Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Arun Sharma; Guang Li; Kuppusamy Rajarajan; Ryoko Hamaguchi; Paul W. Burridge; Sean M. Wu

doi:10.3791/52628

小分子変調分化とその後のグルコース飢餓を用いたヒト人工多能性幹細胞からの高度に精製された心筋細胞...

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Developmental Biology

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JoVE Journal Developmental Biology

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

小分子変調分化とその後のグルコース飢餓を用いたヒト人工多能性幹細胞からの高度に精製された心筋細胞の導出

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11:53 min

March 18, 2015

DOI: 10.3791/52628-v

Arun Sharma*¹, Guang Li*¹, Kuppusamy Rajarajan*¹, Ryoko Hamaguchi¹, Paul W. Burridge¹, Sean M. Wu^1,2

¹Stanford Cardiovascular Institute,Stanford University School of Medicine, ²Institute of Stem Cell Biology and Regenerative Medicine, Cardiovascular Medicine Division, Department of Medicine, Child Health Research Institute,Stanford University School of Medicine

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Overview

This article presents a robust protocol for deriving purified human cardiomyocytes from induced pluripotent stem cells. The method combines small molecule modulation for cardiac differentiation with glucose deprivation for cardiomyocyte purification, facilitating cardiovascular disease modeling and drug screening.

Key Study Components

Area of Science

Cardiovascular research
Stem cell biology
Drug screening methodologies

Background

Human induced pluripotent stem cells (iPSCs) can differentiate into cardiomyocytes.
Cardiomyocytes are essential for studying heart diseases.
Purification of cardiomyocytes is crucial for accurate modeling and drug testing.
Small molecules can effectively direct stem cell differentiation.

Purpose of Study

To develop a reliable protocol for generating purified cardiomyocytes.
To enhance the efficiency of cardiovascular disease modeling.
To improve drug screening processes using purified cell populations.

Methods Used

Culture of human iPSCs in monolayers until 85% confluent.
Application of small molecule compounds to induce mesoderm formation.
Sequential addition of compounds to specify cardiac mesoderm differentiation.
Incubation in low glucose medium for selective purification of cardiomyocytes.

Main Results

Successful differentiation of iPSCs into beating cardiomyocytes.
Purification of cardiomyocytes achieved through glucose deprivation.
Protocol enables the generation of a homogeneous cardiomyocyte population.
Facilitates improved modeling of cardiovascular diseases and drug testing.

Conclusions

The protocol provides a robust method for cardiomyocyte derivation.
Combining differentiation and purification enhances research capabilities.
Potential applications in cardiovascular disease modeling and pharmacology.

Frequently Asked Questions

What are induced pluripotent stem cells?

Induced pluripotent stem cells are adult cells reprogrammed to an embryonic stem cell-like state, allowing them to differentiate into various cell types.

Why is cardiomyocyte purification important?

Purification is crucial for ensuring that the cells used in research are homogeneous, which improves the reliability of experimental results.

How does glucose deprivation aid in purification?

Glucose deprivation selectively promotes the survival of cardiomyocytes while inhibiting the growth of other cell types, leading to a purer population.

What role do small molecules play in this protocol?

Small molecules are used to modulate signaling pathways that direct the differentiation of stem cells into cardiomyocytes.

Can this protocol be applied to other cell types?

While this protocol is specific to cardiomyocytes, similar strategies may be adapted for other cell types with appropriate modifications.

What are the implications of this research?

This research has significant implications for developing new therapies for cardiovascular diseases and improving drug discovery processes.

ここでは、低分子調節心分化とグルコース欠乏媒介心筋細胞精製を組み合わせたヒト心筋細胞誘導のための堅牢なプロトコルについて説明し、心血管疾患のモデリングと薬物スクリーニングの目的で精製された心筋細胞の生産を可能にします。

この手順の全体的な目標は、ヒト誘導多能性幹細胞由来心筋細胞の精製集団を生成することです。まず、ヒト人工多能性幹細胞を細胞単層で約85%コンフルエントになるまで培養します。次に、風細胞シグナル伝達経路を活性化する低分子化合物を添加し、細胞を2日間インキュベートして中胚葉の形成を誘導します。

次に、風細胞シグナル伝達経路を阻害する低分子化合物を細胞に添加して、心臓の中胚葉分化を特定します。その後、細胞をさらに2日間インキュベートします。7日目までに、分化した心筋細胞が不均一な分化細胞集団を打ち負かし、次に低グルコース細胞培養培地でインキュベートして、心筋細胞を選択的に精製します。

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