A sensitive quantitative single‐platform flow cytometry protocol to measure human platelets in mouse peripheral blood

Schipper, Laurus F.; Hensbergen, Yvette van; Fibbe, Willem E.; Brand, Anneke

doi:10.1111/j.1537-2995.2007.01472.x

Cited by 16 publications

(16 citation statements)

References 15 publications

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“…18,19,22 Onset of human platelet formation in CB-CD34 ϩ -injected mice varied from few days to 2 to 3 weeks, albeit modest numbers were reported few days after transplantation. 20,21,23,27,28 The advantage of culturing CB-or PB-CD34 ϩ cells in medium containing cytokine mixtures before transplantation has been demonstrated by several groups up to a few days after transplantation, 20,25,27 whereas no benefits were observed for longer Submitted February 18, 2009; accepted August 18, 2009. Prepublished online as Blood First Edition paper, September 9, 2009; DOI 10.1182 DOI 10.…”

Section: Introductionmentioning

confidence: 99%

Human platelets produced in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice upon transplantation of human cord blood CD34+ cells are functionally active in an ex vivo flow model of thrombosis

Salles¹,

Thijs²,

Brunaud

et al. 2009

Blood

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Xenotransplantation systems have been used with increasing success to better understand human hematopoiesis and thrombopoiesis. In this study, we demonstrate that production of human platelets in nonobese diabetic/severe combined immunodeficient mice after transplantation of unexpanded cord-blood CD34 ؉ cells was detected within 10 days after transplantation, with the number of circulating human platelets peaking at 2 weeks (up to 87 ؋ 10 3 /L). This rapid human platelet production was followed by a second wave of platelet formation 5 weeks after transplantation, with a population of 5% still detected after 8 weeks, attesting for long-term engraftment. Platelets issued from human hematopoietic stem cell progenitors are functional, as assessed by increased CD62P expression and PAC1 binding in response to collagenrelated peptide and thrombin receptoractivating peptide activation and their ability to incorporate into thrombi formed on a collagen-coated surface in an ex vivo flow model of thrombosis. This interaction was abrogated by addition of inhibitory monoclonal antibodies against human glycoprotein Ib␣ (GPIb␣) and GPIIb/IIIa. Thus, our mouse model with production of human platelets may be further explored to study the function of genetically modified platelets, but also to investigate the effect of stimulators or inhibitors of human thrombopoiesis in vivo. (Blood. 2009;114:5044-5051) IntroductionHuman platelets are anucleated cells that not only play a crucial role in primary hemostasis and wound repair, but are also particularly important in pathologic conditions such as thrombosis, vascular remodeling, and inflammation. Platelets originate from megakaryocytes (MK) in the bone marrow (BM) by fragmentation of pseudopodial elongations called proplatelets in a process that consumes the entire cytoplasmic content and is tightly regulated by thrombopoietin. 1,2 Human megakaryopoiesis has been studied ex vivo by measuring colony-forming units (CFUs; eg, CFU-MK, CFU-granulocyte, erythrocyte, macrophage, megakaryocyte), 3,4 MK polyploidy state, 5,6 expression of MK markers, 7 and novel genes expressed during MK differentiation. [8][9][10] Unraveling molecular mechanisms involved in megakaryopoiesis and thrombopoiesis is particularly relevant in the light of thrombocytopenia and pancytopenia associated with widespread use of high-dose chemotherapy for treatment of most cancers, also occurring after stem cell transplantation. Therefore, there has been an increasing interest in generating human platelets from MK in culture as well as in developing animal models of human hematopoiesis.Human platelet production has been described from differentiation of CD34 ϩ progenitor cells, isolated from mobilized peripheral blood (PB) or umbilical cord blood (CB), cultured in medium with a cytokine mixture containing thrombopoietin. 5,[11][12][13][14][15] Such produced platelets are functional, as demonstrated in aggregation assays and by expression of P-selectin on the platelet surface or by activation of glycosylphosphatidylinosito...

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Section: Introductionmentioning

confidence: 99%

Human platelets produced in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice upon transplantation of human cord blood CD34+ cells are functionally active in an ex vivo flow model of thrombosis

Salles¹,

Thijs²,

Brunaud

et al. 2009

Blood

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“…Cells were analyzed for HLA epitope expression before and after expansion with custom-made, in-house human HLA epitope specific monoclonal antibodies [47][48][49][50].…”

Section: Cd34mentioning

confidence: 99%

“…Blood collection and human platelet measurements were performed as described previously [47,48,51]. Briefly, human platelets were stained with noncross reactive mouse anti-human CD41-PE and human CD45 cells were stained with mouse anti-human CD45-PC7 (both Beckman Coulter).…”

Section: Analysis Of Pb After Transplantationmentioning

confidence: 99%

Thrombopoietin Treatment of One Graft in a Double Cord Blood Transplant Provides Early Platelet Recovery While Contributing to Long-Term Engraftment in NSG Mice

