Low Numbers of Megakaryocyte Progenitors in Grafts of Cord Blood Cells May Result in Delayed Platelet Recovery After Cord Blood Cell Transplant

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1 Low Numbers of Megakaryocyte Progenitors in Grafts of Cord Blood Cells May Result in Delayed Platelet Recovery After Cord Blood Cell Transplant SACHIYO KANAMARU, a YOSHIFUMI KAWANO, a TSUTOMU WATANABE, a RYUJI NAKAGAWA, a HIROKO SUZUYA, a TOSHIHIRO ONISHI, a JUN YAMAZAKI, b TAKAYOSHI NAKAYAMA, b YASUHIRO KURODA, a YOICHI TAKAUE c a Department of Pediatrics, University of Tokushima, Tokushima, Japan; b Nakayama Maternity Clinic, Tokushima, Japan; c Stem Cell Transplantation Unit, National Cancer Center Hospital, Tokyo, Japan Key Words. Megakaryocyte progenitors Cord blood Hematopoietic cell transplantation ABSTRACT Delayed platelet recovery is an inherent problem with cord blood cell transplantation (CBCT). To investigate this problem, the number of human megakaryocyte (MK) progenitor cells in cord blood (CB; n = 24) was measured and compared with that in G-CSF-mobilized peripheral blood stem cells (PBSC; n = 25). The median numbers of colony-forming units for MK (CFU-MK) that were detected by a serum-free assay system in CB and peripheral blood (PB) were 26 (range, 6-102)/10 5 nucleated cells (NC) and 37 (2-540)/10 5 mononuclear cells (MNC), respectively. The numbers of colony-forming units for granulocyte/macrophage (CFU-GM) were 88 (33-241)/10 5 NC in CB and 138 (6.3-1,250)/10 5 MNC in PB. The frequencies of CD34 + cells in CB and PB were, respectively, 0.44% ( ) and 0.98% ( ). The numbers of CFU-MK in CB and PBSC were correlated with those of CD34 + cells. The estimated number of infused CFU-MK in CBCT was 1/15 that of PBSC transplantation (PBSCT), based upon the above data and the widely used standard doses for both types of transplants. Further, the numbers of infused CFU-MK in patients who received allogeneic PBSCT at our institute were inversely correlated with the speed of platelet recovery. These data indicate that delayed platelet recovery after CBCT is simply due to the low number of CFU-MK contained in grafts. Stem Cells 2000;18: INTRODUCTION Rapid hematopoietic recovery after hematopoietic stem cell transplantation (HSCT) results in a reduction of transplant-related complications [1]. In this regard, peripheral blood stem cell transplantation (PBSCT) has several advantages over bone marrow transplantation and cord blood cell transplantation (CBCT). Furthermore, it has been shown that rapid platelet recovery after PBSCT has economic benefits by reducing the cost of supportive therapy as well as reducing the risk of fatal bleeding due to severe thrombocytopenia. Estimation of the rate of platelet recovery after HSCT should contribute to the clinical management of such critical patients. On the other hand, in cases of CBCT, which have been developed more recently, the rate of platelet recovery is significantly delayed compared with that of PBSCT, although the number of platelets can be well maintained once engrafted [2-4]. In PBSCT, the rate of the recovery of granulocytes and platelets correlates with the number of CD34 + cells infused, while no similar correlation has been shown after CBCT. With regard to CBCT, the number of nuclear cells given is a reliable indicator for a successful engraftment after transplantation [5]. Based on these considerations, we measured the number of megakaryocyte progenitors (CFU-MK) in cord blood (CB) and G-CSF-mobilized peripheral blood stem cells (PBSC) in Correspondence: Yoshifumi Kawano, M.D., Department of Pediatrics, University of Tokushima, Kuramoto-cho, Tokushima, Japan. Telephone: ; Fax: ; ykawano@clin.med.tokushima-u.ac.jp Received January 10, 2000; accepted for publication March 4, AlphaMed Press /2000/$5.00/0 STEM CELLS 2000;18:

