Engraftment in NOD/SCID Mice of TPO Amplified CB Cells To Promote Platelet Development.

Abstract A major disadvantage of human umbilical cord blood (CB) transplant is the delayed engraftment of platelets, which can lead to hemorrhagic complication, increased platelet transfusion requirements, prolonged alloimmunization and hospitalization. As a strategy for accelerating platelet recovery, the infusion of ex vivo expanded megakaryocytic progenitors to transplant patients has been proposed. NOD/SCID mice constitute an in vivo model to study human megakaryocytopiesis and platelet development after transplantation. CB was obtained from the placenta of full-term newborn, after informed consent provided according to the Declaration of Helsinki, CD34+ progenitors with amplified 100ng/ml thrombopoietin (TPO) were transplanted in NOD/SCID mice, and their ability to promote differentiation of the megakaryocytic lineage, with production of functional platelets was assessed. The engraftment was evaluated on bone marrow cells by flow cytometry analysis using human CD45 MoAb staining. Four weeks after transplantation, a mean value of 3.22% CD45+ human cells was obtained; this percentage significantly increased at week 8 (39.6% hCD45+ cells) and was observed to have remained constant at week 12 (45.6% hCD45+ cells). To evaluate the capacity of engrafted cells to produce platelets, we performed Facs analysis to monitor the appearance of human platelets in mouse peripheral blood. Mouse peripheral blood was collected via retro-orbital bleeding at different times (2, 4, 6 and 8 weeks) in EDTA-coated tubes. Platelet enriched plasma was analyzed after human CD41a-FITC staining by flow cytometry. Of the mice analyzed, 78.8% produced >0.1% human platelets. The results obtained with CB CD34+ cells amplified with TPO were compared with cells amplified in the presence of a cocktail of growth factors (100ng/ml KL, 50ng/ml FL, 10ng/ml IL6) containing TPO at low concentration (10ng/ml) (T10KF6) or high concentration (100ng/ml) (T100KF6). The mean engraftment values were 57.7% in the T10KF6 group and 10.0% in T100KF6 group. However, although reduced engraftment was obtained in T100KF6, a mean of 60.8% of mice produced (>0.1%) human platelets. In T10KF6 this percentage was reduced to 45.2%. Despite the strong variability in platelets production, a mean percentage of 0.49% human platelets was detected at week 2 after transplantation; at week 4, this percentage peaked, reaching 1.26% (which translated into 7.6×109/L total human platelet count). In some mice, a human platelet percentage of 7% (42.2×109/L) was obtained. The platelet count gradually decreased between weeks 6 and 8, with a mean of 1.02% (8.05×109/L) and 0.86% (7.99×109/L) respectively. Our results showed that TPO maintained about 2.2 times higher platelet count than T100KF6 and T10KF6 and that TPO might play an important role in the ex vivo expansion of haematopoietic cells, supporting the hypothesis that Mk lineage engraftment is capable of shortening time of human platelet recovery when transplanted in NOD/SCID mice.

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YM477, a Novel Orally-Active Thrombopoietin Receptor Agonist.

Blood ◽

10.1182/blood.v106.11.2298.2298 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2298-2298 ◽

Cited By ~ 6

Author(s):

Ken-ichi Suzuki ◽

Masaki Abe ◽

Mari Fukushima-Shintani ◽

Keizo Sugasawa ◽

Fukushi Hirayama ◽

...

