Cryopreservation of cord blood CD34+ cells before or after thrombopoietin expansion differentially affects early platelet recovery in NOD SCID mice.

BACKGROUND: Expansion of human cord blood (CB) CD34+ cells with thrombopoietin (TPO) can accelerate delayed platelet (PLT) recovery after transplantation into immunodeficient mice. Clinical implementation, however, will depend on practical and effective protocols. The best timing of TPO expansion in relation to cryopreservation in this respect is unknown. STUDY DESIGN AND METHODS: In this study, we evaluated whether the order of cryopreservation and TPO expansion affected the expansion rate and numbers of clonogenic hematopoietic progenitor cells in vitro or PLT and longer-term hematopoietic repopulation in NOD SCID mice in vivo. RESULTS: Our results demonstrate higher expansion rates and the generation of higher numbers of multilineage and megakaryocytic progenitors (granulocyte, erythrocyte, monocyte, megakaryocyte colony-forming units and megakaryocyte colony-forming units) in vitro when freshly isolated CB CD34+ cells are first cultured with TPO and then cryopreserved and thawed as compared to TPO expansion after CD34+ cell cryopreservation. In contrast, the cells produced with the latter strategy showed higher expression of CD62L and a superior stromal cell-derived factor-1α-mediated migration. This might play a role in an also observed superior early PLT recovery after transplantation of these cells into NOD SCID mice. The hematopoietic engraftment in the marrow 6 weeks after transplantation was not different between the two strategies. CONCLUSION: Although TPO expansion before cryopreservation would yield higher nucleated cell and clonogenic myeloid and megakaryocyte cell numbers and enable earlier availability, CB TPO expansion after cryopreservation is likely to be clinically more effective, despite the lower number of cells obtained after expansion. Moreover, the latter strategy is logistically more feasible.

Double umbilical cord blood transplantation: a study of early engraftment kinetics in leukocyte subsets using HLA-specific monoclonal antibodies.

Single cord blood unit (CBU) predominance is usually established within the first month after double umbilical cord blood transplantation (UCBT). However, the kinetics of engraftment of the different leukocyte subsets and the mechanism of graft predominance is largely unknown. To investigate whether a differential engraftment might reveal a specific subset that could play a key role in the mechanism of graft predominance, we studied early engraftment kinetics of different leukocyte subpopulations by flow cytometry using human monoclonal antigen-specific human leukocyte antigen antibodies, directed against mismatched human leukocyte antigen-A or -B antigens between recipient and CBUs. Twenty-two patients, who had received a double UCBT preceded by a reduced-intensity conditioning regimen, were evaluated at days +11, +18, +25, and +32 posttransplantation. Single CBU predominance in the various leukocyte subsets was established within 18 days posttransplantation. CD4+ T cells of the dominant CBU showed early peripheral blood expansion. Moreover, chimerism in CD4+ and CD8+ T cell and natural killer cell subsets at day +11 was predictive of ultimate graft predominance. These findings show that engraftment kinetics of the various leukocyte subsets vary considerably after double UCBT and may suggest an important role for CD4+ T cells in a presumed alloreactive graft-versus-graft rejection.

Functional characterization of TPO-expanded CD34+ cord blood cells identifies CD34- CD61- cells as platelet-producing cells early after transplantation in NOD/SCID mice and rCD34+ cells as CAFC colony-forming cells.

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.

Validation of human monoclonal HLA Class I antibodies to evaluate the kinetics of donor chimerism in different cell subsets after double-cord-blood transplantation in the NOD/SCID model.