2 Kanamaru, Kawano, Watanabe et al. 191 normal donors for allogeneic transplantation with a newly developed serum-free assay system that enables us to detect platelet-specific glycoprotein IIb/IIIa. The results of this study may help to explain the differences between the two grafts. MATERIALS AND METHODS Cell Preparation A total of 24 human umbilical cord blood samples, obtained from term venous CB before placental delivery, were collected into a citrate phosphate dextrose-containing bag after obtaining consent forms from the mothers. Nuclear cells (NC) were isolated by the red blood cell sedimentation method using hydroxy ethyl starch (HES) [6]. Briefly, 6% HES (Hespan; DuPont Pharma; Wilmington, DE; was added to samples at a final percentage of 1% HES. After centrifugation for 5 min at 50 g to remove cells in the plasma from RBCs, some of the cells were resuspended at 2 to cells/ml in Iscove s modified Dulbecco s medium (IMDM) for evaluation, including WBC counts, CD34 + enumeration, myeloid progenitor assays (colony-forming units-granulocyte/ macrophage [CFU-GM]), and colony-forming unitsmegakaryocyte (CFU-MK) assays. The rest of the cell suspension was mixed with an equal volume of CP-1 medium (Kyokuto Seiyaku Kogyo; Tokyo, Japan) containing 12% HES, 10% dimethylsulfoxide, and 8% human albumin (Yoshitomi Pharmaceutical Co.; Osaka, Japan). The cell suspension was then placed into a deep freezer at 150 C (Legaci, Reyco, Asahi Life Science; Tokyo, Japan). PBSC were mobilized from normal, healthy, sibling donors for allogeneic PBSCT by five days of s.c. administration of G-CSF (10 g/kg/day: filgrastim, Kirin Brewery Co.; Tokyo, Japan; lenograstim, Chugai Pharmaceutical; Tokyo, Japan; or nartograstim, Kyowa-Hakko Kogyo Co.; Tokyo, Japan) with careful monitoring for possible side effects [7-10]. As reported elsewhere [11], PBSC were collected on days 5 and 6 with a Fenwal CS3000 plus (Baxter Healthcare Co.; Irvine, CA; and the total volume of blood processed was 300 ml/kg (maximum 10 liters). The mononuclear cells (MNC) were then enriched by a Percoll separation method [12]. The MNC were washed three times with phosphate buffered saline without Ca2 + or Mg2 + (PBS; Dulbecco s PBS-, Nissui; Tokyo, Japan) and resuspended with IMDM at concentrations of 2 to /ml for further experiments. The remaining MNC were frozen by the same method as for CB. These samples from donors were prepared after obtaining consent forms according to the regulation of the institutional review board. Megakaryocyte Progenitor Cell Assay Serum-free collagen assay was performed with a MegaCult -C kit (StemCell Technologies Inc.; Vancouver, Canada; [13]. As a source of megakaryocyte (MK) colony-stimulating activity, a combination of human recombinant growth factors was used: 1.1 mg/ml collagen, 1% bovine serum albumin (BSA), 10 g/ml bovine pancreatic insulin, 200 g/ml iron-saturated human transferrin, 50 ng/ml thrombopoietin, 10 ng/ml interleukin 6, and 10 ng/ml interleukin 3. The final culture mixture of 1.5 ml was dispensed into the two wells (0.75 ml each) of a chamber slide. Cultures were prepared according to the manufacturer s instructions and incubated in an ESPEC N 2 -O 2 -CO 2 BNP-110 incubator (Tabai ESPEC Co.; Osaka, Japan; which maintained a humid atmosphere of 5% carbon dioxide, 5% oxygen, and 90% nitrogen at 37 C. After 10 days of incubation, immunochemical staining was performed and CFU-MK colonies were counted. The alkaline phosphatase/anti-alkaline phosphatase method was used for staining. MK that express glycoprotein IIb/IIIa (CD41) will appear pink following fixation and staining. Counterstaining with Evan s blue causes the nuclei of all cells to appear pale blue, regardless of lineage. Therefore, we counted groups of cells with a pink membrane and blue nuclei as CFU-MK colonies. CFU-MK colonies ranged in size from three to several hundred cells per colony. CFU-GM Assay The methods have been described in detail previously [12]. Cells were incubated in methylcellulose medium supplemented with 20% fetal bovine serum ([FBS]; Filtron; Brooklyn, Australia), 450 g/ml of human transferrin (Sigma Chemicals T-1147; St. Louis, MO; 2 U/ml of human recombinant erythropoietin (Kirin Brewery Co.), 1% deionized delipidated BSA (Calbiochem 12657, Hoechst Japan; Tokyo, Japan; and a combination of recombinant human G-CSF (filgrastim; Kirin Brewery Co.), IL-3 (Kirin), and stem cell factor (Kirin), as previously reported. These stimulating factors were used at a final concentration of 20 ng/ml, which was the previously determined optimal concentration in our laboratory. Quadruplicate cultures were plated in volumes of 0.4 ml in 24-well tissue culture plates (Corning ; New York, NY; that were then placed in the incubator described above. Cultures were incubated for 13 to 15 days, and CFU-GM colonies were classified as colonies of 50 or more translucent cells using an inverted microscope. The mean number of colonies in four wells was calculated.