Brand

et al. 2015

Stem Cells and Development

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Human cord blood (CB) hematopoietic stem cell (HSC) transplants demonstrate delayed early neutrophil and platelet recovery and delayed longer term immune reconstitution compared to bone marrow and mobilized peripheral blood transplants. Despite advances in enhancing early neutrophil engraftment, platelet recovery after CB transplantation is not significantly altered when compared to contemporaneous controls. Recent studies have identified a platelet-biased murine HSC subset, maintained by thrombopoietin (TPO), which has enhanced capacity for short-and long-term platelet reconstitution, can self-renew, and can give rise to myeloid-and lymphoidbiased HSCs. In previous studies, we have shown that transplantation of human CB CD34+ cells precultured in TPO as a single graft accelerates early platelet recovery as well as yielding long-term repopulation in immunedeficient mice. In this study, using a double CB murine transplant model, we investigated whether TPO cultured human CB CD34+ cells have a competitive advantage or disadvantage over untreated human CB CD34 + cells in terms of (1) short-term and longer term platelet recovery and (2) longer term hematological recovery. Our studies demonstrate that the TPO treated graft shows accelerated early platelet recovery without impairing the platelet engraftment of untreated CD34 + cells. Notably, this was followed by a dominant contribution to platelet production through the untreated CD34 + cell graft over the intermediate to longer term. Furthermore, although the contribution of the TPO treated graft to long-term hematological engraftment was reduced, the TPO treated and untreated grafts both contributed significantly to long-term chimerism in vivo.

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“…Blood collection through a tail vein incision was performed twice weekly during the first 3 weeks after transplantation and once weekly thereafter. Blood collection and human platelets measurements were performed as described previously [41]. Briefly, human platelets were stained with a noncross-reactive mouse anti-human CD41-PE (Beckman Coulter) and erythrocytes were lysed with the IO Test3 Lysing solution FIG.…”

Section: Culture Of Mscsmentioning

confidence: 99%

No Synergistic Effect of Cotransplantation of MSC and Ex Vivo TPO-Expanded CD34⁺Cord Blood Cells on Platelet Recovery and Bone Marrow Engraftment in NOD SCID Mice

Brand

et al. 2015

Stem Cells and Development

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After cord blood (CB) transplantation, early platelet recovery in immune-deficient mice is obtained by expansion of CB CD34(+) cells with thrombopoietin (TPO) as single growth factor. Moreover, improvement of hematopoietic engraftment has been shown by cotransplantation of mesenchymal stem cells (MSC). We investigated whether a combination of both approaches would further enhance the outcome of CB transplantation in NOD SCID mice. NOD SCID mice were transplanted with either CB CD34(+) cells, CD34(+) cells with MSC, TPO-expanded CD34(+) cells or TPO-expanded CD34(+) cells with MSC. We analyzed human platelet recovery in the peripheral blood (PB) from day 4 after transplantation onward and human bone marrow (BM) engraftment at week 6. The different transplants were assessed in vitro for their migration capacity and expression of CXCR4. TPO expansion improved the early platelet recovery in the PB of the mice. Cotransplantation of MSC with CD34(+) cells improved BM engraftment and platelet levels in the PB 6 weeks after transplantation. Combining TPO expansion and MSC cotransplantation, however, neither resulted in a more efficient early platelet recovery, nor in a better BM engraftment, nor even very low or absent BM engraftment occurred. In vitro, MSC boosted the migration of CD34(+) cells, suggesting a possible mechanism for the increase in engraftment. Our results show that cotransplantation of MSC with TPO-expanded CD34(+) cells at most combines, but does not increase the separate advantages of these different strategies. A combination of both strategies even adds a risk of non engraftment.

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A sensitive quantitative single‐platform flow cytometry protocol to measure human platelets in mouse peripheral blood

Cited by 16 publications

References 15 publications

Human platelets produced in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice upon transplantation of human cord blood CD34+ cells are functionally active in an ex vivo flow model of thrombosis

Human platelets produced in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice upon transplantation of human cord blood CD34+ cells are functionally active in an ex vivo flow model of thrombosis

Thrombopoietin Treatment of One Graft in a Double Cord Blood Transplant Provides Early Platelet Recovery While Contributing to Long-Term Engraftment in NSG Mice

No Synergistic Effect of Cotransplantation of MSC and Ex Vivo TPO-Expanded CD34⁺Cord Blood Cells on Platelet Recovery and Bone Marrow Engraftment in NOD SCID Mice

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A sensitive quantitative single‐platform flow cytometry protocol to measure human platelets in mouse peripheral blood

Cited by 16 publications

References 15 publications

Human platelets produced in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice upon transplantation of human cord blood CD34+ cells are functionally active in an ex vivo flow model of thrombosis

Human platelets produced in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice upon transplantation of human cord blood CD34+ cells are functionally active in an ex vivo flow model of thrombosis

Thrombopoietin Treatment of One Graft in a Double Cord Blood Transplant Provides Early Platelet Recovery While Contributing to Long-Term Engraftment in NSG Mice

No Synergistic Effect of Cotransplantation of MSC and Ex Vivo TPO-Expanded CD34+Cord Blood Cells on Platelet Recovery and Bone Marrow Engraftment in NOD SCID Mice

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No Synergistic Effect of Cotransplantation of MSC and Ex Vivo TPO-Expanded CD34⁺Cord Blood Cells on Platelet Recovery and Bone Marrow Engraftment in NOD SCID Mice