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Evaluation of ex vivo expansion potential of cord blood and bone marrow
hematopoietic progenitor cells using cell tracking and limiting dilution
analysis
CM Traycoff, ST Kosak, S Grigsby and EF Srour
Division of Hematology/Oncology, Indiana University School of Medicine,
Indianapolis 46202-5121.
In the absence of conclusive assays capable of determining the
functionality of ex vivo expanded human hematopoietic progenitor cells, we
combined cell tracking with the membrane dye PKH2, immunostaining for CD34,
and limiting dilution analysis to estimate the frequency of long-term
hematopoietic culture-initiating cells (LTHC-ICs) among de novo-generated
CD34+ cells. Umbilical cord blood (CB) and bone marrow (BM) CD34+ cells
were stained with PKH2 on day 0 and cultured with stem cell factor (SCF)
and interleukin-3 (IL-3) in short-term stromal cell- free suspension
cultures. Proliferation of CD34+ cells in culture was tracked through their
PKH2 fluorescence relative to day 0 and the continued expression of CD34.
As such, it was possible to identify cells that had divided while
maintaining the expression of CD34 (CD34+PKH2dim) and others that expressed
CD34 but had not divided (CD34+PKH2bright). In all such cultures, a
fraction of both BM and CB CD34+ cells failed to divide in response to
cytokines and persisted in culture for up to 10 days as CD34+PKH2bright
cells. Between days 5 and 7 of culture, CD34+PKH2bright and CD34+PKH2dim
cells were sorted in a limiting dilution scheme into 96-well plates
prepared with medium, SCF, IL-3, IL-6, granulocyte-macrophage
colony-stimulating factor, and erythropoietin. Cells proliferating in
individual wells were assayed 2 weeks later for their content of clonogenic
progenitors and the percentage of negative wells was used to calculate the
frequency of LTHC-ICs in each population. Among fresh isolated BM and CB
CD34+ cells, the frequencies of LTHC-ICs were 2.01% +/- 0.98% (mean +/-
SEM) and 7.56% +/- 2.48%, respectively. After 5 to 7 days in culture, 3.00%
+/- 0.56% of ex vivo-expanded BM CD34+PKH2bright cells and 4.46% +/- 1.10%
of CD34+PKH2dim cells were LTHC-ICs. In contrast, the frequency of LTHC-IC
in ex vivo expanded CB CD34+ cells declined drastically, such that only
3.87% +/- 2.06% of PKH2bright and 2.29% +/- 1.75% of PKH2dim cells were
determined to be initiating cells after 5 to 7 days in culture. However,
when combined with a calculation of the net change in the number of CD34+
cells in culture, the sum total of LTHC-ICs in both BM and CB cells
declined in comparison to fresh isolated cells, albeit to a different
degree between the two tissues.(ABSTRACT TRUNCATED AT 400 WORDS)
Volume 85,
Issue 8,
pp. 2059-2068,
04/15/1995
Copyright © 1995 by The American Society of Hematology
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