Developing a co-culture system for effective megakaryo/thrombopoiesis from umbilical cord blood hematopoietic stem/progenitor cells (original) (raw)
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Co-infusion of ex-vivo generated megakaryocytic progenitors with hematopoietic stem/progenitor cells (HSC/HPC) may contribute to a faster platelet recovery upon umbilical cord blood (UCB) transplantation. A two stage protocol containing cell expansion and megakaryocyte (Mk) differentiation was established using human UCB CD34+-enriched cells. The expansion stage used a pre-established protocol supported by a human bone marrow mesenchymal stem cells (MSC) feeder layer and the differentiation stage used TPO (100 ng/mL) and IL-3 (10 ng/mL). 18% of culture-derived Mks had higher DNA content (>4 N) and were able to produce platelet-like particles. The proliferation extent of CD34+ cells obtained in the expansion stage (FI-CD34+), rather than expansion duration, determined as a key parameter for efficient megakaryocytic differentiation. A maximum efficiency yield (EY) of 48 ± 7.7 Mks/input CD34+ cells was obtained for a FI-CD34+ of 17 ± 2.5, where a higher FI-CD34+ of 42 ± 13 resulted in a less efficient megakaryocytic differentiation (EY of 22 ± 6.7 and 19 ± 4.6 %CD41).
Journal of Hematotherapy & Stem Cell Research, 2001
The cells were cultured with four cytokines: interleukin-3 (IL-3), thrombopoietin (TPO), stem cell factor (SCF), and Flt-3); five cytokines, IL-3, TPO, SCF, Flt-3 plus granulocyte-macrophage colonystimulating factor (GM-CSF), or erythropoietin (Epo); or all six cytokines in combination. After 16 days, significant expansion of MK precursors (CD41 1 ) and stem cells (CD34 1 and AC133 1 cells) were seen in cells cultured in IL-3, TPO, SCF, and Flt-3 with or without GM-CSF compared to the combinations that contained Epo (p , 0.05). Similar studies were performed using liquid culture medium, and after 14 days the number of MNCs, CD34 1 , AC133 1 , CD41 1 , and CD61 1 cells were higher in the UCB cells cultured in IL-3, TPO, SCF, and Flt-3 compared to those cultured with those four cytokines plus GM-CSF. These results demonstrate that UCB stem cells can be effectively expanded ex vivo and enriched with platelet precursors using TPO, SCF, Flt-3, and IL-3, whereas the addition of Epo and GM-CSF is unnecessary.
Ex Vivo Large-Scale Generation of Human Platelets from Cord Blood CD34 + Cells
Stem Cells, 2006
In the present investigation, we generated platelets (PLTs) from cord blood (CB) CD34 ؉ cells using a three-phase culture system. We first cultured 500 CB CD34 ؉ cells on telomerase gene-transduced human stromal cells (hTERT stroma) in serum-free medium supplemented with stem cell factor (SCF), Flt-3/Flk-2 ligand (FL), and thrombopoietin (TPO) for 14 days. We then transferred the cells to hTERT stroma and cultured for another 14 days with fresh medium containing interleukin-11 (IL-11) in addition to the original cytokine cocktail. Subsequently, we cultured the cells in a liquid culture medium containing SCF, FL, TPO, and IL-11 for another 5 days to recover PLT fractions from the super-natant, which were then gel-filtered to purify the PLTs. The calculated yield of PLTs from 1.0 unit of CB (5 ؋ 10 6 CD34 ؉ cells) was 1.26 ؋ 10 11 ؊1.68 ؋ 10 11 PLTs. These numbers of PLTs are equivalent to 2.5-3.4 units of random donorderived PLTs or 2/5-6/10 of single-apheresis PLTs. The CB-derived PLTs exhibited features quite similar to those from peripheral blood in morphology, as revealed by electron micrographs, and in function, as revealed by fibrinogen/ADP aggregation, with the appearance of P-selectin and activated glycoprotein IIb-IIIa antigens. Thus, this culture system may be applicable for large-scale generation of PLTs for future clinical use.
