Alison Rice

Cord blood is better than bone marrow for generating megakaryocytic progenitor cells

Thrombocytopenia remains an important problem for patients post high-dose chemotherapy and hematopoietic stem cell transplantation. The study of megakaryocytes, the direct precursors of platelets, has been hampered by their relatively low frequency in hematopoietic tissues. In an attempt to obtain a large number of functional megakaryocytic cells, we established a serum-free culture system to grow megakaryocytic progenitor cells derived from normal human bone marrow (BM) and cord blood (CB). Highly purified (purity >95%) CD34+ cells were obtained using magnetic cell sorting (MACS) followed by fluorescence activated cell sorting (FACS). The cells were cultured in a serum-free culture system for 3 weeks in the presence of a single dose of MGDF (50 ng/mL). On days 0, 5, 8, 12, 14, 18, and 21 of culture, the cellularity and morphology were examined. Megakaryocytic cells were monitored by detecting the expression of GPIIIa (CD61), GPIIb/IIIa (CD41) and GPIb (CD42b), and the distribution of megakaryocyte (MK) ploidy was analyzed by two-color flow cytometry. MGDF alone induced maximal nucleated cell expansion at day 14, resulting in a 38.20+/-10.47-fold increase in cell number for CB and a 5.08+/-1.30-fold increase in cell number for BM. On day 14 of the culture, the percentage of CD41-/CD14- cells derived from CB reached 73.54%+/-6.01% giving an absolute number of CD41+/CD14- cells of 27.25+/-2.23 x 10(4)/mL (27,250-fold increase), whilst the percentage of CD41+/CD14- cells derived from BM was only 29.21%+/-5.63% with an absolute number of 1.36+/-0.26 x 10(4)/mL (680-fold increase). Increased expression of GPIIIa occurred the earliest in culture, followed by GPIIb/IIIa, and then GPIb. The majority (81.6%-92.6%) of megakaryocytes (CD41+ cells) on day 14 of culture were 2N, although we did detect some 4N, 8N and greater ploidy cells. In conclusion, CD34+ cells stimulated by MGDF alone generated highly enriched MK progenitor cells at day 14 of serum-free culture. CB stem and progenitor cells have a greater proliferative response to MGDF alone than those derived from BM and may, therefore, prove to be a better source of cells for MK expansion.

Characterization of cytokine interactions by flow cytometry and factorial analysis.

BACKGROUND Multiple cytokines are required for the growth and development of hematopoietic cells. The effect of many cytokines depends on the activity of other signaling pathways. These interactions are quantified using factorial experimental design and analysis. METHODS Human umbilical cord blood (HUCB) CD34+ cells were cultured in fully defined media containing various combinations of recombinant cytokines as defined by resolution IV factorial (2(7-3)(IV)) or full factorial (2(4)) design experiments. The cytokines studied were stem cell factor (SCF), interleukin (IL)-3, megakaryocyte growth and development factor (MGDF), granulocyte-colony stimulating factor (G-CSF), Flt-3 ligand, IL-6, IL-11, and erythropoietin (EPO). In vitro cell divisions were tracked by staining CD34+ cells with 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester, followed by flow cytometric analysis at 4 days of culture. In separate experiments, lineage commitment and differentiation were determined at 7 days by immunophenotype. RESULTS In addition to the main effects of single cytokines, cytokine interactions were identified. There was a negative interaction between IL-3 and MGDF that resulted in a less than additive effect of these factors on erythroid and megakaryocytic development. The effect of Flt-3 ligand and SCF factor on CD34+ cell production was also less than additive, although the response to both cytokines was greater than single cytokines. The only positive interaction that was identified was between EPO and SCF, which resulted in the synergistic production of erythroid cells. CONCLUSIONS Factorial analysis provides a powerful methodology to study the integration of multiple signals at the cellular and molecular level.

Comparative study of the in vitro behavior of cord blood subpopulations after short-term cytokine exposure.

We investigated the effect of short-term cytokine exposure on defined cord blood subpopulations. CD34+Thy1+, CD34+Thy1−, CD34+38−, CD34+38+, CD34+DR+, CD34+DR−, CD34+Rhodamine123 (Rh123)− and CD34+Rh123+ cells were incubated for 7 days in IMDM + 10% FCS + IL3 + IL6 + G-CSF + SCF (36GS) ± flt3L. We evaluated LTHC-IC, immunophenotype and nucleated cell count for each cell population before and after cytokine exposure. Short-term exposure of CD34+38+, CD34+Thy1−, CD34+DR+, CD34+DR− and CD34+Rh123+ cells to 36GS causes a significant increase in cell number, whereas CD34+38−, CD34+Thy1+, and CD34+Rh123− cells show only a limited increase. CD34 status post cytokine incubation shows that CD34+38+, CD34+Thy1−, CD34+DR+, and CD34+Rh123+ fractions have a lower proportion of cells remaining CD34+ than CD34+38− CD34+Thy1+, CD34+DR− and CD34+Rh123− fractions. LTHC-IC analyses among input subpopulations show a higher frequency among CD34+38+, CD34+Thy1−, CD34+DR+, CD34+DR−and CD34+Rh123+ cells as compared with CD34+38−, CD34+Thy1+ and CD34+Rh123− cells. However, when LTHC-IC were evaluated after cytokine exposure, CD34+38−, CD34+Thy1+, and CD34+Rh123− cells showed a higher frequency of LTHC-IC as compared with other subpopulations. Addition of flt3L to 36GS doubled the numbers in all subpopulations without altering the proportion of CD34+ cells. Results suggest that CD34+38−, CD34+Thy1+ and CD34+Rh123− cells have a limited proliferative response to cytokines, the stem cell component of these populations is largely maintained and that expansion is derived from mature cell populations.

Hematopoietic stem cell expansion

This review addresses two issues: (1) cytokine-mediated expansion of functional end cells for the abrogation of short term neutropenia and thrombopenia following high dose chemotherapy and (2) cytokine-driven increase in absolute numbers of functional stem cells. The literature suggests that the short term exposure of CD34+ cells to cytokines produces mature progenitors which in turn give rise to functional neutrophils and platelets. The expansion of functional stem cells is a more complex issue, as primitive stem cells are quiescent and their growth requirements are less clearly defined.

Prior Cryopreservation of Ex Vivo-Expanded Cord Blood Cells Is Not Detrimental to Engraftment as Measured in the NOD-SCID Mouse Model

Cytokine-mediated expansion has been proposed and successfully used to facilitate engraftment post transplantation. This study examined whether cryopreservation following expansion has a detrimental effect on the ability of cells to engraft, using the NOD-SCID mouse model. Cord blood (CB) CD34(+) cells were incubated for 7 days with stem cell factor (SCF), flt-3 ligand (FL), and megakaryocyte growth and development factor (MGDF). Expanded CD34(+) cells were transplanted into NOD-SCID mice either fresh or following cryopreservation and thawing. After thawing, recovery of nucleated cells was 94%, of CD34 cells was 63%, and of day-14 progenitors was 17%. The loss of day-14 progenitor cells among the thawed expanded cells did not influence the kinetics of human engraftment in the mouse. Bone marrow (BM) of mice transplanted with thawed expanded CD34(+) cells (14 +/- 3.9%) showed significantly higher levels of human engraftment than mice transplanted with fresh expanded CD34(+) cells (1.5 +/- 0.5%, p = 0.0064). Thawed expanded CD34(+) cells had significantly higher SCID Engrafting Potential (SEP) than freshly expanded CD34(+) cells (p < 0.001). Results suggest that prior cryopreservation does not prevent expanded cells engrafting in NOD-SCID mice.