Amélie Robert

Distinct cell death pathways triggered by the adenovirus early region 4 ORF 4 protein

In transformed cells, induction of apoptosis by adenovirus type 2 (Ad2) early region 4 ORF 4 (E4orf4) correlates with accumulation of E4orf4 in the cell membrane–cytoskeleton fraction. However, E4orf4 is largely expressed in nuclear regions before the onset of apoptosis. To determine the relative contribution of nuclear E4orf4 versus membrane-associated E4orf4 to cell death signaling, we engineered green fluorescent fusion proteins to target E4orf4 to specific cell compartments. The targeting of Ad2 E4orf4 to cell membranes through a CAAX-box or a myristylation consensus signal sufficed to mimic the fast Src-dependent apoptotic program induced by wild-type E4orf4. In marked contrast, the nuclear targeting of E4orf4 abolished the early induction of extranuclear apoptosis. However, nuclear E4orf4 still induced a delayed cell death response independent of Src-like activity and of E4orf4 tyrosine phosphorylation. The zVAD.fmk-inhibitable caspases were dispensable for execution of both cell death programs. Nevertheless, both pathways led to caspase activation in some cell types through the mitochondrial pathway. Finally, our data support a critical role for calpains upstream in the death effector pathway triggered by the Src-mediated cytoplasmic death signal. We conclude that Ad2 E4orf4 induces two distinct cell death responses, whose relative contributions to cell killing may be determined by the genetic background.

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

BACKGROUND AIMS Expansion 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. METHODS We 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. RESULTS These 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. CONCLUSIONS Collectively, 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.

Megakaryocyte and Platelet Production from Human Cord Blood Stem Cells

The cloning of thrombopoietin together with advances in the culture of hematopoietic stem cells have paved the way for the study of megakaryopoiesis, ongoing clinical trials and, in the future, for the potential therapeutic use of ex vivo produced blood substitutes, such as platelets. This chapter describes a 14-day culture protocol for the production of human megakaryocytes (MKs) and platelets, and assays that can be used to characterize the functional properties of the platelets produced ex vivo. CD34(+) cells isolated from cord blood cells are grown in a serum-free medium supplemented with newly developed cytokine cocktails optimized for MK differentiation, expansion, and maturation. Detailed methodologies for flow cytometry analysis of MKs and platelets, for the purification of platelets and functional assays, are presented together with supporting figures. The chapter also provides a brief review on megakaryocytic differentiation and ex vivo MK cultures.

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.

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.