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 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.
Juopperi
Tarja A
TA
Graduate Program of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD 21231, USA.
Schuler
William
W
Yuan
Xuan
X
Collector
Michael I
MI
Dang
Chi V
CV
Sharkis
Saul J
SJ
D000444:Aldehyde Dehydrogenase / Q000276:immunology; D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000201:enzymology / Q000528:radiation effects; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000379:methods; D004789:Enzyme Activation; D005260:Female; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000201:enzymology / Q000528:radiation effects; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D015996:Survival Rate; D019172:Transplantation Conditioning; D014184:Transplantation, Homologous; D014916:Whole-Body Irradiation
D000444:Aldehyde Dehydrogenase