Keyword(s):

Stem Cells ◽

Platelet Count ◽

Hematopoietic Stem Cells ◽

Peripheral Blood ◽

Receptor Agonist ◽

Human Platelet ◽

Human Platelets ◽

Scid Mice ◽

Hematopoietic Stem ◽

Orally Active

Abstract Thrombopoietin (TPO) is the principal physiologic regulator of platelet production. The search for an orally-active nonpeptidyl small molecule TPO receptor agonist has resulted in the discovery of YM477. YM477 acted specifically on the TPO receptor and stimulated megakaryocytopoiesis throughout the development and maturation of megakaryocytes just as TPO does. YM477, however, was shown to have high species specificity, effective in only humans and chimpanzees. Recently, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were characterized as an efficient engraftment model for human hematopoietic stem cells, as this model results in the production of human platelets. In this way, we examined the in vivo platelet-increasing effect of YM477 in human platelet-producing NOD/SCID mice in which human hematopoietic stem cells were transplanted. In this study, we used commercially available cryopreserved human fetal liver CD34+ cells as a source of human hematopoietic stem cells. The cells were transplanted into sublethally irradiated (240 cGy) NOD/SCID mice. Human platelets started to appear in peripheral blood of these mice 4 weeks after transplantation. The production of human platelets continued up to one year post-transplant. Various doses of YM477 (0, 0.3, 1, and 3 mg/kg/day) were orally administered for 14 days to NOD/SCID mice that had been confirmed to produce human platelets stably. Oral administration of YM477 dose-dependently increased the number of human platelets produced by these mice, with significance at 1 mg/kg/day and above. The increase in the human platelet count reached about 2.7-fold at 1 mg/kg/day and about 3.0-fold at 3 mg/kg/day on day 14. Withdrawal of YM477 administration caused the human platelet count to return to the pretreatment level. The number of murine platelets did not change during the study period. Next, to evaluate the function of human platelets produced in peripheral blood of these mice, the expression of activation-dependent marker CD62P (P-selectin) on human platelets stimulated with thrombin receptor agonist peptide (TRAP) were examined. CD62P expression on human platelets was induced by the stimulation of blood from transplanted mice with TRAP, suggesting that human platelets produced in NOD/SCID mice were functional. Furthermore, the maximum response of CD62P expression on human platelets induced by TRAP was evaluated before and after administration of YM477 at 3 mg/kg/day for 14 days. CD62P expression was not changed by administration of YM477, which was similar to the results obtained with a vehicle group. These results suggest that YM477 is an orally active TPO receptor agonist useful for treating patients with thrombocytopenia.

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Temperature Cycling Improves In Vivo recovery of Cold Stored Human Platelets

Blood ◽

10.1182/blood.v118.21.720.720 ◽

2011 ◽

Vol 118 (21) ◽

pp. 720-720

Author(s):

Fei Xu ◽

Monique Gelderman-Fuhrmann ◽

John Farrell ◽

Jaroslav Vostal

Keyword(s):

