A comparative analysis of the transplant potential of umbilical cord blood versus mobilized peripheral blood stem cells

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1 J Nippon Med Sch 1997; 64 (4) (307)21 Originals A comparative analysis of the transplant potential of umbilical cord blood versus mobilized peripheral blood stem cells Seiji Gomi, Setsuo Hasegawa, Kazuo Dan and Ichiji Wakabayashi Third Department of Internal Medicine, Nippon Medical School Abstract Human umbilical cord blood (UCB) is currently considered as a third source of hematopoietic stem cells for transplantation, following bone marrow and growth-factormobilized peripheral blood (MPB). To evaluate the potential benefits of UCB, we performed a comparative study of the properties of the stem cells in UCB and MPB samples. CD 34+ cell determination and CFU-GM colony assay showed a lower frequency of committed progenitors in UCB than in MPB. In contrast, a higher level of the CD 34+ CD 38- subset in UCB suggested that more primitive, multipotent progenitors are enriched in UCB than in MPB. Phenotypic analysis of UCB lymphocytes revealed a reduced level of T cell subsets, especially cytotoxic CD 8+ lymphocytes, which might minimize graft versus host disease in clinical practice. In conclusion, UCB is an attractive alternative source for stem cell transplantation, but ex vivo expansion of stem/progenitor cells could be effective for attaining rapid and safer hemopoietic reconstitution. (J Nippon Med Sch 1997 ; 64: ) Key words: umbilical cord blood, mobilized peripheral blood, stem cells, hematopoietic progenitors, transplantation Introduction Bone marrow has for years been the principal source of transplantable hematopoietic stem cells and progenitor cells. There has been much interest in other ways of obtaining hematopoietic stem cells for transplantation. In recent years, autologous peripheral blood stem cell transplantation (PBSCT) has attracted considerable interest, mainly because it results in more rapid recovery of neutrophils and platelets than autologous bone marrow transplantation. In the last few years, human umbilical cord blood (UCB) has been shown to contain large numbers of hematopoietic stem cells and to be a suitable source of hematopoietic stem cells for transplantation. In fact, UCB has been successfully applied as a source of clinically transplantable stem cells. Given its general availability, ease of procurement, and progenitor content, UCB is an attractive alternative to bone marrow or growth factor mobilized peripheral blood as a source of transplantable hematopoietic tissue. With UCB, there is virtually no risk to the donor. Another advantage that cord blood seems to have over the other sources of donated hematopoietic cells is the immunological naivety of the cells, suggesting reduced severity of graft-versus-host disease (GVHD). Furthermore, the use of UCB might also reduce the risk of transmission of some viral infections to the recipient. There is, however, a finite number of hematopoietic progenitor cells in a donation of UCB. It remains unclear whether a single collection of UCB contains enough progenitors to allow successful engraftment in adult patients, which ultimately depends on the quality of UCB as a source of stem cells. With the aim of evaluating the transplant potential of UCB, we performed flowcytometric Correspondence to Seiji Gomi, Third Department of Internal Medicine, Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo, 113 Japan

2 22(308) phenotypic analysis of early hematopoietic progenitors in UCB compared to MPB, which established its position as a source of stem cell transplantation. We also determined the correlation of the frequency of CD 34 + cells with clonogenic progenitors based on colony assays using methylcellulose. Further, a comparative analysis of the lymphocyte phenotypes in UCB and MPB was performed to assess their immunological naivety relevant to GVHD. Materials and Methods Collection and fractionation of UCB Umbilical cord venous blood samples were collected, during 20 normal full-term (from 37 to 41 gestational weeks) deliveries, in syringes containing preservative-free sodium heparin at a final concentration of 20 U/ml. These studies were approved by the institutional review board of Nippon Medical School. Although cord blood samples were collected from placentas destined for discard after delivery, informed consent was obtained. Light density mononuclear cells were isolated by Ficoll-Hypaque (Sigma, USA) gradient separation at a density of g/ml. Mononuclear fractions were washed twice and suspended in Iscove's modified Dulbecco' s Medium (IMDM) (Gibco, USA). Collection and fractionation of MPB Ten MPB samples were obtained from eight patients (two males and six females, age range: years) undergoing autologous peripheral blood stem cell transplantation, including four cases of non-hodgkin lymphoma (NHL), two cases of acute lymphocytic leukemia (ALL) and two cases of acute myeloid leukemia (AML). After intensive chemotherapy, rhg-csf (Chugai, Japan) (75,ug/day, s.c.) was used to mobilize stem cells until the last day of harvest. MPB was collected on two consecutive days after the peripheral white blood cell count reached 10,000/ƒÊl, using a Fresenius AS 104 continuous flow cell separator (Fresenius, Germany). In