Isolation of Bone Marrow–Derived Stem Cells using Density-Gradient Separation

Objective Our laboratory has established two unique methods to isolate murine hematopoietic stem cells on the basis of functional characteristics such as the ability of stem cells to home to bone marrow and aldehyde dehydrogenase (ALDH) activity. An essential component of both protocols is the separ...

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Bibliographic Details
Published in:Experimental hematology 2007-02, Vol.35 (2), p.335-341
Main Authors: Juopperi, Tarja A, Schuler, William, Yuan, Xuan, Collector, Michael I, Dang, Chi V, Sharkis, Saul J
Format: Article
Language:English
Subjects:
Advanced Basic Science
Aldehyde Dehydrogenase - immunology
Animals
Bone Marrow Cells - cytology
Bone Marrow Cells - enzymology
Bone Marrow Cells - radiation effects
Cell Separation - methods
Centrifugation, Density Gradient - methods
Enzyme Activation
Female
Hematology, Oncology and Palliative Medicine
Hematopoietic Stem Cell Transplantation
Hematopoietic Stem Cells - cytology
Hematopoietic Stem Cells - enzymology
Hematopoietic Stem Cells - radiation effects
Male
Mice
Mice, Inbred C57BL
Survival Rate
Transplantation Conditioning
Transplantation, Homologous
Whole-Body Irradiation
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Summary:Objective Our laboratory has established two unique methods to isolate murine hematopoietic stem cells on the basis of functional characteristics such as the ability of stem cells to home to bone marrow and aldehyde dehydrogenase (ALDH) activity. An essential component of both protocols is the separation of whole bone marrow into small-sized cells by counter-flow elutriation. We sought to provide the scientific community with an alternate approach to acquire our stem cells by replacing elutriation with the use of density-gradient centrifugation. Methods The elutriated fraction 25 population was characterized based on density using a discontinuous gradient. The long-term reconstituting potential of whole bone marrow cells collected at each density interface was determined by subjecting the fractions to the two-day homing protocol, transplanting them into lethally irradiated recipient mice, and assessing peripheral blood chimerism. We also investigated the ability of high-density bone marrow cells isolated in conjunction with the ALDH protocol to repopulate the hematopoietic system of myeloablated recipients. Results Bone marrow cells collected at the high-density interface of 1.081/1.087 g/mL (fraction 3) had the capacity for homing to marrow and the ability to provide long-term hematopoietic reconstitution. Fraction three lineage-depleted ALDH-bright cells could also engraft and provide long-term hematopoiesis at limiting dilutions. Conclusions Density-gradient centrifugation can be used in conjunction with either of our stem cell isolation protocols to obtain cells with long-term reconstitution ability. We anticipate that this strategy will encourage and enable investigators to study the biology of HSCs isolated using functional characteristics.
ISSN:0301-472X
1873-2399
DOI:10.1016/j.exphem.2006.09.014