Flow Cytometry ◽

Cold Storage ◽

Room Temperature ◽

Human Platelets ◽

Scid Mice ◽

Temperature Cycling ◽

Post Injection ◽

Platelet Recovery ◽

In Vivo Recovery

Abstract Abstract 720 Platelets are currently limited to 5 days of storage at room temperature to prevent growth of bacteria to high levels. Cold storage of platelets could reduce bacterial proliferation but platelets stored in cold for over 48 hours are cleared rapidly from circulation through the hepatocyte Ashwell-Morell (AM) receptor thus limiting the applicability of cold temperatures to platelet storage. We used a temperature cycling method to store human platelets in the cold without decreasing their in vivo recovery in an immunodeficient (SCID) animal model of transfusion. Temperature cycled (TC) apheresis human platelets were stored in the cold (4°C) for 12 hours and then incubated at 37°C for 30 minutes before returning back to cold storage. The TC (37°C pulses for 30 minutes at 12 hour intervals) was continued for 2, 5 and 7 days. Human platelets stored either at room temperature (RT), cold or TC for 2, 5 and 7 days were infused into 6 to 8 SCID mice per group and their in vivo recovery in circulation was determined at 5, 20 and 60 minutes after transfusion by flow cytometry. Carbohydrate exposure on the surface of the platelets was analyzed for galactose by Erythrina cristagalli agglutinin (ECA), and for β-GlnNAc by succinyl wheat germ agglutinin (sWGA) using flow cytometry. Involvement of the AM receptor was examined by monitoring clearance of cold stored platelets in the presence of asialofetuin, a competitive ligand for the receptor. In vivo recovery of human platelets stored for two-days in SCID mice circulation is shown in Figure 1. As expected, cold platelets had significantly decreased recovery compared to RT platelets, from 22.1±2.5% to 11.1±3.3% (P<0.01), 11.5±2.9% to 5.5±3.6% (P<0.01) and 11.2±1.4% to 6.2±1.8% (P<0.01) respectively at 5, 20 and 60 min post platelets injection. Compared to cold platelets, TC platelets recovery increased significantly from 11.1±3.3% to 15.9±4.4% (P<0.01), 5.5±3.6+% to 10.5±4.7% (P<0.01) and 6.2±1.8% to 9.5±2.2% (P<0.05) respectively at 5, 20 and 60 min post platelets injection. At 20 and 60 min post injection, the TC platelets have recovery of 10.5±4.7% and 9.5±2.2% respectively, that are comparable (P>0.05%) to RT platelet recoveries of 11.5±2.9% and 11.2±1.4% for the same time points. Similar increases of in vivo recovery for TC platelets as compared to cold platelets were obtained for at 5 and 7 days.Figure 1Human Platelet Recovery (% of total platelets circulating) * p< 0.05, ** p< 0.01, *** p< 0.001Figure 1. Human Platelet Recovery (% of total platelets circulating) * p< 0.05, ** p< 0.01, *** p< 0.001 Binding of the galactose specific lectin, ECA, was increased by 142±22% from RT to cold platelets (P<0.01) as previously reported. However, binding of ECA was also increased by 134±16% from RT to TC platelets (P<0.01). β-GlnNAc exposure, as measured by sWGA lectin binding, was increased after cold and TC storage by 222±65% (P<0.01) and 197±14% (P<0.01), respectively, when compared to RT platelets. Platelets stored in the cold for >48 hours have been reported to be cleared through the hepatic AM receptor which recognizes asialocarbohydrates. Co-injection of asialofetuin significantly improved the recovery of two-day cold stored platelets from 9.5±5.1% to 18.4±7.3% (P<0.05) and 4.8±3.7% to 12.1±4.9% (P<0.01), at 5 min and 20 min post injection, respectively. Native fetuin did not alter the clearance of cold platelets. However, there was no significant increase in the recovery of TC platelets in the presence of asialofetuin as compared to fetuin injection (P>0.28), even though the TC platelets, like cold platelets, have significantly increased β-galactose exposure. Our results indicate that ‘temperature cycling' during cold storage of platelets may be an effective method to store human platelets up to 7 days without loss of in vivo recovery after transfusion when compared to RT platelets. Temperature cycling does not alter the cold induced increases in β-gal or β-GlcNAc expression which suggests that there are other mechanisms besides binding to the AM receptor that mediate clearance of platelets stored in the cold for >48 hours. The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. Disclosures: No relevant conflicts of interest to declare.

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A SCID Mouse Model for Monitoring in Vivo Viability of Human Platelet Concentrates Using Flow Cytometry

Blood ◽

10.1182/blood.v112.11.4549.4549 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4549-4549

Author(s):

Jia-hua Ding ◽

Cheng-yin Huang ◽

Shiyun Xu

Keyword(s):