Experimental Hematology, 2008
Delayed platelet recovery post–cord blood (CB) transplantation might be due to CB characteristics: low maturity of stem cell compartment, poor production of CD34+/CD41+ cells when induced to differentiate along the megakaryocytic (MK) lineage, retention of a low ploidy in the expanded MKs. Ex vivo expansion of CB hematopoietic progenitor cells for reconstitution of different human hematopoietic lineages has already been developed in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. However, optimal conditions for MK–progenitor engraftment to reduce hemorrhaging risk still to be developed. This study assesses the hypothesis that CB–CD34+ amplification with thrombopoietin (TPO) can be applied to a portion of a CB transplant unit to stimulate recovery along MK differentiation program.Human CB–CD34+ cells were amplified in a serum–free, clinical grade medium with 100 ng/mL TPO alone and in addition to other cytokines (Kit ligand, interleukin–6, and Flt–3 ligand). Seven–day cultured cells were transplanted into irradiated NOD/SCID mice and engraftment, megakaryocytopoiesis, and platelet production were assessed.Platelet release was successful and continuously present for at least 8 weeks in NOD/SCID mice transplanted with CB cells stimulated by TPO. Thrombocytopoiesis was more effective with transplanted TPO–amplified cells than with the cytokine cocktails.Platelet number obtained is within the minimum level considered sufficient for hemostasis. Furthermore, amplified cells maintain their self–renewal capacity and multilineage potential differentiation. Thus, transplantation of TPO–expanded CB cells has the potential favoring both platelet recovery and human engraftment.
Journal of Immunological Methods, 2008
The aim of this study was to determine whether the implementation of such expansion phase in a two-phase culture strategy prior to the induction of megakaryocyte (Mk) differentiation would increase the yield of Mks produced in cultures. Toward this end, we first characterized the functional properties of five cytokine cocktails to be tested in the expansion phase on the growth and differentiation kinetics of CD34+-enriched cells, and on their capacity to expand clonogenic progenitors in cultures. Three of these cocktails were chosen based on their reported ability to induce HPC expansion ex vivo, while the other two represented new cytokine combinations. These analyses revealed that none of the cocktails tested could prevent the differentiation of CD34+ cells and the rapid expansion of lineage-positive cells. Hence, we sought to determine the optimum length of time for the expansion phase that would lead to the best final Mk yields. Despite greater expansion of CD34+ cells and overall cell growth with a longer expansion phase, the optimal length for the expansion phase that provided greater Mk yield at near maximal purity was found to be 5 days. Under such settings, two functionally divergent cocktails were found to significantly increase the final yield of Mks. Surprisingly, these cocktails were either deprived of thrombopoietin or of stem cell factor, two cytokines known to favor megakaryopoiesis and HPC expansion, respectively. Based on these results, a short resource-efficient two-phase culture protocol for the production of Mks near purity (N 95%) from human CD34+ CB cells has been established.
Haematologica, 2003
Megakaryocyte (Mk) engraftment is often poor and delayed after cord blood (CB) transplantation. Ex vivo manipulations of the cells that will be infused may be a way to achieve better Mk engraftment. In this study we investigated the ability of different hematopoietic growth factor combinations to generate large numbers of Mk cells ex vivo. To find the best cytokine combination capable of generating large numbers of Mks, baseline CB CD34+ (bCD34+) cells and CD34+ and CD34- cells, immunoselected after 4 weeks of expansion with thrombopoietin (TPO), stem cell factor (SCF) and Flt-3 ligand (FL) (eCD34+, eCD34-), were further cultured in the presence of different cytokine combinations (containing interleukin(IL)-3, SCF, TPO and IL-6). To evaluate Mk reconstitution in vivo, Mk-committed cells, generated during 10 days of in vitro culture, were injected into NOD/SCID mice and the kinetics of human platelet production was evaluated. TPO and SCF together were found to be sufficient to genera...
