Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

Kyle  M. Loh; Amrita Palaria; Lay Teng Ang

doi:10.3791/58975

Differenziazione efficiente delle cellule staminali pluripotenti umane nelle cellule del fegato

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Developmental Biology

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JoVE Journal Developmental Biology

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

Differenziazione efficiente delle cellule staminali pluripotenti umane nelle cellule del fegato

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07:37 min

June 11, 2019

DOI: 10.3791/58975-v

Kyle M. Loh^1,2, Amrita Palaria¹, Lay Teng Ang¹

¹Institute for Stem Cell Biology & Regenerative Medicine, Stanford-UC Berkeley Siebel Stem Cell Institute,Stanford University School of Medicine, ²Department of Developmental Biology,Stanford University School of Medicine

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Overview

This protocol describes a serum-free method for generating hepatocyte-like cells from human pluripotent stem cells (hPSCs) in just 18 days. The process involves six steps of differentiation through various intermediate cell types.

Key Study Components

Area of Science

Stem Cell Biology
Regenerative Medicine
Cell Differentiation

Background

Human liver cells are crucial for studying liver diseases and drug metabolism.
Current methods for generating liver cells often yield low purity.
Efficient generation of hepatocyte-like cells can advance regenerative therapies.
This protocol aims to improve the purity and yield of liver progenitor cells.

Purpose of Study

To develop a high-purity method for generating hepatocyte-like cells from hPSCs.
To enhance the understanding of liver cell differentiation pathways.
To provide a reliable source of liver cells for research and therapeutic applications.

Methods Used

Sequential differentiation of hPSCs through defined stages.
Control of developmental signaling pathways to promote liver differentiation.
Use of serum-free culture conditions to enhance cell purity.
Preparation of PVA stock for medium formulation.

Main Results

High purity of hepatocyte-like cells achieved within 18 days.
Efficient generation of liver bud progenitors.
Demonstrated control over unwanted cell type formation.
Potential applications in regenerative medicine highlighted.

Conclusions

This method provides a reliable approach to generate liver cells from hPSCs.
High purity and yield can facilitate research in liver biology.
Future studies may explore therapeutic applications of these cells.

Frequently Asked Questions

What are hepatocyte-like cells?

Hepatocyte-like cells are cells that mimic the function and characteristics of liver cells, derived from stem cells.

How long does the differentiation process take?

The differentiation process takes 18 days to generate hepatocyte-like cells.

What is the significance of using serum-free conditions?

Serum-free conditions help improve the purity of the generated liver cells and reduce variability.

Can this method be applied to other cell types?

While this method is specific for liver cells, similar techniques may be adapted for other cell types.

What are the potential applications of these hepatocyte-like cells?

They can be used in drug testing, disease modeling, and regenerative medicine.

What are the main challenges in generating liver cells from hPSCs?

Challenges include achieving high purity and controlling differentiation pathways effectively.

Questo protocollo descrive in dettaglio un metodo monostrato e privo di siero-per generare in modo efficiente cellule simili a epatociti da cellule staminali pluripotenti umane (hPSC) in 18 giorni. Ciò comporta sei passaggi in quanto gli hPSC si differenziano in sequenza in tipi di cellule intermedie come la striscia primitiva, l'endoderma definitivo, il foregut posteriore e i progenitori del germoglio epatico prima di formare cellule simili a epatociti.

Questo metodo consente la generazione di un gran numero di progenitori di gemme epatiche umane e cellule simili a epatociti con elevata purezza. La capacità di generare queste cellule del fegato potrebbe essere importante per la medicina rigenerativa e altri campi. Controllando le vie di segnalazione dello sviluppo per promuovere la differenziazione epatica e sopprimere la formazione di tipi di cellule indesiderate, questo metodo consente una generazione efficiente di progenitori di gemme epatiche umane e cellule simili a epatociti rispettivamente di sei e 18 giorni di differenziazione.

Il principale vantaggio di questa tecnica è l'elevata purezza delle cellule epatiche umane generate. Per essere questa procedura, aggiungere 50 millilitri di IMDM a un pallone conico contenente una barra di agitazione. Riscaldare il mezzo a 50 gradi Celsius e aggiungere 0,5 grammi di PVA mescolando continuamente per preparare uno stock di PVA a una concentrazione di 10 milligrammi per millilitro.

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