Flow Cytometry ◽

Whole Blood ◽

Human Platelet ◽

Human Platelets ◽

Scid Mice ◽

Test Method ◽

Platelet Concentrates ◽

Tail Vein

Abstract Objective To develop an animal test method for evaluating the in vivo quality of human platelet concentrates. Methods Human platelets were transfused to mice by tail vein with a 1mL insulin syring fitted with a 29-gauge ultra-fine needle. Blood samples were taken at 30 minutes,2,4,6,8,12, and 24hours after infusion with a tail vein nick technique, whole blood was collected into heparinized capillary tubes. Human platelets in mouse whole blood were detected by flow cytometry with monoclonal anti-human CD61-PE–conjugated antibodies. All subsequent recoveries were calculated as a percentage of the initial collection. Results The survival time of human platelets were significantly prolonged in SCID than in BALB/c,FVB mice. Recoveries at 4 hours after transfusion in SCID, BALB/c,FVB mice were 68.6%±8.1%(n =10),29.9%±6.5%(n =8),28.1%±5.5%(n =8), respectively, and with a T½ estimate of 8 hours for SCID, 2.5 hours for BALB/c and 2 hours for FVB mice. platelet storage lesions either by chemical treatment or by suboptimal conditions storage exhibited decreased recoveries in SCID mice. Conclusion The quality of platelet Products can be evaluated by assessing the survival of human platelets in SCID mice using flow cytometry.

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Mesenchymal Stem Cells Promote the Engraftment of Cord Blood CD34+ Cells but Do Not Accelarate Platelet Recovery NOD/SCID Mice.

Blood ◽

10.1182/blood.v106.11.1267.1267 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1267-1267

Author(s):

Yvette van Hensbergen ◽

Helen de Boer ◽

Manon C. Slot ◽

Laurus F. Schipper ◽

Anneke Brand ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Cord Blood ◽

Ex Vivo ◽

Human Platelets ◽

Scid Mice ◽

Post Transplantation ◽

Platelet Recovery ◽

Cd34 Cells ◽

Cord Blood Cd34

Abstract Aim: Delayed platelet reconstitution in the peripheral blood (PB) remains a problem in transplantation with umbilical cord blood (CB)-derived stem cells. Previously, we have shown that transplantation with ex-vivo expanded CB CD34+ cells (CD34exp) with thrombopoietin for 10 days, results in an accelerated platelet reconstitution in NOD/SCID mice. It has been shown that mesenchymal stem cells (MSC) are able to enhance the overall engraftment when co-transplanted with CB CD34+ cells. Therefore, we investigated whether co-transplantation of MSC with CD34+ cells or CD34exp cells may have an additive effect in shortening the time to platelet recovery and on the total number of platelets in the PB at 6 weeks after transplantation. Methods: To evaluate the time to platelet recovery and the total number of platelets at 6 weeks after transplantation, we used 4 groups of irradiated NOD/SCID mice, divided according to the transplant received: 1) CD34+ 2) MSC+CD34+ 3) CD34exp 4) MSC+CD34exp. Human platelet recovery was measured twice a week for the first three weeks and once a week thereafter, using an assay that reliably detects 1x106plt/L. The percentage of human CD45+ cells in the bone marrow (BM) was evaluated at 6 weeks after transplantation. Results: In accordance with previous experiments, platelet recovery started earlier in mice transplanted with CD34exp cells compared to CD34+ cells (Table 1). Co-transplantation of MSC with CD34+ cells did not result in an accelerated platelet recovery during the first 2 weeks after transplantation, as was observed for expanded cells. However, co-transplantation of MSC did enhance the number of platelets at 6 weeks after transplantation (454.2±264.5 plt/μ l for MSC+CD34+ vs. 101.9±78.4 plt/μ l for CD34+). MSC had no affect on either the time to platelet recovery nor the total number of human platelets at 6 weeks after transplantation when co-transplanted with CD34exp cells. To assess the overall efficacy of the MSC on the engraftment of human CB cells, we evaluated the percentage of human CD45+ cells in the BM of the NOD/SCID mice at 6 weeks after transplantation. In mice transplanted with MSC+CD34+, the percentage of human CD45+ cells was higher compared to controls transplanted with CD34+ cells only (30.4% for MSC+CD34+ vs. 17.8% for CD34+). No further engraftment enhancing effect of MSC was observed following transplantation of CD34exp cells only (32.1% for CD34exp vs. 35.7% for MSC+CD34exp). Conclusion: Our results show that transplantation with CD34exp cells results in an accelerated platelet recovery in NOD/SCID mice, an effect that can not be achieved by co-transplantation of MSC+CD34+ cells. However, at 6 weeks after transplantation co-transplantation with MSC+CD34+ cells results in a higher number of platelets in the PB. In addition, the level of engraftment of human CD45+ cells in the BM of NOD/SCID mice is increased by co-transplantation of MSC+CD34+ cells. In contrast, MSC did not affect the time to platelet recovery, the number of human platelets at 6 weeks after transplantation, or the engraftment of human CD45+ cells in the BM when co-transplanted with CD34exp. Table 1: % of mice with ≥ 1x106 platelets/L in the PB Days post transplantation 6 9 13 16 CD34+ 0% 20% 67% 100% MSC+CD34+ 20% 0% 80% 100% CD34exp 83% 100% 100% 100% MSC+CD34exp 60% 100% 100% 100%

