RESUMEN
Transplantation of thrombopoietin (TPO)-expanded cord blood CD34(+) cells accelerates human platelet recovery in NOD/SCID mice. It is unknown which subpopulations of the TPO-expanded cells mediate accelerated platelet recovery and bone marrow (BM) engraftment. In this study, the contribution of these subpopulations to human platelet appearance in the blood and BM engraftment was studied in NOD/SCID mice. Following transplantation of CD34(-) /CD61(-)/lineage(-) cells (Lin(-)), human platelets were detected in the blood of recipient mice from day 4. Both time to platelet recovery and blood platelet counts at 6 weeks after transplantation showed Lin(-) dose dependence. The Lin(-) population was virtually negative for lineage marker expression and lacked CD42b expression but was heterogeneous with regard to CD36 and CD38 expression, reflecting a population in transit but not fully committed toward the megakaryocyte (MK) lineage. Although no definitive phenotype could be established of the cells generating prompt platelet production and cells generating platelets 6 weeks after transplantation, this relatively heterogeneous Lin(-) population is prerequisite to accelerate platelet recovery in vivo. The interval to platelet recovery after transplantation of the CD34(+) cells remaining after expansion (rCD34(+)) was similar to mice transplanted with nonexpanded CD34(+) cells, although the total platelet counts and the engraftment levels in the BM were lower. Cobblestone area-forming cell colony-forming cells resided mostly in the rCD34(+) population. The pro-MK CD61(+) cells did not contribute to human platelet recovery or engraftment in the BM. Our study shows that not all expanded cells appear critical for transplantation. These data support that functional characterization of the expanded cell populations is warranted to make future expansion protocols suitable for clinical application.
Asunto(s)
Antígenos CD34 , Trasplante de Células Madre de Sangre del Cordón Umbilical , Sangre Fetal/metabolismo , Integrina beta3 , Células Progenitoras de Megacariocitos/metabolismo , Megacariocitos/metabolismo , Trombopoyetina/farmacología , Animales , Plaquetas/citología , Plaquetas/metabolismo , Técnicas de Cultivo de Célula , Células Cultivadas , Femenino , Sangre Fetal/citología , Humanos , Masculino , Células Progenitoras de Megacariocitos/citología , Megacariocitos/citología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante HeterólogoRESUMEN
BACKGROUND: The NOD/SCID mouse is a widely used model for human cord blood (CB) transplantation. Engraftment is generally estimated with semiquantitative methods, measuring the percentage of human cells among mouse cells. To compare protocols aiming to improve hematopoietic recovery, quantitative methods to enumerate human cells would be preferred. This study describes a single-platform protocol to count human platelets (hPLTs) after transfusion and CB transplantation in the peripheral blood (PB) of the mouse. METHODS: With an anti-human CD41 antibody against hPLTs and counting beads, the sensitivity to detect hPLTs in mouse blood by flow cytometry was validated. PLT recovery after hPLT transfusions and PLT kinetics after transplantation with CB CD34+ cells was followed in time in NOD/SCID mice. RESULTS: hPLTs could be reliably detected to a level as low as 1 PLT per microL with this single-platform protocol, what appeared to be at least 10 times more sensitive than detection with the dual-platform protocol. To verify the applicability for mouse studies, hPLTs were measured serially in transfusion and transplantation studies in NOD/SCID mice. The results showed that earlier detection of PLT recovery was feasible with the single-platform protocol. CONCLUSION: A single-platform flow cytometry method can repeatedly measure low numbers of circulating hPLTs in the PB of the same mouse. This method may be helpful in search of new protocols aiming at accelerating PLT recovery after CB transplantation, but also in a number of clinical settings, such as monitoring PLT reconstitution after hematopoietic stem cell transplantation.
Asunto(s)
Plaquetas/citología , Sangre Fetal/citología , Citometría de Flujo/métodos , Animales , Antígenos CD34/análisis , Plaquetas/inmunología , Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Femenino , Sangre Fetal/inmunología , Sangre Fetal/trasplante , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Modelos Animales , Recuento de Plaquetas , Transfusión de Plaquetas/métodos , Reproducibilidad de los Resultados , Trasplante HeterólogoRESUMEN
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/microL were observed, respectively. At similar time intervals 0.0 and 1.5 +/- 0.2 plt/microL 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.
Asunto(s)
Antígenos CD34/inmunología , Plaquetas/citología , Sangre Fetal/citología , Animales , Linaje de la Célula , Células Cultivadas , Femenino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Modelos AnimalesRESUMEN
OBJECTIVE: In comparison with stem cell transplantation using bone marrow or cytokine-mobilized peripheral blood, cord blood transplantation is characterized by delayed engraftment, in particular platelet recovery. The differences in the kinetics of engraftment may be related to quantitative differences in the numbers of stem cells and megakaryocyte progenitor cells and/or to qualitative differences between megakaryocyte progenitor cells in these grafts. We compared the hematopoietic composition of these grafts and determined the distribution of mature and immature megakaryocyte progenitor cells in cord blood and mobilized peripheral blood and their in vitro kinetic behavior. METHODS: Megakaryocyte progenitor cell subpopulations from cord blood (CB) and mobilized peripheral blood (PBSC) were expanded in vitro in the presence of mpl-ligand. The developmental differences during expansion of megakaryocyte progenitors were analyzed by flow cytometry and progenitor assays. RESULTS: We found that the immature (CD34(+)/CD41(-)) subpopulation from CB contains more than 98% of all megakaryocyte progenitor cells, responsible for 99% of all megakaryocytic cells cultured during 2 weeks. The CB CD34(+)/CD41(+) subpopulation shows no contribution to megakaryocytic cell formation. In contrast, in PBSC the mature (CD34(+)/CD41(+)) subpopulation contains 7% of all megakaryocyte progenitor cells. Moreover, CD34(+) cells from CB and PBSC also showed distinct phenotypic differences during maturation in vitro. PBSC megakaryocyte progenitor cells transiently express both CD34 and CD41 during maturation in vitro, whereas CB progenitor cells transiently lack expression of both markers before differention into (CD34(-)/CD41(+)) megakaryocytic cells. CONCLUSION: The in vitro data indicate the presence of different developmental stages of megakaryocyte progenitor cells in CB as compared to PBSC. These differences in composition and maturation between CB and PBSC may be related to the different kinetics of engraftment following transplantation of these stem cell sources.