Experimental Hematology, 2006
Objectives. Hematopoietic recovery, in particular platelet reconstitution, can be severely delayed after transplantation with cord blood (CB) stem cells (SC). Expansion of CB SC may be one way to improve the recovery, but there is concern that ex vivo expansion compromises the repopulating ability of SC. Methods. We used a short-term expansion protocol with TPO as single growth factor. The expanded cells were tested in the NOD/SCID mouse model and both platelet recovery and repopulation capacity were examined and compared with unexpanded CD34 + CB cells of the same CB donor. Results. Platelet recovery started 1 week earlier in mice transplanted with TPO-expanded CD34 + cells and at days 5 and 8 after transplantation, 6.2 ± 2.6 and 13.9 ± 6.7 plt/mL were observed, respectively. At similar time intervals 0.0 and 1.5 ± 0.2 plt/mL respectively were detected in mice receiving the unmanipulated CD34 + grafts. This was accompanied by a higher number of CFU-Mk in the bone marrow (BM) 7 days after transplantation. Moreover, the BM engraftment and the lineage differentiation of human cells at 6 weeks after transplantation was similar, suggesting that long-term engraftment was not compromised by the expansion procedure. Conclusion. Ex vivo expansion with TPO as single growth factor results in an accelerated platelet recovery in NOD/SCID mice and appears not to affect the long-term repopulation capacity. Ó
Bone Marrow Transplantation, 2001
Infusion of ex vivo expanded megakaryocytic (MK) progenitor cells is a strategy for shortening the duration of thrombocytopenia after haematopoietic stem cell transplantation. The cell dose after expansion has emerged as a critical factor for achieving the desired clinical outcomes. This study aimed to establish efficient conditions for the expansion of the MK lineage from enriched CD34 + cells of umbilical cord blood and to investigate the effect of platelet-derived growth factor (PDGF) in this system. Our results demonstrated that thrombopoietin (TPO) alone produced a high proportion of CD61 + CD41 + cells but a low total cell count and high cell death, resulting in an inferior expansion. The addition of interleukin-1 (IL-1), Flt-3 ligand (Flt-3L) and to a lesser extent IL-3 improved the expansion outcome. The treatment groups with three to five cytokines produced efficient expansions of CFU-MK up to 400fold with the highest yield observed in the presence of TPO, IL-1, IL-3, IL-6 and Flt-3L. CD34 + cells were expanded by five to 22-fold. PDGF improved the expansion of all cell types with CD61 + CD41 + cells, CFU-MK and CD34 + cells increased by 101%, 134% and 70%, respectively. On day 14, the CD61 + population consisted of diploid (86.5%), tetraploid (11.8%) and polyploid (8N-32N; 1.69%) cells. Their levels were not affected by PDGF. TPO, IL-1, IL-3, IL-6, Flt-3L and PDGF represented an effective cytokine combination for expanding MK progenitors while maintaining a moderate increase of CD34 + cells. This study showed, for the first time, that PDGF enhanced the ex vivo expansion of the MK lineage, without promoting their in vitro maturation. PDGF might be a suitable growth factor to improve the ex vivo expansion of MK progenitors for clinical applications.
Large generation of megakaryocytes from serum-free expanded human CD34+ cells
Biochemical and Biophysical Research Communications, 2009
Ex vivo generation of megakaryocytes from hematopoietic stem cells (HSCs) is crucial to HSC research and has important clinical potential for thrombocytopenia patients to rapid platelet reconstruction. In this study, factorial design and steepest ascent method were used to screen and optimize the effective cytokines (10.2 ng/ml TPO, 4.3 ng/ml IL-3, 15.0 ng/ml SCF, 5.6 ng/ml IL-6, 2.8 ng/ml FL, 2.8 ng/ml IL-9, and 2.8 ng/ml GM-CSF) in megakaryocyte induction medium that facilitate ex vivo megakaryopoiesis from CD34(+) cells. After induction, the maximum fold expansion for accumulated megakaryocytes was almost 5000-fold, and the induced megakaryocytes were characterized by analysis of gene expression, polyploidy and platelet activation ability. Furthermore, the combination of megakaryocyte induction medium and HSC expansion medium can induce and expand a large amount of functional megakaryocytes efficiently, and might be a promising source of megakaryocytes and platelets for cell therapy in the future.