RESUMO
Stem cell-derived platelets have the potential to replace donor platelets for transfusion. Defining the platelet-producing megakaryocytes (MKs) within the heterogeneous MK culture may help to optimize the in vitro generation of platelets. Using 2 human stem cell models of megakaryopoiesis, we identified novel MK populations corresponding to distinct maturation stages. An immature, low granular (LG) MK pool (defined by side scatter on flow cytometry) gives rise to a mature high granular (HG) pool, which then becomes damaged by apoptosis and glycoprotein Ib α chain (CD42b) shedding. We define an undamaged HG/CD42b+ MK subpopulation, which endocytoses fluorescently labeled coagulation factor V (FV) from the media into α-granules and releases functional FV+CD42b+ human platelet-like particles in vitro and when infused into immunodeficient mice. Importantly, these FV+ particles have the same size distribution as infused human donor platelets and are preferentially incorporated into clots after laser injury. Using drugs to protect HG MKs from apoptosis and CD42b shedding, we also demonstrate that apoptosis precedes CD42b shedding and that apoptosis inhibition enriches the FV+ HG/CD42b+ MKs, leading to increased platelet yield in vivo, but not in vitro. These studies identify a transition between distinct MK populations in vitro, including one that is primed for platelet release. Technologies to optimize and select these platelet-ready MKs may be important to efficiently generate functional platelets from in vitro-grown MKs.
Assuntos
Plaquetas/citologia , Células da Medula Óssea/imunologia , Fator V/genética , Células Progenitoras de Megacariócitos/citologia , Megacariócitos/citologia , Animais , Apoptose/efeitos dos fármacos , Arteríolas/efeitos dos fármacos , Arteríolas/imunologia , Arteríolas/lesões , Biomarcadores/sangue , Plaquetas/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular , Linhagem da Célula/imunologia , Endocitose , Fator V/imunologia , Fator V/farmacologia , Citometria de Fluxo , Expressão Gênica , Humanos , Imunofenotipagem , Lasers , Células Progenitoras de Megacariócitos/imunologia , Megacariócitos/imunologia , Camundongos , Camundongos SCID , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/imunologiaRESUMO
Platelets are the most prominent elements of blood tissue involved in hemostasis at sites of blood vessel injury. Platelet cytoskeleton is responsible for their shape modifications observed during activation and adhesion to the substratum; therefore the interactions between cytoskeleton and plasma membrane are critical to modulate blood platelet functions. Several cytoskeletal components and binding partners, as well as enzymes that regulate the cytoskeleton, localize to membrane/lipid rafts (MLR) and regulate lateral diffusion of membrane proteins and lipids. Resting, thrombin-activated, and adherent human platelets were processed for biochemical studies including western-blot and immunprecipitation assays and confocal analysis were performed to characterize the interaction of MLR with the main cytoskeleton elements and ß-dystroglycan as well as with the association of caveolin-1 PY14 with focal adhesion proteins. We transfected a megakaryoblast cell line (Meg-01) to deplete ß-dystroglycan, subsequent to their differentiation to the platelet progenitors. Our data showed a direct interaction of the MLR with cytoskeleton to regulate platelet shape, while an association of caveolin-1 PY14 with vinculin is needed to establish focal adhesions, which are modulated for ß-dystroglycan. In conclusion, caveolin-1 PY14 in association with platelet cytoskeleton participate in focal adhesions dynamics.
Assuntos
Plaquetas/citologia , Caveolina 1/metabolismo , Citoesqueleto/metabolismo , Microdomínios da Membrana/metabolismo , Vinculina/metabolismo , Plaquetas/metabolismo , Adesão Celular , Diferenciação Celular , Linhagem Celular , Distroglicanas/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Células Progenitoras de Megacariócitos/citologia , Trombina/metabolismoRESUMO
BACKGROUND: Hematopoietic stem cells (HSC) have been characterized by CD34+ expression and an adequate dose of CD34+ cells is associated with a complete engraftment. CD133 is a more specific marker of HSC. MATERIALS AND METHODS: We studied the relationship between graft content of CD34+, CD133+, and CD38+ cells and trilineage engraftment after autologous stem cell transplantation in patients with different hematological disorders. Blood samples were obtained before and after mobilization with recombinant granulocyte-colony stimulating factor (G-CSF, 16 µg/kg), from apheresis collections, and after transplantation. RESULTS: Cell subsets were quantified by flow cytometry, and the dose of each population infused was correlated with success of engraftment. G-CSF induced mobilization of CD133+CD38+ cells (12.6-fold) and CD133+CD34+ cells (14.7-fold). A correlation was observed between the infused dose of CD133+CD34+ and CD133+CD38+ cells and platelet engraftment. CONCLUSION: CD133+CD34+ and CD133+CD38+ cells were mobilized with G-CSF and these cell subsets were correlated with platelet engraftment.