Lucie Boyer

Preferential Ex Vivo Expansion of Megakaryocytes from Human Cord Blood CD34+-Enriched Cells in the Presence of Thrombopoietin and Limiting Amounts of Stem Cell Factor and Flt-3 Ligand

The high proliferative potential of cord blood (CB) stem cells and the identification of the key factor of megakaryopoiesis, thrombopoietin (TPO), permit the ex vivo expansion of megakaryocytes (MKs) for possible use in early post-transplant support of patients and the production of functional platelets for transfusion. However, culture conditions for the generation of adequate MKs for this purpose are not yet optimized. Therefore, we sought to define the mixture of early-acting cytokines and TPO that would promote the expansion of MK progenitors over other lineages and result in overall better MK expansion and platelet yields. CB CD34(+)-enriched cells were cultured in serum-free medium for 17 days in presence of TPO alone or in various combinations with early-acting cytokines used at different concentrations and addition times. MK expansion and polyploidy and platelet production were monitored by flow cytometry analysis using specific surface markers (CD41 and CD42b) and propidium iodide labeling. Our results showed that the use of high concentrations of stem cell factor (SCF) and Flt-3 ligand (FL) in early CB TPO-supplemented cultures was more favorable to monocytic and granulocytic cell expansion. However, we observed that their presence in limiting amounts was required for the preferential expansion of MK progenitors. The addition of SCF, FL, TPO, and interleukin-6 (IL-6) at high concentrations in secondary cultures of these expanded MKs resulted in optimal MK proportion (approximately 25% of MKs) and expansion (>300 MK per seeded cell), highest proportions of polyploid MKs (22% of mature MKs > or = 8N), and best platelet yields. Our results indicate that TPO-induced MK progenitors are more sensitive to early-acting cytokines than non-MK cells. We propose that MKs generated in the optimized conditions, in combination with immature stem/progenitor cells, could prove useful for the short-term platelet recovery following CB transplantation.

Individual and synergistic cytokine effects controlling the expansion of cord blood CD34+ cells and megakaryocyte progenitors in culture

BACKGROUND AIMSnExpansion of hematopoietic progenitors ex vivo is currently investigated as a means of reducing cytopenia following stem cell transplantation. The principal objective of this study was to develop a new cytokine cocktail that would maximize the expansion of megakaryocyte (Mk) progenitors that could be used to reduce periods of thrombocytopenia.nnnMETHODSnWe measured the individual and synergistic effects of six cytokines [stem cell factor (SCF), FLT-3 ligand (FL), interleukin (IL)-3, IL-6, IL-9 and IL-11] commonly used to expand cord blood (CB) CD34(+) cells on the expansion of CB Mk progenitors and major myeloid populations by factorial design.nnnRESULTSnThese results revealed an elaborate array of cytokine individual effects complemented by a large number of synergistic and antagonistic interaction effects. Notably, strong interactions with SCF were observed with most cytokines and its concentration level was the most influential factor for the expansion and differentiation kinetics of CB CD34(+) cells. A response surface methodology was then applied to optimize the concentrations of the selected cytokines. The newly developed cocktail composed of SCF, thrombopoietin (TPO) and FL increased the expansion of Mk progenitors and maintained efficient expansion of clonogenic progenitors and CD34(+) cells. CB cells expanded with the new cocktail were shown to provide good short- and long-term human platelet recovery and lymphomyeloid reconstitution in NOD/SCID mice.nnnCONCLUSIONSnCollectively, these results define a complex cytokine network that regulates the growth and differentiation of immature and committed hematopoietic cells in culture, and confirm that cytokine interactions have major influences on the fate of hematopoietic cells.

Increased production of megakaryocytes near purity from cord blood CD34+ cells using a short two-phase culture system.

Expansion of hematopoietic progenitor cells (HPC) ex vivo remains an important focus in fundamental and clinical research. 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 (>95%) from human CD34+ CB cells has been established.

Characterization of the effects and potential mechanisms leading to increased megakaryocytic differentiation under mild hyperthermia.