3 192 Megakaryocyte Progenitors in Grafts of Cord Blood Cells Flow Cytometry Analysis of CD34 + Cells The details of this procedure have been reported previously [14]. Briefly, aliquots ( ml) of cell suspension ( cells/ml) were mixed with 1.5 ml IMDM supplemented with 10% FBS and stored at 4 C for flow cytometry analysis performed within 48 h. Cells were stained with a fluorescein isothiocyanate (FITC)-conjugated CD45 antibody that detects all isoforms and glycoforms of the CD45 antigen family, and a phycoerythrin (PE)-conjugated CD34 antibody (HPCA-2, Becton Dickinson; Mountain View, CA; at a concentration of 1 g/10 6 cells. Control samples were stained with CD45-FITC and an IgG1-PE isotype. Red blood cells in the sample were lysed with a solution of 0.826% (w/v) NH 4 Cl, 0.1% KHCO 3, and 0.004% EDTA-4Na. Samples were analyzed with a FACScan flow cytometer (Becton Dickinson). A total of 50,000 CD45 + events were counted to identify the mononuclear cell fraction. The flow cytometric data were analyzed using a gated analysis via a set of SSC-FL parameters for the CD34 + cells to calculate the percentage of positive cells. Transplantation Procedure The speed of platelet recovery after PBSCT was evaluated in patients treated at our institute. The patients characteristics and the data for transplanted cells were presented in a previous report [15]. Briefly, the patients (four males, five females; median age eight years, range 2 to 14 years) were diagnosed with acute lymphoblastic leukemia (n = 5), acute myelogenous leukemia (n = 2), myelodysplastic syndrome (n = 1), or granulocytic sarcoma (n = 1), and underwent allogeneic PBSCT. The nine corresponding donors (four males, five females), aged 4 to 16 years (median eight years), were HLA-matched siblings. A combination of busulfan (4 mg/kg/day or 150 mg/m 2 /day, orally for 4 days) and melphalan (70 mg/m 2 /day i.v. for three days) was administered to all of the patients as a preparative regimen. Cryopreserved apheresis products were thawed and infused 48 h after conditioning. The number of CD34 + cells infused was 5.3 ( ) 10 6 /kg. Cyclosporin A and methylprednisolone were used for graft-versus-host disease prophylaxis. Statistical Analysis Statistical analysis was performed using the StatView program (Version 4.5; Abacus Concepts, Inc.; Berkeley, CA; for a Macintosh computer. Differences in CFU-GM, CD34 + cells, and CFU- MK between cord blood and mobilized PBSC were evaluated by the Mann-Whitney test or Student s t-test, as appropriate. RESULTS Cord Blood (n = 24) The median volume of CB collected was 78 ml (range, ml) and the median number of NC obtained after RBC sedimentation was 5.1 ( ) The numbers of NC did not correlate with the blood volumes harvested in our target samples (r = 0.215). The percentage of CD34 + cells in CB was 0.44 ( ), and the numbers of CFU-GM and CFU-MK per 10 5 NC were 88 (33-241) and 26 (6-102), respectively. Although the numbers of CFU-GM and CFU- MK per 10 5 NC did not correlate with those of CD34 + cells per 10 5 NC, the numbers of CFU-GM and CFU-MK per ml of CB were positively correlated with the number of CD34 + cells per ml of CB (Fig. 1). The ratios of CFU-MK to CD34 + cells and to CFU-GM were ( ) and ( ), respectively. G-CSF-Mobilized PBSC (n = 