Judy Carol Young

CD34+ cells from mobilized peripheral blood retain fetal bone marrow repopulating capacity within the Thy-1+ subset following cell division ex vivo

Ex vivo cell cycling of hematopoietic stem cells (HSC), a subset of primitive hematopoietic progenitors (PHP) with engrafting capacity, is required for transduction with retroviral vectors and to increase transplantable HSC numbers. However, induction of division of HSC ex vivo also may lead to differentiation and loss of in vivo marrow repopulating potential. We evaluated mobilized peripheral blood (MPB) PHP for maintenance of stem cell function after ex vivo culture under conditions that we show can induce cycling of a majority of PHP with minimal differentiation. The following methods were combined: cell labeling with the division tracking dye carboxyfluorescein-diacetate succinimidylester (CFSE), analysis of primitive cell surface marker expression, an ex vivo PHP assay, and an in vivo marrow repopulating assay. MPB-purified CD34+ Thy-1+ cells were labeled with CFSE dye and cultured for 112 hours in serum-deprived medium in the presence of the cytokine combinations of thrombopoietin (TPO), flt3 ligand (FL), and c-kit ligand (KL), or TPO, FL, and interleukin 6 (IL-6). Both cytokine combinations supported division of greater than 95% of cells within 112 hours with an average 2.1-fold (TPO, FL, KL) or 1.3-fold (TPO, FL, IL-6) increase in total cell numbers. An average of 21.6% (TPO, FL, KL) and 27.4% (TPO, FL, IL-6) of the divided cells still expressed the Thy-1 marker after 112 hours. Functional assays were performed to compare cultured and uncultured cells. CD34+ Thy-1+ CFSElo (post division) cells showed maintenance of cobblestone area-forming cell (CAFC) frequency (a mean of 1/9.0) relative to the starting population of uncultured CD34+ Thy-1+ cells (a mean of 1/8.4). In contrast, CD34+ cells that had lost Thy-1 expression during culture (CD34+ Thy-1 CFSElo) showed a mean 5.8-fold reduction in CAFC frequency (a mean of 1/52.5). Only the Thy-1-expressing fraction of cells post culture could engraft in vivo in the SCID-hu bone assay. Because the majority of HSC functional activity post culture was found in the CD34+ Thy-1+ fraction, we focused on this fraction for subsequent analysis. CFSE labeling allows segregation and purification by flow cytometry of cells having undergone discrete numbers of divisions during culture. Very few cells that divided more than four times in culture still expressed Thy-1. Cells that retained expression of Thy-1 during culture retained CAFC activity relative to fresh CD34+ Thy-1+ cells, after undergoing at least two divisions. CAFC frequency decreased after four divisions in culture with TPO, FL, and KL or after three divisions in TPO, FL, and IL-6. We then compared populations of Thy-1+ cells that had undergone sequential numbers of divisions in culture for their ability to engraft in the SCID-hu bone assay. Engrafting ability was retained throughout four divisions in both cytokine combinations. These data demonstrate that primitive MPB CD34+ cells maintain HSC function coincident with Thy-1 expression while undergoing two to four divisions under these culture conditions. Essentially all CD34+ Thy-1+ cells divided under the conditions tested, promoting susceptibility to retroviral transduction.

Investigation into an engraftment defect induced by culturing primitive hematopoietic cells with cytokines

BACKGROUND Strategies for transplanting primitive hematopoietic progenitor (PHP) cells are under development that require in vitro manipulation of cells for several hours to several days prior to transplantation. This applies to gene-therapy protocols involving transduction with adenoviral or lentiviral vectors (typically 1 day of ex vivo culture) or retroviral vectors (up to 3 days of culture). METHODS Human mobilized peripheral blood (MPB) CD34(+) cells were cultured with the cytokines thrombopoietin mimetic peptide (mTPO), flt3 ligand (FL), and c-kit ligand (KL). Equal numbers of CD34(+) cells, either uncultured or cultured for various time periods up to 5 days, were tested for engraftment in sublethally irradiated 8-10 week-old NOD/SCID mice. Cells were also compared for expression and function of several key surface molecules. RESULTS At a limiting dose of 1 million cells, mice receiving uncultured cells had a mean of 20% CD45(+) (human) cells in their BM 6 weeks post-transplantation, versus 3% for mice receiving 3-5 day cultured cells. Analysis of 10 surface molecules, CD11a, CD18, CD29, CD49d, CD49e, CXCR-4, CD62L, CD31, CD43, and CD44 over a 5-day culture period showed that their expression levels were either maintained or up-regulated on CD34(+) cells and the primitive Thy-1(+) subset. Similar percentages of uncultured and 3-day cultured MPB CD34(+) cells bound to plates coated with vascular cell adhesion molecule-1 (VCAM-1) under both static and physiological flow conditions, and chemotaxis of cultured cells towards stromal-derived factor-1 (SDF-1) was not impaired, suggesting that VLA-4 and CXCR-4 were functional on cultured cells. CD34(+) Thy-1(+) MPB cells cultured with cytokines expressed increasing levels of Fas receptor beginning at 20 h in culture, with peak expression levels after 3 days (mean Day 0 expression, 39%; mean Day 3 expression, 86%), without increased apoptosis. Including inhibitors of caspases in the media of cells cultured for 24-48 h significantly improved their engraftment in a SCID-hu bone-engraftment model. DISCUSSION Increased susceptibility to apoptosis upon in vivo injection may contribute to impaired engraftment of in vitro manipulated cells. Inhibitors of apoptosis may increase their engrafting capacity in clinical settings.

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 Iscoves 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)). DISCUSSION 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.