The physical culture parameters have important influences on the proliferation and differentiation fate of hematopoietic stem cells. Recently, we have demonstrated that CD34+ cord blood (CB) cells undergo accelerated and increased megakaryocyte (Mk) differentiation when incubated under mild hyperthermic conditions (i.e., 39 degrees C). In this study, we investigated in detail the impacts of mild hyperthermia on Mk differentiation and maturation, and explored potential mechanisms responsible for these phenomena. Our results demonstrate that the qualitative and quantitative effects on Mk differentiation at 39 degrees C appear rapidly within 7 days, and that early transient culture at 39 degrees C led to even greater Mk yields (p<0.03). Surprisingly, cell viability was only found to be significantly reduced in the early stages of culture, suggesting that CB cells are able with time to acclimatize themselves to 39 degrees C. Although mild hyperthermia accelerated differentiation and maturation of CB-derived Mks, it failed to promote their polyploidization further but rather led to a small reduction in the proportion of polyploid Mks (p=0.01). Conversely, gene arrays analysis demonstrated that Mks derived at 39 degrees C have a normal gene expression program consistent with an advanced maturation state. Finally, two independent mechanisms that could account for the accelerated Mk differentiation were investigated. Our results suggest that the accelerated and increased Mk differentiation induced by mild hyperthermia is not mediated by cell-secreted factors but could perhaps be mediated by the increased expression of Mk transcription factors.

Polyploid megakaryocytes can complete cytokinesis.

Megakaryocytes (MK) undergo polyploidization through endomitosis, a mitotic process that ends prematurely due to aborted cytokinesis. To better understand this and other events associated with MK differentiation, we performed long-term and large-field live cell imaging of human MKs derived in cord blood (CB) and bone marrow (BM) CD34+ cell cultures. Polyploid level of imaged cells was evaluated using three complementary approaches; cell history, cell size and ploidy correlation and nuclei staining. This system and strategy enabled the direct observation of the development of a large number of MKs (n=4865) and to quantify their fates. The most significant finding of this study is that a considerable proportion of polyploid MKs could complete cytokinesis. This unexpected process gave rise to polyploid daughter cell(s) with normal fates and contributed significantly to the expansion of polyploid MKs. Further analyses revealed that the proliferation rate amongst polyploid MKs was inversely correlated to their ploidy level, and that this phenomenon was much more frequent in CB- than BM-derived MKs. Accordingly, endomitosis was identified as the dominant fate of polyploid BM-MKs, while this was less accentuated for polyploid CB-MKs. These findings explain partially why CB-derived MKs remain in lower ploidy class. In conclusion, this study demonstrates that the development of polyploid MK results from the failure and/or success of cytokinesis and brings a new paradigm to the field of megakaryopoiesis.

Glycoprotein Ibα receptor instability is associated with loss of quality in platelets produced in culture.

The development of culture processes for hematopoietic progenitors could lead to the development of a complementary source of platelets for therapeutic purposes. However, functional characterization of culture-derived platelets remains limited, which raises some uncertainties about the quality of platelets produced in vitro. The aim of this study was to define the proportion of functional platelets produced in cord blood CD34+ cell cultures. Toward this, the morphological and functional properties of culture-derived platelet-like particles (PLPs) were critically compared to that of blood platelets. Flow cytometry combined with transmission electron microscopy analyses revealed that PLPs formed a more heterogeneous population of platelets at a different stage of maturation than blood platelets. The majority of PLPs harbored the fibrinogen receptor αIIbβ3, but a significant proportion failed to maintain glycoprotein (GP)Ibα surface expression, a component of the vWF receptor essential for platelet functions. Importantly, GPIbα extracellular expression correlated closely with platelet function, as the GPIIb+ GPIbα+ PLP subfraction responded normally to agonist stimulation as evidenced by α-granule release, adhesion, spreading, and aggregation. In contrast, the GPIIb+ GPIbα⁻ subfraction was unresponsive in most functional assays and appeared to be metabolically inactive. The present study confirms that functional platelets can be generated in cord blood CD34+ cell cultures, though these are highly susceptible to ectodomain shedding of receptors associated with loss of function. Optimization of culture conditions to prevent these deleterious effects and to homogenize PLPs is necessary to improve the quality and yields of culture-derived platelets before they can be recognized as a suitable complementary source for therapeutic purposes.

Medium conditioned with mesenchymal stromal cell–derived osteoblasts improves the expansion and engraftment properties of cord blood progenitors