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Transplantation of Human Peripheral Blood CD34-Positive Cells in Combination with Ex Vivo Expanded Megakaryocytes Results in Fast Platelet Formation in NOD/SCID Mice.

Blood ◽

10.1182/blood.v108.11.3203.3203 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3203-3203

Author(s):

Marloes R. Tijssen ◽

Paula B. Van Hennik ◽

Franca Di Summa ◽

Peter L. Hordijk ◽

Jaap Jan Zwaginga ◽

...

Keyword(s):

Peripheral Blood ◽

Ex Vivo ◽

Human Platelets ◽

Human Cells ◽

Scid Mice ◽

Combination Group ◽

High Dose ◽

Severe Thrombocytopenia ◽

Cd34 Cells ◽

Group A

Abstract The period of severe thrombocytopenia after high dose chemotherapy may be shortened by the addition of ex vivo expanded megakaryocytes (MKs, CD41+ cells) to the graft. This has been shown for expanded cord blood stem cells in mouse models, but hardly any effect has been observed in clinical trials in PBSC transplantation. The aim of the present study is to show the fate, engraftment ability and platelet production capacity of ex vivo expanded megakaryocytes from human mobilized peripheral blood (MPB) in NOD/SCID mice. MPB CD34+ cells were cultured for 7 days in the presence of Tpo (100 ng/ml) and IL-1 (10 ng/ml). Cell numbers increased approximately 6-fold after 7 days of culture. Over 50% of the expanded cells expressed CD41, and only a limited number of cells still expressed CD34. After sublethal irradiation (3.5 Gy), NOD/SCID mice were transplanted with unmanipulated CD34+ cells (group A), expanded MKs (Group E) or a combination (group B, C, D) [Table 1]. As a control, the mice of group F did not receive any cells after irradiation. Blood was collected at day 3, 7, 10, 14, 21, 28, and 35 after transplantation. After three days, human platelets could be detected in the blood of the mice from groups C and E. After 7 days, human platelets were detected in the blood of the mice from all groups that received a transplant, except for group A. In the mice of the groups A, B, C and D platelet numbers increased till day 14 (to an average of 6.9 × 10^6/ml blood) with a small decrease toward day 35 (1.3 × 10^6/ml). The mice of group E reached a maximum of 3.4 × 10^5 human platelets per ml blood at day 10 and numbers declined from thereon to be below threshold at day 21. At day 35, mice were sacrificed and the presence of human cells in the blood, lung, spleen and BM was determined. In the BM, the chimerism (CD45+ cells) correlated with the number of unmanipulated CD34+ cells transplanted per group. This was also observed for all other lineages tested in the BM and a similar pattern was found in the spleen and blood. However, the percentages of CD45+ cells in spleen and blood were lower than in BM. No human cells were detected in the lung. In summary, IL-1- and Tpo-expanded MKs can significantly contribute to thrombocytopoiesis during the first days after transplantation. This indicates that the period of thrombocytopenia after intensive chemotherapy can be overcome by the co-transplantation of ex vivo generated human MPB-derived MKs. It may therefore be interesting to extend our culture protocol to a clinical setting. Group Cells Cells transplanted Mice (n) A MPB CD34-positive 4.5×106 NC/mouse 100% 4 B MPB CD34-positive + expanded CD34-positive 4.05×106 NC + 0.45×106 NC input culture/mouse 90% + 10% 4 C MPB CD34-positive + expanded CD34-positive 4.5×106 NC + 4.5×106 NC input culture/mouse 100% + 100% 4 D MPB CD34-positive + expanded CD34-positive 2.25×106 NC + 2.25×106 NC input culture/mouse 50% + 50% 4 E Expanded CD34-positive 4.5×106 NC input culture/mouse 100% 4 F Control - 3