two patients (one NHL, one AML), harvests were performed twice with an interval of more than one month. Determination of CD 34+ cells Analysis of CD 34+ cells in the UCB and MPB mononuclear cells was performed by flowcytometry using a FACScan (Beckton Dickinson Immunocytometry System (BDIS), USA). Briefly, erythrocytes in samples were lysed with a lysing buffer (prepared by dissolving 8.26 g of NH,CI, 1.00 g of KHCO3 and 0.04 g of EDTA-4 Na in 1 liter of distilled water adjusted to ph 7.3) at room temperature for 10 min. The cell concentration was adjusted to 1 } 106 cells/ml with phosphate-buffered saline (PBS, 0.1% BSA, 0.1% sodium azide). The cell suspension was incubated with PBS containing 1.0% mouse serum (ICN Immuno Biologica, USA) at 4 C for 10 min to block non-specific reactions. A monoclonal antibody was then added to the cell suspension, and this was incubated at 4 Ž for 30 min, protected from light. The isotype controls used were mouse immunoglobulins IgG 1 and IgG 2 a conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). After incubation the cells were washed twice with PBS, fixed with 1.0% paraformaldehyde in PBS, and analyzed by flowcytometry. The monoclonal antibody to the CD 34 antigen was obtained from Becton Dickinson (USA). Phenotypic analysis of CD 34+ cells Analysis of the CD 34+ subpopulations in UCB and MPB mononuclear cells was performed by flowcytometry with immunofluorescence staining by the dual-color direct immunofluorescence method. All antibodies used in this study, including anti- CD 38 antibody, anti-cd 33 antibody and anti-hla- DR antibody, were obtained from Becton Dickinson (USA). The relative percentages of expression of CD 38(-), CD 33(-) and HLA-DR(-) on CD 34 positive cells were calculated. Clonogenic assays Clonogenic progenitors were assayed according to a modified form of the method of Dan et al. Mononuclear cells were plated in semisolid cultures; cell numbers varied from 3-5 ~ 105/ml. Clonogenic assays in methylcellulose for CFU-GM were performed in quadruplicate with 100 ng/ml G-CSF (Kyowa, Japan) and 100 ng/ml stem cell factor (SCF) (Kirin, Japan). Each culture dish contained 1 ml IMDM, 15% fetal calf serum (filton, Australia) and 0.8% methylcellulose (Sigma, USA). After 12 days of incubation in a humidified atmosphere at 37 Ž and 5% CO2 colonies (>40 cells) were counted using an inverted microscope. Clonogenic assays for BFU-E were also performed in quadruplicate with 2 U/ml erythropoietin (Chugai, Japan), 100 ng/ml

3 (309)23 ous population of immature and committed progenitor cells. The more immature precursor cells can be distinguished from their committed progenitors based on the expression or lack of expression of some antigens. The relative percentages of expression of CD 38(-), CD 33(-) and HLA-DR(-) on CD 34+ cells are presented in Table 1. The relative percentages of CD 38(-) and HLA-DR(-) on CD 34+ cells were significantly elevated in UCB compared to MPB. This observation indicates that UCB contains a significantly larger number of immature progenitor cells. In contrast, the absence of coexpression of CD 34 with CD 33 was not statistically different between UCB and MPB. SCF and 100 ng/ml IL-3 (Kirin, Japan). Each culture dish contained 1 ml IMDM, 2 ~ 10-4 mol/l 2- mercaptoethanol (Wako Pure Chemical Industries, Japan), 20% fetal calf serum, 1% bovine serum albumin (Sigma, USA) and 0.8% methylcellulose. After 14 days of incubation in a humidified atmosphere at 37 Ž and 5% CO2, burst-forming colonies were counted. Phenotypic analysis of lymphocytes Phenotypic analysis of UCB and MPB was performed by flowcytometry according to the method described above. The anti-cd 2, CD 3, CD 4, CD 5, CD 8, CD 10, CD 19, CD 20, CD 16 and CD 56 monoclonal antibodies were obtained from Becton Dickinson (USA). Appropriate isotype controls were performed for each sample. Comparison of numbers of clonogenic progenitors in UCB and MPB Statistical analysis To evaluate the engraftmentpotential in clinical To assess significant differences in all of the results for the UCB and MPB samples, the Mann- Whitney U-test was used. A standard significance practice, we performed a clonogenic assay of CFU- GM and BFU-E. The numbers of CFU-GM and BFU-E per 1 ~ 104 of mononuclear cells in UCB and level of p<0.05 was chosen. Results Comparison of UCB and MPB CD34 + cells The CD 34 antigen is stage-specific and identifies cells in the earlier stages of hematopoietic differentiation. Fig. 1 shows the percentages of CD 34+ cells in two different preparations of mononuclear cells as determined by flowcytometry. The average CD 34+ cell percentage was 0.90 (± 0.58)% in UCB and 1.79 ( } 2.20)% in MPB, without a statistically significant difference. Comparison of UCB and MPB CD34 + cell subpopulations The cells expressing the CD 34 antigen are enriched for progenitor cells but represent a heterogene- Fig. 1 Comparison of the percentage of CD34+ cells in mononuclear cell preparations from UCB and MPB. Abbreviations are the same as described in the legend for Table 1. Horizontal bars show 10 percentile, 25 percentile, 50 percentile, 75 percentile and 90 percentile. Table 1 Comparison of CD 34+ subpopulations in UCB and MPB Data repressent the mean }SD of relative percentages UCB : umbilical cord blood, MPB : mobilized peripheral blood.