Strategies to enhance the expansion of umbilical cord blood hematopoietic stem and progenitor cells (HSPCs) are crucial to enable their widespread application to adults and to overcome important limitations, such as delayed engraftment. Osteoblasts regulate HSPCs under steady-state and also under stress conditions, when HSPCs undergo numerous cycles of expansion. We hypothesized that osteoblasts could provide better stimulation for the expansion of multipotent HSPCs and subsequent hematopoietic recovery than mesenchymal stromal cells. Hence, we assessed the growth and engraftment modulatory activities of mesenchymal stromal cell-derived osteoblasts (M-OSTs) on hematopoietic progenitors. Mesenchymal stromal cells and M-OSTs favored the maintenance of CD34(+) cells. The expansion of cord blood CD34(+) cells and myeloid progenitors was highest in cultures supplemented with unfiltered M-OST-conditioned medium (M-OST CM). In addition, increased expression of cell surface receptors important for the homing of progenitors to the bone marrow, C-X-C chemokine receptor type 4 and lymphocyte function-associated antigen 1, was observed in CM-based cultures. Additionally, M-OST CM positively modulated the engraftment properties of expanded progenitors. Most notably, although human platelet levels remained steady in the first 2xa0weeks in mice transplanted with HSPCs expanded in standard medium, levels in mice transplanted with M-OST CM HSPCs rose continuously. Consistent with this, short-term human progenitor reconstitution was consistently greater in M-OST recipients. Finally, cytokine array-based profiling revealed increases in insulin-like growth factor binding protein 2, chemokines, and myeloid stimulating cytokines in M-OST CM. In conclusion, this study suggests that M-OSTs represent a new underappreciated source of feeder cells for the expansion of HSPCs with enhanced thrombopoietic activity.

Ex Vivo Differentiation of Cord Blood Stem Cells into Megakaryocytes and Platelets

Megakaryocytes (MK) are hematopoietic cells present in the bone marrow that are responsible for the production and release of platelets in the circulation. Given their very low frequency (<1%), human MK often need to be derived in culture to study their development or to generate sufficient material for biological studies. This chapter describes a simplified 14-day culture protocol that efficiently leads to the production of MK and platelets from cord blood enriched progenitor cells. A serum-free medium is suggested for the growth of the CB cells together with an optimized cytokine cocktail developed specifically for MK differentiation, expansion, and maturation. Methodologies for flow cytometry analysis, MK and platelets estimation, and MK progenitor assay are also presented.

Cotransplantation of ex vivo expanded progenitors with nonexpanded cord blood cells improves platelet recovery.

Umbilical cord blood (UCB) transplantation is associated with prolonged periods of cytopenia. Ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs) is currently investigated as a mean to accelerate hematological recovery. Contrary to neutrophils, platelet recovery remains problematic. For this reason, we have developed a culture protocol promoting the expansion of megakaryocyte (Mk) progenitors. The objective of this work was to determine whether the expanded (E) UCB HSPCs could accelerate platelet recovery in vivo using a murine HSPC transplantation model. The thrombopoietic activity of UCB and mobilized peripheral blood CD34(+) cells expanded under mild hyperthermia (MH, ie, 39°C) with the optimized megakaryocyte progenitor cocktail (OMPC) diverged significantly from the nonexpanded (NE) cells of origin; E cells provided rapid platelet release, while NE cells strongly contributed to platelet production past 10 days of transplantation. Consequently, the complementary of both cell sources was investigated. Cotransplantation of NE with E UCB cells significantly improved the recovery of human platelets (hPLTs) in vivo due to their complementary and synergistic thrombopoietic activities. Moreover, short-term human bone marrow (BM) reconstitution was also improved. Finally, we show that early hPLT release is dependent on Mk-primed cells and that E cells do not act as accessory cells, but have a more active role. In conclusion, hPLT recovery and short-term BM engraftment can be efficiently improved by the cotransplantation of Mk-primed UCB cells with NE HSPCs in a murine transplantation model.

Mild Hyperthermia Promotes and Accelerates Development and Maturation of Erythroid Cells

Hyperthermia treatment has at times been associated with increased platelet levels in humans. The heat shock protein HSP70, which can be induced by hyperthermia in megakaryocytes and erythrocytes, was recently shown to protect GATA-1 from degradation and to be required for erythroid differentiation. Based on these findings, we hypothesize that mild hyperthermia (MH), such as fever (39°C), could impact the differentiation of hematopoietic progenitors into erythrocytes and their subsequent maturation. Cell growth and erythroid differentiation increased dramatically in cord blood CD34(+) cell cultures incubated under MH. Erythroid maturation was also strongly promoted, which resulted in an increased proportion of hemoglobinized and enucleated erythroids. The rise in erythroid development was traced to a strong synergistic activity between MH and erythropoietin (EPO). The molecular basis for this potent synergy appears to originate from the capacity of MH to increase the basal activation of several signaling molecules downstream of the EPO receptor and the transcriptional activity of GATA-1. Moreover, the potent impact of MH on erythroid development was found be dependent on increased intracellular levels of reactive oxygen species. Thus, fever-like temperatures can promote the differentiation of progenitors along the erythroid lineage and accelerate their maturation through normal regulatory circuitry.