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Xenotransplantation of immunodeficient mice with mobilized human blood CD34+ cells provides an in vivo model for human megakaryocytopoiesis and platelet production

Blood ◽

10.1182/blood.v97.6.1635 ◽

2001 ◽

Vol 97 (6) ◽

pp. 1635-1643 ◽

Cited By ~ 31

Author(s):

Lia E. Perez ◽

Henry M. Rinder ◽

Chao Wang ◽

Jayne B. Tracey ◽

Noel Maun ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Ex Vivo ◽

Human Platelets ◽

Scid Mice ◽

In Vivo Model ◽

Specific Flow ◽

Platelet Production

The study of megakaryocytopoiesis has been based largely on in vitro assays. We characterize an in vivo model of megakaryocyte and platelet development in which human peripheral blood stem cells (PBSCs) differentiate along megakaryocytic as well as myeloid/lymphoid lineages in sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD-SCID) mice. Human hematopoiesis preferentially occurs in the bone marrow of the murine recipients, and engraftment is independent of exogenous cytokines. Human colony-forming units–megakaryocyte (CFU-MK) develop predominantly in the bone marrow, and their presence correlates with the overall degree of human cell engraftment. Using a sensitive and specific flow cytometric assay, human platelets are detected in the peripheral blood from weeks 1 to 8 after transplantation. The number of circulating human platelets peaks at week 3 with a mean of 20 × 109/L. These human platelets are functional as assessed by CD62P expression in response to thrombin stimulation in vitro. Exogenous cytokines have a detrimental effect on CFU-MK production after 2 weeks, and animals treated with these cytokines have no circulating platelets 8 weeks after transplantation. Although cytokine stimulation of human PBSCs ex vivo led to a significant increase in CFU-MK, CD34+/41+, and CD41+ cells, these ex vivo expanded cells provided only delayed and transient platelet production in vivo, and no CFU-MK developed in vivo after transplantation. In conclusion, xenogeneic transplantation of human PBSCs into NOD/SCID mice provides an excellent in vivo model to study human megakaryocytopoiesis and platelet production.

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Effects of a Monoclonal Antibody to Glycoprotein IIb/IIIa (P256) and of Enzymically Derived Fragments of P256 on Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648166 ◽

1991 ◽

Vol 65 (04) ◽

pp. 432-437 ◽

Cited By ~ 9

Author(s):

A W J Stuttle ◽

M J Powling ◽

J M Ritter ◽

R M Hardisty

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Platelet Aggregation ◽

Calcium Ions ◽

Human Platelet ◽

Human Platelets ◽

In Vivo Detection ◽

Fibrinogen Binding ◽

Specific Antagonist

SummaryThe anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10−9−3 × 10−8 M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10−7 M. P256–induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In–111–labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications

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Platelet Decrease and Efficacy of Platelet-Rich Plasma Return for Peripheral Blood Stem Cell Apheresis

Blood ◽

10.1182/blood-2020-142316 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 29-30

Author(s):

Takahiro Shima ◽

Teppei Sakoda ◽

Tomoko Henzan ◽

Yuya Kunisaki ◽

Takahiro Maeda ◽

...

