Evaluation of ex vivo expansion potential of cord blood and bone marrow hematopoietic progenitor cells using cell tracking and limiting dilution analysis

Abstract

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)

Primitive human HPCs are better maintained and expanded in vitro at 1 percent oxygen than at 20 percent

“…This increased macrophage and megakaryocyte differentiation in hypoxia could be of interest for the in vitro amplification of committed progenitors and maturing cells for autografting. Quiescent cytokine-unresponsive cells, [18][19][20] characterized by the CD34+PKH2 bright phenotype after 7 days of liquid culture, correspond to primitive HPCs surviving an in vitro treatment with 5FU for several days. 21,22 It is surprising that their number was lower at 1-percent O 2 than at 20percent O 2 .…”

“…Membrane dye PKH2 was used essentially as described previously. 18 Briefly, thawed PBPCs were incubated for 5 minutes with PKH2 dye (Sigma, St. Louis, MO) according to the manufacturer's instructions, washed extensively, and resuspended in serum-free medium (StemBIO-A). PKH2 fluorescence of total viable cells, as well as of CD34+ cells, was measured with a flow cytometer (XL) immediately after labeling.…”

The anti‐apoptotic genes Bcl‐XL and Bcl‐2 are over‐expressed and contribute to chemoresistance of non‐proliferating leukaemic CD34+ cells

“…Cell division analysis. AML or normal CD34 + cells were stained with PKH-26-GL (Sigma Co.) as per manufacturer's instructions and as previously described (Traycoff et al, 1995). Briefly, cells were suspended in 1 ml of diluent and immediately transferred into a polypropylene tube containing 1 ml of 4 · 10 )6 mol/l PKH26 in diluent at room temperature.…”

The purification of CD34+ cells from human cord blood: comparison of separation techniques and cytokine requirements for optimal growth of clonogenic progenitors

“…Moreover, they appear to be appropriate candidates for transduction using retroviral vectors (Lu et al, 1993) where high multiplicity of infection and cytokine-induced entry into cycle can be achieved. CD34 cells are also appropriate targets for precursor and progenitor amplification with eventual increase in the CD34 cell compartment itself (Traycoff et al, 1994(Traycoff et al, , 1995. The aim of this study was to evaluate different means of separating CD34 cells that could be later used in a clinical setting at reasonable cost.…”