25) The numbers of CFU-GM and CFU-MK in PBSC were 138 (6.3-1,250)/10 5 MNC and 37 (2-540)/10 5 MNC, respectively. The percentage of CD34 + cells in PBSC was Numbers of progenitors CFU-GM ( 10 3 /ml) CFU-MK ( 10 3 /ml) r = p < r = p < CD34 + cells ( 10 4 /ml) Figure 1. Relationships between the numbers of CD34 + cells and colony-forming units for megakaryocyte (CFU-MK) (upper) or colony-forming units for granulocyte/macrophage (CFU-GM) (lower) in 1 ml of cord blood. Significant positive correlations were observed.

4 Kanamaru, Kawano, Watanabe et al. 193 Numbers of progenitors CFU-GM ( 10 5 MNC) CFU-MK ( 10 5 MNC) , r = p < r = p < Log 10 (Infused CFU-MK/kg) r = p = Platelets > /l (days) r = p = CD34 + cells ( 10 3 /10 5 MNC) Platelets > /l (days) Figure 2. Relationships between the numbers of CD34 + cells and colony-forming units for megakaryocyte (CFU-MK) (upper) or colony-forming units for granulocyte/macrophage (CFU-GM) (lower) in 10 5 mononuclear cells (MNC) of G-CSF-mobilized blood. Significant positive correlations were observed ( ), which was statistically identical to that of CB. The numbers of CFU-GM and CFU-MK per 10 5 MNC were correlated with those of CD34 + cells per 10 5 MNC (Fig. 2). The ratios of CFU-MK to CD34 + cells and to CFU- GM were ( ) and ( ), respectively, which were statistically identical to those in CB. Platelet Recovery After Allo-PBSCT and Estimation After CBCT A total of nine patients were treated with allo-pbsct. The number of CFU-MK infused was inversely correlated with the days to achieve a platelet count of >20 or /l (Fig. 3). The number of CFU-MK in those grafts was 3.37 ( ) 10 5 /kg. If patients are infused with /kg of CD34 + cells in allo-pbsct, the number of CFU-MK is estimated to be 1.41 ( ) 10 5 /kg using the above data. On the other hand, the estimated number of infused CFU-MK is ( ) 10 5 /kg, based on the hypothesis that NC per kg is the threshold dose for safe CBCT (Fig. 4). Figure 3. Relationship between the numbers of infused colony-forming units for megakaryocyte (CFU-MK) and the rate of platelet recovery. Days to achieve platelet counts of >20 or /l were inversely correlated with the doses of infused CFU-MK in nine pediatric patients who underwent allogeneic blood stem cell transplantation. DISCUSSION Ensuring rapid hematopoietic recovery after HSCT is very important for reducing the morbidity and mortality related to transplant-associated complications such as infection or bleeding. The recovery of hematopoiesis is related to the quantity of cells infused. Although relationships have been previously established between the recovery of granulocytes or platelets and the number of infused CFU-GM or CD34 + cells in PBSCT [16-18], this has not been seriously tested in CBCT. Moreover, studies that evaluated the relationships between the number of CFU-MK infused and the rate of platelet recovery after PBSCT have used individual methods, which makes it difficult to compare data [19-23]. On the other hand, there have been several reports concerning the relationship between subsets of CD34 + cells and the speed of platelet recovery after autologous transplantation [24, 25]. In this study, we compared the number of CFU-MK between PBSC and CB using recently developed sophisticated culture methods.

