Isolation Method for Long-Term and Short-Term Hematopoietic Stem Cells

Katsuyuki Nishi; Akiomi Nagasaka; Taro Sakamaki; Kay Sadaoka; Masanori Miyanishi

doi:10.3791/64488

Método de Isolamento de Células-Tronco Hematopoéticas de Longa e Curta Duração

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Isolation Method for Long-Term and Short-Term Hematopoietic Stem Cells

Método de Isolamento de Células-Tronco Hematopoéticas de Longa e Curta Duração

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06:41 min

May 19, 2023

DOI: 10.3791/64488-v

Katsuyuki Nishi^1,2, Akiomi Nagasaka^1,2, Taro Sakamaki^1,2, Kay Sadaoka^1,2, Masanori Miyanishi^1,2

¹Hematopoietic Stem Cell Biology and Medical Innovation (HSCBMI), Department of Pediatrics,Kobe University Graduate School of Medicine, ²RIKEN Center for Biosystems Dynamics Research

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Overview

This study presents a detailed protocol for isolating long-term hematopoietic stem cells (LT-HSCs) and short-term HSCs (ST-HSCs) using a Hoxb5 reporter system. The isolation method enhances the understanding of cell renewal mechanisms and the biological basis of heterogeneity within the HSC compartment.

Key Study Components

Research Area

Hematopoietic stem cell biology
Cell renewal mechanisms
Stem cell heterogeneity

Background

Hoxb5 identified as a potential exclusive marker for LT-HSCs.
Establishment of Hoxb5-reporter mice for targeted isolation.
Importance of isolating specific HSC populations for better understanding.

Methods Used

Bone marrow isolation techniques.
Flow cytometry for cell sorting.
P93-based analysis for assessing self-renewal capacity.

Main Results

Successful isolation of Hoxb5-positive and Hoxb5-negative pHSCs.
Demonstrated the self-renewal capacity differences post-transplantation.
Evidence that Hoxb5-negative pHSCs lose hematopoietic potential over time.

Conclusions

This protocol aids in studying HSC characteristics and behavior.
Highlights the significance of Hoxb5 in understanding stem cell functionality.

Frequently Asked Questions

What are hematopoietic stem cells?

Hematopoietic stem cells (HSCs) are the progenitor cells that give rise to all blood cell types.

Why is the Hoxb5 marker significant?

Hoxb5 is potentially an exclusive marker of long-term hematopoietic stem cells, aiding in their identification and isolation.

How long does the isolation procedure take?

The protocol typically takes 9 to 12 hours to complete.

What is the relevance of self-renewal in stem cells?

Self-renewal is critical for maintaining the stem cell population and ensuring continual blood cell production.

How does temperature affect cell viability during the protocol?

Maintaining samples at 4 degrees Celsius helps to preserve cell viability throughout the isolation process.

What technologies are used in this research?

The study utilizes flow cytometry for cell sorting and analysis.

Apresentamos um protocolo passo a passo para o isolamento de células-tronco hematopoéticas de longa duração (CT-HSCs) e CTHs de curta duração (CTH-ST) utilizando o sistema repórter Hoxb5.

O método de isolamento de células-tronco hematopoéticas ou HSC de longo e curto prazo ajudará os pesquisadores a entender melhor os mecanismos de renovação celular e a base biológica da heterogeneidade no compartimento de CTH. Um fator de transcrição, Hoxb5, que pode ser um marcador exclusivo de HSCs de longa duração, foi identificado. Com base nisso, uma linhagem de camundongos Hoxb5-repórter foi estabelecida e as HSCs de longo e curto prazo foram isoladas com sucesso.

Para iniciar a coleta das células da medula óssea do doador, esterilize uma argamassa e pilão com etanol 70% e deixe-os secar completamente. Equilibre aqueles com tampão de coloração celular composto por suplementos contendo PBS livre de cálcio e magnésio. Coloque os ossos isolados do camundongo Hoxb5-tri-mCherry macho doador na argamassa e adicione três mililitros do tampão de coloração celular.

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