Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

Maria  G. Garone; Valeria de Turris; Alessandro Soloperto; Carlo Brighi; Riccardo De Santis; Francesca Pagani; Silvia Di Angelantonio; Alessandro Rosa

doi:10.3791/59321

Conversión de células madre pluripotentes inducidas por humanos (IPSC) en neuronas motoras cranea...

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JoVE Journal Neuroscience

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

Conversión de células madre pluripotentes inducidas por humanos (IPSC) en neuronas motoras craneales y espinales funcionales usando vectores PiggyBac

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07:33 min

May 1, 2019

DOI: 10.3791/59321-v

Maria G. Garone¹, Valeria de Turris², Alessandro Soloperto², Carlo Brighi², Riccardo De Santis³, Francesca Pagani^2,4, Silvia Di Angelantonio^2,4, Alessandro Rosa^1,2

¹Department of Biology and Biotechnology Charles Darwin,Sapienza University of Rome, Italy, ²Center for Life Nano Science,Istituto Italiano di Tecnologia, Italy, ³Laboratory of Stem Cell biology and Molecular Embryology,The Rockefeller University, USA, ⁴Department of Physiology and Pharmacology,Sapienza University of Rome, Italy

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Overview

This study presents a protocol for the rapid conversion of induced pluripotent stem cells (iPSCs) into motor neurons with specific spinal or cranial identities. The method simplifies drug screening for motor neuron diseases and allows for the generation of cell populations without purification steps.

Key Study Components

Area of Science

Neuroscience
Cell Biology
Stem Cell Research

Background

Motor neuron diseases, such as amyotrophic lateral sclerosis (ALS), require efficient models for study.
iPSCs can be differentiated into various cell types, providing valuable tools for research.
The study focuses on transforming iPSCs into motor neurons for modeling and drug testing.

Purpose of Study

To establish a method for generating motor neurons from iPSCs.
To provide a rapid approach for screening potential drugs for motor neuron diseases.
To explore functional properties of derived motor neurons.

Methods Used

Cell culture techniques for iPSCs were employed to generate motor neurons.
Transfection was achieved using piggyBac transposons for effective gene delivery.
Key steps included cell dissociation, transgene selection, and differentiation into motor neurons.

Main Results

The method produced functional motor neurons that exhibited voltage-dependent currents and firing properties typical of mature neurons.
80% of derived motor neurons showed the ability to trigger spike trains under stimulation.
The study demonstrated successful differentiation and functional maturation of motor neurons from iPSCs.

Conclusions

This protocol enhances the functionality of iPSCs for modeling motor neuron diseases and facilitates drug discovery.
The generated cell lines can be utilized to better understand the progression of ALS.
The findings support the use of iPSC-derived motor neurons as valuable resources in neuroscience research.

Frequently Asked Questions

What advantages does this method offer for studying motor neurons?

The method allows for rapid generation of motor neurons without the need for lengthy purification steps, facilitating drug screening and modeling of diseases.

How are induced pluripotent stem cells differentiated into motor neurons?

iPSCs are transfected with specific transcription factors via piggyBac vectors and cultured under defined conditions to promote differentiation into motor neurons.

What types of outcomes can be assessed using this protocol?

The protocol enables the assessment of functional properties such as excitability, voltage-dependent currents, and morphological characteristics of motor neurons.

Can this method be adapted for other cell types?

Yes, the inducible expression of transcription factors can also be used to generate other cell types like glial cells and skeletal muscle cells from iPSCs.

What are some key limitations of this approach?

The approach may lead to mixed populations of cells, and stably transfected clones may require additional isolation for specific applications.

How can the findings of this study contribute to understanding ALS?

The derived motor neurons can be used to model the disease's progression and to screen potential therapeutic compounds.

Este protocolo permite la conversión rápida y eficiente de células madre pluripotentes inducidas en neuronas motoras con una identidad espinal o craneal, mediante la expresión ectópica de factores de transcripción de vectores piggyBac inducibles.

La posibilidad de generar diferentes subtipos de neuronas motoras es particularmente relevante para modelar enfermedades modernas de las neuronas motoras como la esclerosis lateral amiotrófica. Este método permite la adquisición de poblaciones celulares que son altamente rígidas para las neuronas motoras con una identidad espinal o craneal en un corto período de tiempo y sin el paso de purificación. La generación de neuronas motoras humanas a partir de células madre pluripotentes inducidas o iPSC bajo una condición de cultivo simplificada puede facilitar la detección de fármacos para enfermedades de las neuronas motoras.

La expresión inducible del factor de transcripción de programación se puede utilizar para generar otros tipos de células como neuronal, músculo esquelético y células gliales desde un repositorio iPSC. Para la generación de líneas iPSC NIL y NIP, enjuague el cultivo humano de iPSC con PBS libre de calcio y magnesio antes de tratar las células con reactivo de disociación celular durante cinco a 10 minutos a 37 grados centígrados. Cuando las células hayan comenzado a desprenderse del recipiente de cultivo, utilice una pipeta P1000 para disociar manualmente las células de tres a cuatro veces.

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