BACKGROUND: Double-cord-blood transplantation (DCBT) in patients is typically accompanied by predominance of a single unit. The causative mechanism, however, is unknown. Identifying the dynamics of mixed donor chimerism in general and in specific subpopulations may help to resolve this question. We conducted studies in a mouse model to develop a new analytic method using anti-human HLA Class I allele-specific monoclonal antibodies (HLA-MoAbs) in flow cytometry. STUDY DESIGN AND METHODS: Single-cord-blood transplantation or DCBT from HLA-mismatched donors was performed in NOD/SCID mice. Bone marrow (BM) and peripheral blood were collected from 3 to 20 weeks after transplantation. Donor chimerism was determined quantitatively within human platelets (hPLTs), human CD45+ (hCD45+) cells, and human myeloid and lymphocyte subsets by flow cytometry. RESULTS: Both cord donors stably engrafted in NOD/SCID. The sensitivity to detect chimerism measured with all HLA-MoAbs was 1% (>10 cells/µL). In mouse BM, the percentage of human cells measured with hCD45+ versus HLA-MoAbs correlated excellently (r = 0.999). Donor origin could be defined with HLA-MoAbs for nearly all (>93.6%) human cells in mouse peripheral blood and BM in all lineages. Chimerism of hPLTs in peripheral blood correlated well with hCD45+ cells in BM enabling frequent measurement of chimerism from early after transplantation onward. CONCLUSION: This approach using HLA-MoAbs enables longitudinal analysis of double-mixed human chimeric populations despite low absolute concentrations of human hematopoietic cell subsets in peripheral blood and BM in mice. Lacking reactivity with mouse cells, the HLA-MoAbs are suitable for use in other mouse models and in humans to identify the mechanisms involved in DCBT.

Exploring the use of expanded erythroid cells for autologous transfusion for anemia of prematurity.

BACKGROUND: Autologous cord blood (CB) red blood cells (RBCs) can partly substitute transfusion needs in premature infants suffering from anemia. To explore whether expanded CB cells could provide additional autologous cells suitable for transfusion, we set up a simple one-step protocol to expand premature CB cells. STUDY DESIGN AND METHODS: CB buffy coat cells and isolated CD34-positive (CD34(pos) ) cells from premature and full-term CB and adult blood were tested with several combinations of growth factors while omitting xenogeneic proteins from the culture medium. Cell differentiation was analyzed serially during 21 days using flow cytometry, progenitor assays, and high-performance liquid chromatography. RESULTS: Expanded CB buffy coat cells resulted in a threefold higher number of erythroblasts than the isolated CD34(pos) cells. However, the RBCs contaminating the buffy coat remained present during the culture with uncertain quality. Premature and full-term CB CD34(pos) cells had similar fold expansion capacity and erythroid differentiation. With the use of interleukin-3, stem cell factor, and erythropoietin, the fold increases of all CD34(pos) cell sources were similar: CB 3942 ± 1554, adult peripheral mobilized blood 4702 ± 1826, and bone marrow (BM) 4143 ± 1908. The proportion of CD235a expression indicating erythroblast presence on Day 21 was slightly higher in the adult CD34(pos) cell sources: peripheral blood stem cells (96.7 ± 0.8%) and BM (98.9 ± 0.5%) compared to CB (87.7 ± 2.7%; p = 0.002). We were not able to induce further erythroid maturation in vitro. CONCLUSION: This explorative study showed that fairly pure autologous erythroid-expanded cell populations could be obtained by a simple culture method, which should be optimized. Future challenges comprise obtaining ex vivo enucleation of RBCs with the use of a minimal manipulating approach, which can add up to autologous RBCs derived from CB in the treatment of anemia of prematurity.

Gata1 expression driven by the alternative HS2 enhancer in the spleen rescues the hematopoietic failure induced by the hypomorphic Gata1low mutation.

Rigorously defined reconstitution assays developed in recent years have allowed recognition of the delicate relationship that exists between hematopoietic stem cells and their niches. This balance ensures that hematopoiesis occurs in the marrow under steady-state conditions. However, during development, recovery from hematopoietic stress and in myeloproliferative disorders, hematopoiesis occurs in extramedullary sites whose microenvironments are still poorly defined. The hypomorphic Gata1(low) mutation deletes the regulatory sequences of the gene necessary for its expression in hematopoietic cells generated in the marrow. By analyzing the mechanism that rescues hematopoiesis in mice carrying this mutation, we provide evidence that extramedullary microenvironments sustain maturation of stem cells that would be otherwise incapable of maturing in the marrow.

