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Related Experiment Videos

Isolation of bone marrow-derived stem cells using density-gradient separation.

Tarja A Juopperi1, William Schuler, Xuan Yuan

  • 1Graduate Program of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.

Experimental Hematology
|January 30, 2007
PubMed
Summary
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Density-gradient centrifugation offers an alternative to elutriation for isolating functional hematopoietic stem cells (HSCs). This method successfully isolates HSCs with long-term reconstitution potential, enabling further study of these vital cells.

Area of Science:

  • Hematology
  • Stem Cell Biology
  • Immunology

Background:

  • Established methods for isolating murine hematopoietic stem cells (HSCs) rely on functional characteristics like homing ability and aldehyde dehydrogenase (ALDH) activity.
  • A key step in these protocols involves separating whole bone marrow into small-sized cells using counter-flow elutriation.

Purpose of the Study:

  • To develop an alternative method for isolating functional HSCs by replacing counter-flow elutriation with density-gradient centrifugation.
  • To validate the efficacy of density-gradient centrifugation in isolating HSCs with long-term reconstituting potential.

Main Methods:

  • Whole bone marrow cells were separated using discontinuous density-gradient centrifugation.
  • Fractions were assessed for their ability to home to bone marrow and provide long-term hematopoietic reconstitution in recipient mice.

Related Experiment Videos

  • The ALDH protocol was combined with density-gradient centrifugation to evaluate the repopulation capacity of high-density bone marrow cells.

    • Main Results:

    • Bone marrow cells isolated at a high-density interface (1.081/1.087 g/mL, fraction 3) demonstrated homing capacity and long-term hematopoietic reconstitution potential.
    • Lineage-depleted, ALDH-bright cells from fraction three also successfully engrafted and provided long-term hematopoiesis.

    Conclusions:

    • Density-gradient centrifugation is a viable alternative to elutriation for isolating HSCs with long-term reconstitution ability.
    • This simplified approach facilitates the study of HSCs isolated based on functional characteristics, promoting broader research in the field.