4 24(310) Table 2 Comparison of the numbers of clonogenic progenitors in UCB and MPB Data show colony number (mean }SD) per 1 ~ 104 mononuclear cells CFU-GM : colony-forming unit-granulocyte-macrophage, BFU-E : burst-forming unit-erythroid. Other abbreviations are the same as described in the legend for Table 1. MPB are presented in Table 2. Boh of these committed progenitors were more numerous in MPB than in UCB samples. Comparison of the numbers of colonies generated per CD 34+ cell revealed a significant difference in CFU-GM (MPB> UCB) but not in BFU-E (Fig. 2). Comparison of lymphocyte subpopulations in UCB and MPB Characterization of the lymphocyte subpopulations in UCB compared with MPB was performed by flowcytometric analysis (Table 3). The percentages of T cell subsets, including CD 2 and CD 3, were significantly decreased in UCB compared to MPB. Although there was no difference in the numbers of CD 4 subset (helper T cells), the percentage of CD 8 (cytotoxic T cells) was reduced almost by half in UCB, resulting in a higher rate of CD 4/ CD 8 compared with in MPB (Fig. 3). In contrast, the percentages of B cell subsets, including CD 10, CD 19 and CD 20, were markedly elevated in UCB. There was no significant difference in the NK subpopulations expressing the CD 16 or CD 56 antigen. Discussion Fig. 2 Comparison of number of colonies per CD34+ cell in UCB and MPB. Colony numbers were divided by CD34 values. a: CFU- GM; b: BFU-E. Abbreviations are the same as described in the legend for Table 1 and Fig. 1. In this study, we performed a comparative analysis of the characteristics of hematopoietic progenitors in UCB and MPB samples. Our data showed that the percentage of CD 34 + cells is lower and the number of CFU-GM colonies is significantly reduced in UCB compared to MPB. However, these findings do not mean that UCB has inferior engraftment potential as a source of hematopoietic stem cells. CFU-GM are considered not as multipotent progenitors but as myeloid-committed progenitors. Further, hematopoietic cells expressing the CD 34 antigen are enriched for progenitor cells but represent a heterogeneous population of immature and committed progenitor cells", '" The immature precursor cells can be distin-

5 (311)25 Table 3 Comparison of lymphocyte subpopulations in UCB and MPB Data represent the mean }SD of percentages. Abbreviations are the same as described in the legend for Table 1. Fig. 3 CD4/CD8 ratios in UCB and MPB. Abbreviations are the same as described in the legend for Table 1 and Fig. 1. guished from their committed progenitors based on their lack of expression of some antigens associated with commitment of hematopoietic CD 34+ stem cells, including the CD 38, CD 33 and HLA-DR antigens, or their expression of some antigens such as thy-1 Currently, the most promising marker of CD 34+ cell differentiation seems to be the CD 38 antigen, which appears to occur as an early event in the differentiation of hematopoietic cells into erythroid, myeloid, B and T lymphoid lineages18. The CD 34+ CD 38- cells thus represent very immature progenitors, which have conserved the capacity for self -renewal. In our study, the expression of CD 38, CD 33 and HLA-DR antigens on CD 34+ cells was analyzed and compared for the UCB and MPB samples. As a result, CD 34 + CD 38- and CD 34+ HLA-DR-subpopulations were found to be significantly increased in UCB compared to MPB. These data suggest that UCB contains more immature, multipotent progenitors, which play a critical role in hematopoietic reconstitution. Characterization of the UCB lymphocytes revealed lower levels of T cell subsets and reciprocal elevation of B cell subsets compared to the MPB lymphocytes. It should be noted that UCB contains approximately one-half the number of CD 8+ cells as MPB, suggesting reduced cytotoxic activity of UCB lymphocytes. Recently, it