Photodynamic treatment with mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin for pathogen inactivation in cord blood stem cell products.

BACKGROUND: Hematopoietic stem cell transplants and culture of hematopoietic progenitor cells require pathogen-free conditions. The application of a method of pathogen inactivation in red blood cells using photodynamic treatment (PDT) was investigated for the decontamination of cord blood stem cell (CBSC) products. STUDY DESIGN AND METHODS: CBSC products, spiked with Gram-positive and Gram-negative bacteria, were treated with PDT using mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin (Tri-P(4)) and red light. After PDT, in vitro and in vivo evaluation of the CBSC functions were performed. RESULTS: PDT of CBSC products resulted in the inactivation of the bacteria, with Staphylococcus aureus being the most resistant. Complete decontamination was achieved when CBSC products were contaminated with low titers of bacteria. PDT had no effect on white blood cell viability, the ex vivo expansion potential of the progenitor cells, and their capacity to differentiate to various hematopoietic cell lineages. However, PDT reduced the engraftment of human CBSCs in NOD/SCID mice, particularly affecting the B-cell lineage engraftment. CONCLUSION: Pathogen inactivation of CBSC with Tri-P(4)-mediated PDT is feasible at contamination level up to 10 to 20 colony-forming units per mL and can be considered when ex vivo expansion culture is anticipated. However, this treatment is not recommended for transplantation purposes at this time. Further investigations may elucidate why engraftment is diminished.

Ex vivo culture of human CD34+ cord blood cells with thrombopoietin (TPO) accelerates platelet engraftment in a NOD/SCID mouse model.

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.

Soluble aminopeptidase N/CD13 in malignant and nonmalignant effusions and intratumoral fluid.

PURPOSE: On the basis of the finding of marked overexpression in angiogenic microvessels, aminopeptidase N/CD13 has recently been suggested to play a prominent role in tumor angiogenesis. A soluble form of CD13 (sCD13) is present in human plasma, but its role in cancer has not been addressed. We hypothesized that sCD13 would be shed by tumor cells and/or endothelial cells lining tumor vessels, giving high levels of sCD13 in intratumoral fluid (TF) deposits and in malignant effusions. If so, sCD13 could be a convenient potential marker for tumor load and/or activated tumor endothelium. EXPERIMENTAL DESIGN: We have measured the specific sCD13 activity in effusions from 90 cancer patients and 12 patients with a nonmalignant condition, and studied its relationship with other major (anti-)angiogenic factors. In a separate group of patients (n = 41), the relationship of sCD13 activity in plasma with tumor load was studied. RESULTS: The sCD13 activity was highest in plasma from cancer patients 71.9 (fmol/ml/s hydrolyzed substrate) versus 42.4 for healthy subjects. In TF, malignant effusions, and nonmalignant effusions, the activities were 52.8, 33.5, and 18.6, respectively. We further studied the relationship of sCD13 with tumor load as well as with vascular endothelial growth factor (VEGF), endostatin, matrix metalloproteinase (MMP)-2, MMP-9, urokinase-type plasminogen activator, and plasmin. A significant correlation of sCD13 activity in plasma was found with tumor load (r = 0.68; P = 0.01), suggesting that plasma sCD13 is, at least, partly originating from tumor(-endothelium). The concentrations of VEGF and endostatin and the activities of urokinase-type plasminogen activator and MMP-9, but not MMP-2, were significantly higher in TF compared with all other effusions. In TF, a correlation between sCD13 and VEGF was found (r = 0.67; P = 0.03). No correlation of sCD13 with the other protease activities was found. CONCLUSION: The sCD13 activity is elevated in plasma and effusions of cancer patients. A strong correlation of plasma sCD13 with tumor load was found. On the basis of these results, the potential of sCD13 activity as a tumor and/or angiogenesis marker warrants further investigation.