5 194 Megakaryocyte Progenitors in Grafts of Cord Blood Cells Infused CFU-MK (per Kg) CB PB Figure 4. Numbers of colony-forming units for megakaryocyte (CFU-MK) contained in grafts of cord blood and G-CSF-mobilized blood. The numbers of CFU-MK were estimated utilizing the widely used standard doses of nucleated cells per kg in cord blood cell transplantation and CD34 + cells/kg in peripheral blood stem cell transplantation. CBCT, which has attracted attention as a rich source of hematopoietic stem cells compared to bone marrow and PBSC, is particularly plagued by poor platelet recovery [2-4]. Hassan et al. reported that the delayed platelet recovery after allogeneic CBCT could be due to an inadequate number of CD34 + CD41 + megakaryocytic progenitor cells in the CB allografts. Although they compared the number of CD34 + CD41 + cells per ml of CB with that of BM, in contrast to our colony assay, we have drawn almost the same conclusion [26]. After confirming the relationship between the number of CFU-MK infused and the rate of platelet recovery in allo-pbsct, we tried to calculate the number of CFU-MK infused in both types of transplants. If we consider the threshold numbers of transfused cells to be /kg of CD34 + cells in PBSCT [27] and /kg of NC [5] in CBCT in allogeneic transplant settings, the estimated number of CFU-MK infused in CBCT was 1 /15 of that in PBSCT. Thus, we confirmed that the number of transfused CFU-MK in CBCT was significantly lower than that in PBSCT. Our data suggest that a consideration of the significance of such colony assays is necessary for safe clinical management after CBCT, if CBCT is to be successful with the present standard of /kg of NC. In conclusion, we confirmed that the delayed platelet recovery after CBCT is due to a lower number of CFU- MK infused. Further investigations are needed to establish a system for rapidly counting CFU-MK to make CBCT clinically safer. ACKNOWLEDGMENTS The authors are grateful to Ms. Yasuda for her technical assistance. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, and by a Grant-in-Aid for the Second-Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare. REFERENCES 1 Hassan HT, Zander AR. Thrombocytopenia after high-dose chemotherapy and autologous stem cell transplantation: an unresolved problem and possible approaches to resolve it. J Hematother 1996;5: Locatelli F, Maccario R, Comoli P et al. Hematopoietic and immune recovery after transplantation of cord blood progenitor cells in children. Bone Marrow Transplant 1996;18: Wagner JE. Umbilical cord blood transplantation: overview of the clinical experience. Blood Cells 1994;20: Nishihara H, Ohnuma K, Ikuta K et al. Unrelated umbilical cord-blood stem cell transplantation: a report from Kanagawa Cord Blood Bank, Japan. Int J Hematol 1998;68: Gluckman E, Rocha V, Boyer-Chammard A et al. Outcome of cord-blood transplantation from related and unrelated donors. N Engl J Med 1997;337: Regidor C, Posada M, Monteagudo D et al. Umbilical cord blood banking for unrelated transplantation: evaluation of cell separation and storage methods. Exp Hematol 1999;27: Anderlini P, Körbling M, Dale D et al. Allogeneic blood stem cell transplantation: considerations for donors. Blood 1997;90: Kitamura S, Kinouchi K, Fukumitsu K et al. A risk of pulmonary edema associated with G-CSF pretreatment. Masui 1997;46: Falzetti F, Aversa F, Minelli O et al. Spontaneous rupture of spleen during peripheral blood stem-cell mobilisation in a healthy donor. Lancet 1999;353: Becker PS, Wagle M, Matous S et al. Spontaneous splenic rupture following administration of granulocyte colony-stimulating factor (G-CSF): occurrence in an allogeneic donor of peripheral blood stem cells. Biol Blood Marrow Transplant 1997;3: Takaue Y, Kawano Y, Abe T et al. Collection and transplantation of peripheral blood in stem cells in very small children weighing 20 kg or less. Blood 1995;86: Takaue Y, Watanabe T, Kawano Y et al. Isolation and storage of peripheral blood hematopoietic stem cells for the autotransplantation in cancer children. Blood 1989;74:

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