Keyword(s):

Multivariate Analysis ◽

Stem Cell ◽

Platelet Count ◽

Peripheral Blood ◽

Peripheral Blood Stem Cell ◽

Platelet Rich Plasma ◽

Blood Stem Cell ◽

Platelet Recovery ◽

Platelet Counts ◽

Cd34 Cells

Peripheral blood stem cell (PBSC) transplantation is a key treatment option for hematological diseases and widely performed in clinical practice. Platelet loss is the major complication of PBSC apheresis, and platelet-rich plasma (PRP) return is recommended in case of severe platelet decrease following apheresis; however, little is known about the frequency and severity of platelet loss nor the efficacy of PRP return post-apheresis. To address these questions, we assessed changes in platelet counts following PBSC-related apheresis in 270 allogeneic (allo)- and 105 autologous (auto)-PBSC settings. We also evaluated efficacy of PRP transfusion on platelet recovery post-apheresis. Platelet counts reduced up to 70% post-apheresis in both allo- and auto-PBSC settings, while severe platelet count decrease (< 50 x 109/L) was only observed in auto-PBSC patients (Figure 1). We next analyzed the relationship between severe platelet (< 50 x 109/L) after apheresis and several clinical factors by using univariate and multivariate analysis for auto-PBSC patients. As shown in Table 1, in univariate analysis, severe platelet counts following auto-PBSC apheresis was found more frequently in patients with lower platelet count, lower percentage of CD34+ cells in PB at pre-apheresis, repeated round of apheresis, and smaller number of collected CD34+ cells. On the other hand, in multivariate analysis, the white blood cell (WBC) counts pre-apheresis was the only significant risk factor of severe platelet count following apheresis (p = 0.038). We finally analyzed the transitions of platelet counts in the setting of apheresis. The median platelet counts at pre-apheresis, post-apheresis, and post-PRP return were 187.0 x 109/L, 132.0 x 109/L, and 154.0 x 109/L for allo-PBSC apheresis, and 147.0 x 109/L, 111.0 x 109/L, and 127.0 x 109/L for auto-PBSC apheresis (p < 0.0001 for all, allo-PBSC donors and auto-PBSC patients, respectively) (Figure 2), indicating that PRP return post-apheresis facilitated a rapid platelet recovery in both allo- and auto-settings. Collectively, our data suggest that WBC counts pre-apheresis is a useful predictor for severe platelet decrease following auto-PBSC apheresis and that PRP return is an effective mean to facilitate platelet recovery post-apheresis. Disclosures No relevant conflicts of interest to declare.

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CD34-positive cells isolated from cryopreserved peripheral-blood progenitor cells can be expanded ex vivo and used for transplantation with little or no toxicity.

Journal of Clinical Oncology ◽

10.1200/jco.1996.14.6.1839 ◽

1996 ◽

Vol 14 (6) ◽

pp. 1839-1847 ◽

Cited By ~ 80

Author(s):

M J Alcorn ◽

T L Holyoake ◽

L Richmond ◽

C Pearson ◽

E Farrell ◽

...

Keyword(s):