Reduced growth, increased vascular area, and reduced response to cisplatin in CD13-overexpressing human ovarian cancer xenografts.

PURPOSE: Expression of aminopeptidase N/CD13 can be detected in several solid tumor types. Thus far, the role of CD13 in ovarian cancer has not been studied. We have investigated the expression pattern and biological function of CD13 in ovarian cancer. EXPERIMENTAL DESIGN: First, we studied the expression of CD13 in ovarian cancer tissue of 15 patients representing three different histological types (5 patients each) by immunohistochemistry. We then stably transfected the IGROV-1 human ovarian cancer cell line with a CD13 expression vector and examined the biological effect of CD13 in vitro and in vivo. RESULTS: The expression of CD13 in ovarian cancer was associated with the histological subtype: CD13 expression in tumor cells was observed in 80-100% of the patients with a serous or mucinous carcinoma and in only 20% of the clear cell carcinoma patients. In all patients' tumor samples, CD13-positive blood vessels were present. CD13 overexpression in IGROV-1 cells did not affect in vitro cell growth and sensitivity to doxorubicin, cisplatin, or gemcitabine. CD13 overexpression reduced invasion in Matrigel, which appeared to be independent of the aminopeptidase activity of CD13. Furthermore, the growth rate of IGROV-1/CD13 xenografts was reduced. The area of the vessel lumens was enlarged in a small percentage of vessels in the CD13-overexpressing xenografts. In addition, the CD13-overexpressing tumors were less sensitive to cisplatin. CONCLUSIONS: CD13 is expressed in tumor as well as endothelial cells in human ovarian cancer. Our results suggest that CD13 overexpression affects ovarian cancer growth, vascular architecture, and response to chemotherapy. Further elucidation of the mechanism of the observed effects of CD13 is warranted to better understand its role in the pathophysiology of ovarian cancer.

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.

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.

Thrombopoietin treatment of one graft in a double cord blood transplant provides early platelet recovery while contributing to long-term engraftment in NSG mice.

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 lymphoid-biased 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 immune-deficient 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.

Possible role of minor h antigens in the persistence of donor chimerism after stem cell transplantation; relevance for sustained leukemia remission.

Persistent complete donor chimerism is an important clinical indicator for remissions of hematological malignancies after HLA-matched allogeneic stem cell transplantation (SCT). However, the mechanisms mediating the persistence of complete donor chimerism are poorly understood. The frequent coincidence of complete donor chimerism with graft-versus-leukemia effects and graft-versus-host disease suggests that immune responses against minor histocompatibility antigens (mHags) are playing an important role in suppressing the host hematopoiesis after allogeneic SCT. Here, we investigated a possible relationship between donor immune responses against the hematopoiesis-restricted mHag HA-1 and the long-term kinetics of host hematopoietic chimerism in a cohort of 10 patients after allogeneic HLA-matched, HA-1 mismatched SCT. Functional HA-1 specific CTLs (HA-1 CTLs) were detectable in 6/10 patients lysing host-type hematopoietic cells in vitro. Presence of HA-1 CTLs in the peripheral blood coincided with low host hematopoiesis levels quantified by highly sensitive mHag specific PCR. Additionally, co-incubation of host type CD34+ cells with HA-1 CTLs isolated after allogeneic SCT prevented progenitor and cobblestone area forming cell growth in vitro and human hematopoietic engraftment in immunodeficient mice. Conversely, absence or loss of HA-1 CTLs mostly coincided with high host hematopoiesis levels and/or relapse. In summary, in this first study, presence of HA-1 CTLs paralleled low host hematopoiesis levels. This coincidence might be supported by the capacity of HA-1 CTLs isolated after allogeneic SCT to specifically eliminate host type hematopoietic stem/progenitor cells. Additional studies involving multiple mismatched mHags in more patients are required to confirm this novel characteristic of mHag CTLs as factor for the persistence of complete donor chimerism and leukemia remission after allogeneic SCT.

