Isolation and Maintenance of Hematopoietic Stem Cell

Hematopoietic stem cells (HSCs) play a crucial role in replenishing the entire blood system throughout an individual's lifetime. These rare and multipotent cells have the remarkable ability to self-renew and differentiate into various blood cell lineages. The isolation and maintenance of HSCs are of paramount importance for understanding their biology, studying hematopoiesis, and advancing therapeutic applications.

Creative Biolabs offers a comprehensive protocol for the isolation and maintenance of HSCs. This protocol provides a detailed guide for the successful isolation and long-term maintenance of HSCs, ensuring their optimal functionality.

Materials Required

• Mouse or other bone marrow samples
• Cell culture media
• Fetal bovine serum
• Penicillin-streptomycin solution
• L-Glutamine
• Heparin
• Antibodies: anti-CD34, anti-CD45, anti-Sca-1, anti-c-Kit (CD117), anti-CD48, anti-CD150, anti-CD16/32, and appropriate isotype controls.
• Lymphocyte separation medium (LSM)
• Phosphate-buffered saline (PBS)

Procedure

Isolation of HSCs

Prepare the cell suspension by flushing the bone marrow. Centrifuge the cell suspension and carefully aspirate the supernatant to resuspend the cell pellets in PBS. Cover the cell suspension with an equal volume of LSM in a sterile tube. Centrifuge again, carefully collect the mononuclear cell layer and transfer it to a new tube. Wash the collected cells with PBS, centrifuge and discard the supernatant, and resuspend the cell pellets in PBS.

Immunophenotyping of HSCs

Aliquot the cell suspensions into different tubes according to the desired antibody plate. Add the appropriate antibody and isotype control to each tube and incubate in the dark. Then centrifuge and wash several times. Then, resuspend the cell pellet in PBS and perform flow cytometry analysis using a FACS machine to identify the hematopoietic stem cell population according to the specific antibody plate.

Isolation of HSCs by Fluorescence-Activated Cell Sorting

Aliquot the cell suspension into different tubes, taking into account the desired purity and cell concentration. Add antibodies against hematopoietic stem cell-specific surface markers and incubate in the dark. Then centrifuge and wash several times. Then resuspend the cell pellets in the appropriate medium. Sort the hematopoietic stem cell population according to the expression pattern of specific surface markers using a FACS machine.

Maintenance of HSCs

Load the sorted hematopoietic stem cells into culture dishes, such as 6-well plates or culture flasks, and coat with the appropriate extracellular matrix. Incubate the culture dishes in a humid environment. Refresh the medium every 2-3 days and replace half of the medium with fresh complete cell culture medium.

Notes

• Handle the bone marrow samples and isolated cells under aseptic conditions to prevent contamination.
• Maintain consistent temperature and humidity conditions during cell culture.
• Always use appropriate isotype controls to set appropriate gating and compensate for fluorescence overlap.
• Avoid excessive pipetting or shaking, as it may compromise HSC viability and functionality.
• Characterization and differentiation experiments are essential to confirm the identity and functionality of glial stem cells.

Researchers can follow this protocol and obtain purified HSC populations and establish long-term cultures, ultimately advancing our understanding of hematopoiesis and facilitating therapeutic developments in the field of regenerative medicine.

If you have any questions or comments, please do not hesitate to contact us.

References

1. Morrison S J and Scadden D T. The bone marrow niche for haematopoietic stem cells. Nature, 2014, 505(7483): 327-334.
2. Laurenti E and Göttgens B. From haematopoietic stem cells to complex differentiation landscapes. Nature, 2018, 553(7689): 418-426.

For Research Use Only. Not For Clinical Use.