and differentiation of cells.

How long does the conversion process take?

The conversion process takes approximately 20 days from infection to selection of INPC colonies.

Generation of induced pluripotent stem cells provides fascinating prospects for the derivation of autologous transplants. However, progression through a pluripotent state and laborious re-differentiation still hinders clinical translation. Here we describe the derivation of adult human fibroblasts and their direct conversion into induced neural progenitor cells and the subsequent differentiation into neural lineages.

The overall goal of this procedure is to shorten the classical Yamanaka IPS approach by directly generating induced neural progenitor cells from a patient's skin biopsy for therapeutic applications. This is accomplished by first performing a punch biopsy from the patient to derive primary fibroblast cells that can be cultured in vitro. The second step is to expand and subsequently infect cultured fibroblasts with viruses that encode reprogramming factors to induce trans differentiation.

Next, the putative converted induced neural progenitor cell or INPC colonies must be carefully selected by visual validation and subsequently isolated by manual picking approximately 20 days after infection. The final step is to monoclonal, expand the IPCs in neuro induction medium, ultimately targeted in vitro differentiation as well as immunofluorescence microscopy is used to show that patients own directly converted IPCs are able to differentiate into neuronal as well as glial cells making them a virtually unlimited source for biomedical applications. The advantage of this direct conversion technique over existing methods like the Yamanaka type derivation of induced blue potent stem cells and their subsequent differentiation is twofold.

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