Aminopeptidase inhibitor bestatin stimulates microvascular endothelial cell invasion in a fibrin matrix.

The aminopeptidase inhibitor bestatin has been shown to have anti-angiogenic effects in a number of model systems. These effects are thought to result from inhibition of CD13 activity. Because tumor angiogenesis can evolve in a fibrin-rich stroma matrix we have studied for the first time the effects of bestatin on microvascular endothelial capillary-like tube formation in a fibrin matrix. Bestatin enhanced the formation of capillary-like tubes dose-dependently. Its effects were apparent at 8 micro M; the increase was 3.7-fold at 125 micro M; while high concentrations (>250 micro M), that were shown to have anti-angiogenic effects in other systems, caused extensive matrix degradation. Specific CD13-blocking antibodies WM15 and MY-7, and the aminopeptidase inhibitors amastatin and actinonin also enhanced capillary-like tube formation (maximally 1.5-fold), but these effects did not reach statistical significance. The effect of bestatin was not due to a change in uPAR availability because the relative involvement of the u-PA/u-PAR activity was not altered by bestatin. In view of the present findings we hypothesize that aminopeptidases other than CD13 predominantly contribute to the observed pro-angiogenic effect of bestatin in a fibrin matrix. The identification of this novel effect of bestatin is important in the light of the proposed use of bestatin as anti-angiogenic and/or anti-tumor agent.

A doxorubicin-CNGRC-peptide conjugate with prodrug properties.

There is increasing interest in the exploitation of molecular addresses for the targeting of tumor imaging or therapeutic agents. A recent study demonstrated anticancer activity in human xenografts of doxorubicin (DOX)-peptide conjugates targeted to the tumor vascular endothelium, among them DOX coupled to the cyclic pentapeptide CNGRC [Science 279 (1998) 377]. In order to learn more about the mechanism of action of this type of DOX-peptide conjugates, we have studied the interaction of DOX-CNGRC with primary human umbilical cord vein endothelial cells (HUVEC) and tumor cells under defined in vitro conditions. We used a DOX conjugate, in which the cyclic CNGRC peptide, for which an in vivo endothelial address has recently been identified as aminopeptidase N (APN)/CD13, has been coupled via a hydrolysable spacer to the C-14 anthracycline-side chain. First we determined that the t(1/2) of DOX-CNGRC conjugate in human blood was 442 min (at 37 degrees ) allowing sufficient time for endothelial targeting when administered i.v. When cultured cells were exposed for 30 min to DOX-CNGRC a more cytoplasmic localization of fluorescent drug was seen when compared to DOX exposure and intracellular DOX-CNGRC was identified after extraction from the cells. This revealed differences in the cellular uptake process of the conjugate compared to DOX. The antiproliferative effect of DOX-CNGRC was determined by 30 min exposure in medium with a high protein content in order to mimick the in vivo targeting situation. In this medium, the IC(50) was 1.1 microM for highly CD13 expressing HT-1080, 1.45 microM for CD13 negative SK-UT-1 sarcoma cells and 6.5 microM for CD13 positive HUVEC. The IC(50) of DOX for these cells were 1.0, 2.0 and 7.3 microM, respectively. Although DOX-CNGRC inhibited the peptidase activity of CD13 up to 50%, our data do not favor an important role for the enzyme inhibition in the cytotoxic effect of the conjugate. The antitumor activity was tested in nude mice bearing human ovarian cancer xenografts (OVCAR-3). A weekly i.v. administration (3mg/kg DOX-equivalent, 3x) showed a minor (40%) growth delay, which does not indicate efficacy better than that expected for free DOX. In conclusion, this study indicates that the antiproliferative and anti-angiogenic effects of DOX-CNGRC as reported before, are likely caused by the cytostatic effects of intracellularly released parent drug DOX, independent of CD13 expression/activity. More research is needed to identify the optimal specific chemical configuration of DOX-peptide conjugates for in vivo targeting and receptor-mediated cellular uptake.

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

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.