Quantitation of the proliferative potential of highly enriched human primitive hematopoietic progenitor cells using a stroma-free limiting dilution assay with automated scoring.
BACKGROUND
Increasing use of phenotypically-enriched stem cell populations for clinical hematopoietic transplants has led to an urgent demand for a reliable, rapid and simple functional assay which would provide an estimation of the reconstituting potential of cells prior to transplantation.


METHODS
We have developed a 2-week quantitative, stroma-free assay to measure the frequency of primitive progenitors within hematopoietic cell samples. This relatively short-term assay provides frequency information which correlates with that measured by a 5-week stroma-dependent CAFC assay. Cells with the phenotype CD34+Thy-1+ were purified by fluorescence-activated cell sorting from peripheral blood apheresis products of multiple myeloma patients mobilized with cytoxan and GM-CSF. CD34+Thy-1+ cells were plated at limiting dilution into microtiter wells and cultured in an Iscove's based serum-deprived culture medium, supplemented with the cytokines, interleukin (IL)-3, IL-6, G-CSF, Flk2/Flt3 ligand (FL) and Kit ligand (KL). After 2 weeks, cell proliferation in individual wells was quantified by microscopy and bright-field imaging, or by using a fluorescent nucleic acid-binding dye and fluorimetry. Poisson statistics were used to calculate the frequency of wells containing cells with high proliferative potential (wells containing > or = 500 cells).


RESULTS
Progenitor cell frequencies generated using this assay were compared by linear regression analysis to those generated from 32 parallel CAFC and CFU-C assays performed on the same patient samples. Correlations were r = 0.80, r2 = 0.65, and r = 0.76, r2 = 0.58, respectively; these correlations were highly significant (p < 10(-7)).


CONCLUSIONS
This limiting dilution assay should more directly quantitate the potential of primitive hematopoietic cells than a CFU-C assay. It also has advantages over both the CAFC and the CFU-C assay, in that scoring has been automated, making it simple, rapid, and objective compared with manual cobblestone area or colony counting. The described limiting dilution assay may provide a useful alternative to assays currently used to evaluate the viability and proliferative potential of purified hematopoietic cells intended for transplant.
Young
J C
JC
SyStemix Inc., Palo Alto, CA, USA.
Wu
S
S
Travis
M
M
Luens
K M
KM
Osborne
L
L
Scollay
R
R
Hill
B
B
D018952:Antigens, CD34 / Q000032:analysis; D001781:Blood Component Removal; D049109:Cell Proliferation; D002469:Cell Separation; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay / Q000379:methods*; D003520:Cyclophosphamide / Q000627:therapeutic use; D005434:Flow Cytometry; D005470:Fluorometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000627:therapeutic use; D019650:Hematopoietic Stem Cell Mobilization; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000276:immunology; D006801:Humans; D007090:Image Interpretation, Computer-Assisted; D016130:Immunophenotyping; D008853:Microscopy; D009101:Multiple Myeloma / Q000097:blood / Q000276:immunology / Q000628:therapy*; D018800:Thy-1 Antigens / Q000032:analysis; D013997:Time Factors
D018952:Antigens, CD34; D018800:Thy-1 Antigens; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D003520:Cyclophosphamide