Progenitor Cells ◽

Peripheral Blood ◽

Large Scale ◽

Ex Vivo ◽

Interleukin 1 ◽

Cell Number ◽

Autologous Serum ◽

Platelet Recovery ◽

Peripheral Blood Progenitor Cells ◽

Historical Controls

PURPOSE The objectives of this phase I study were to assess the feasibility of using cryopreserved peripheral-blood progenitor cells (PBPC) for large-scale CD34 selection and subsequent expansion, and the safety of their use for reinfusion following chemoradiotherapy. PATIENTS AND METHODS For 10 patients with nonmyeloid malignancy, an aliquot from a PBPC harvest was recovered from liquid nitrogen, and CD34 selected using the Isolex system (Baxter Healthcare, Newbury, United Kingdom) and expanded for 8 days ex vivo in a medium free of animal proteins but supplemented with autologous serum, stemcell factor (SCF), interleukin-1 beta (IL-1 beta), IL-3, IL-6, and erythropoietin. RESULTS The mean increase for cell number was 21-fold, for colony-forming units-granulocyte/macrophage (CFU-GM) 139-fold, and for burst-forming units-erythroid (BFU-E) 114-fold. The expanded cells were reinfused in tandem with unmanipulated material (> or = 25 x 10(4) CFU-GM/kg). The patients did not experience any adverse effects immediately on cell infusion or within 48 hours. The 10 index patients were compared with 10 historical controls for parameters of myelosuppressive morbidity. In this small study, there were no differences in either neutrophil or platelet recovery between the patients who received expanded cells and historical controls. CONCLUSION These data demonstrate that CD34 cells can successfully be selected from cryopreserved material, expanded ex vivo on a large scale, and safely reinfused following myeloablative conditioning regimens.

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Characterization of ‘Early’ vs ‘Late’ Endothelial Progenitor Cells (EPCs) Which Are Derived from Human Umbilical Cord Blood (HCB) during Ex Vivo Expansion.

Blood ◽

10.1182/blood.v106.11.1706.1706 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1706-1706

Author(s):

Eun-Sun Yoo ◽

Jee-Young Ahn ◽

Yun-Kyung Bae ◽

Seung-Eun Lee ◽

Sang min Lee ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Ex Vivo ◽

Tube Formation ◽

Ex Vivo Expansion ◽

Human Umbilical Cord ◽

Vascular Injuries ◽

Rt Pcr ◽

Different Types

Abstract EPCs have been isolated from adult peripheral blood and bone marrow. Recently, several groups reported that two types (‘early’ & ‘late’) of EPC could be isolated from peripheral blood and bone marrow when pertinent cocktails of cytokines were used. Interestingly, early and late EPCs are different in terms of expression of surface markers, the abilities of tube formation in vitro and the capabilities of re-vascularization on hind limb ischemia models in mice. We found EPC formation during ex vivo expansion of HCB and one EPC could be found from 314 CD34+ cells from HCB based on limiting dilutional assay (ref. Stem Cells; 2003, Yoo et al). However, little is known about the characteristics of ‘early’ and ‘late’ EPCs that are derived from HCB. In this study, our aims are to isolate the ‘early’ and ‘late’ EPCs from HCB during ex-vivo HCB expansion period and to characterize the biologic properties between ‘early’ and ‘late’ EPCs. 1 x 108 mononuclear cells were plated on a 100mm culture dish coated with 50ug/ml of human fibronectin (Calbiochem) and cultured in EGM-2 BulletKit system (Clonetics). Endothelial cells were assessed by colony counts, flow cytometry, proliferation assay, RT-PCR and in vitro tube formation in Matrigel plate. Migration of EPCs were also measured by in vitro transmigration assay in the presence of VEGF and SDF-1. In results, early spindle-shaped cells (‘early’ EPCs) which were grown at first week of culture were positive for CD31, CD14 and CXCR-4. Cobblestone shaped cells (‘late’ EPC) were in peak growth at second and third weeks of culture and were also positive using above antibodies except CD14. Early EPCs had not expressed mRNA of KDR, vWF and VE-Cadherin by RT-PCR. However, late EPCs expressed high level of mRNA of those endothelial marker genes. Both early and late EPCs expressed mRNA of eNOS. Late EPC produced more nitric oxide and formed more capillary tubes than those of early spindle-shaped cells. Early EPCs were readily migrated by VEGF and SDF-1 compared with those of late EPCs. In conclusions, we have found two different types of EPCs with different biologic properties during HCB ex vivo expansion. These findings may have potential clinical applications for “cell therapy” on vascular injuries (ie, hindlimb ischemia and myocardial infacrtion). Murine models for vascular injuries are being established to test the efficacy of different types of EPCs from HCB in our Lab.

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