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

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

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Biology

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JoVE Journal Biology

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

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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

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.

We present a step-by-step protocol for the isolation of long-term hematopoietic stem cells (LT-HSCs) and short-term HSCs (ST-HSCs) using the Hoxb5 reporter system.

The long-term and short-term hematopoietic stem cell or HSC isolation method will help researchers better understand the cell renewal mechanisms and biological basis of heterogeneity in HSC compartment. A transcription factor, Hoxb5, which may be an exclusive marker of long-term HSCs, was identified. Based on this, a Hoxb5-reporter mice line was established and the long-term and the short-term HSCs were successfully isolated.

To begin the collection of the donor bone marrow cells, sterilize a mortar and pestle with 70%ethanol and let them dry completely. Equilibrate those with cell-staining buffer comprised of calcium and magnesium-free PBS-containing supplements. Put the bones isolated from the donor male Hoxb5-tri-mCherry mouse in the mortar and add three milliliters of the cell-staining buffer.

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