was reported that cord blood T cells generate a vigorous proliferative response, yet produce little antigen-specific cytotoxity8-10. UCB immune cells are presumed not to be fully mature, which may minimize GVHD. Kurtzberg et al.s and Wagner et al. recently reported that HLA-mismatched UCB from unrelated donors can be an effective alternative source of stem cells for hematopoietic reconstitution. The low frequency and mild degree of GVHD may be related to the immunological naivety of the UCB lymphocytes and macrophages. In conclusion, UCB contains more primitive, multipotential stem cells and immature lymphocytes than MPB, which could reinforce the feasibility of UCB transplantatin. It remains to be clarified whether UCB contains enough stem cells to reproduce the hematopoietic system of an adult. Recently, Laporte et al. described an adult case with chronic myelogenous leukemia who underwent successful transplantation of UCB stem cells. However, the lower level of committed progenitors in UCB may cause delayed hematopoietic reconstitution. Thus, ex vivo expansion of UCB stem/progenitor cells with various cytokines could be an effective

6 26(312) means to ensure safer transplantation, especially for adult patients. This approach is now under investigation in our laboratory and various other institutions. Acknowledgments: We wish to thank Prof. T. Araki and the doctors of the Department of Obstetrics and Gynecology of Nippon Medical School for the supply of UCB. We would also like to express our great appreciation to all the nursing staff of the obstetric ward for their assistance in UCB collection. References 1. Sheridan WP: Effects of peripheral blood progenitor cells mobilized by filgrastim (G-CSF) on platelet recovery after high-dose chemotherapy. Lancet 1992; 1339: Teshima T, Harada M, Takamatsu Y, Makino K, Taniguchi S, Inaba S, Kondo S, Tanaka T, Akashi K, Minamishima I, Ishii E, Nishimura J, Niho Y: Cytotoxic and cytotoxic/g-csf mobilization of peripheral blood stem cells and their autografting. Bone Marrow Transplant 1992; 10: Broxmeyer HE, Douglas GW, Hangoc G, Cooper S, Bard J, English D, Arny M, Thomas L, Boyse EA: Human umbilical cord blood as a potential source of transplantable hematopoeitic stem/progenitor cells. Proc Natl Acad Sci USA 1989; 86: Hirao A, Kawano Y, Takaue Y, Suzue T, Abe T, Sato J, Saito S, Okamoto Y, Makimoto A, Kawahito M, Kuroda Y: Engraftment potential of peripheral and cord blood stem cells evaluated by a longterm culture system. Exp Hematol 1994; 22: Gluckman E, Broxmeyer HE, Auerbach AD, Friedman HS, Douglas GW, Devergie A, Esperou H, Thierry D, Socie G, Lehn P, Cooper S, English D, Kurtzberg J, Bard J, Boyse EA: Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical-cord blood from an HLAidentical sibling. New Eng J Med 1989; 321: Wagner JE, Kernan NA, Steinbuch M, Broxmeyer HE, Gluckman E: Allogeneic sibling umbilical-cordblood transplantation in children with malignant and non-malignant disease. Lancet 1995; 346: Kurtzberg J, Laughlin M, Graham ML, Smith C, Olson JF, Halperin EC, Ciocci G, Carrier C, Stevens CE, Rubinstein P: Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. New Eng J Med 1996; 335: Harris DT, Schumacher MJ, Locascio J, Besebcon FJ, Olson GB, DeLica D, Shenker L, Brad J, Boyse EA: Phenotypic and functional immaturity of human umbilical cord blood T lymphocytes. Proc Natl acad Sci USA 1992; 89: Harris DT, Locascio J, Besencon FJ: Analysis of the alloreactive capacity of human umbilical cord blood: Implications for graft-versus-host disease. one Marrow Transplant 1994; 14: Han P, Hodge G, Story C, Xu X: Phenotypic analysis of functional T-lymphocyte subtypes and natural killer cells in human cord blood: Relevance to umbilical cord blood transplantation. Brit J Haematol 1995; 89: Harris DT: in vitro and in vivo assessment of the graft-versus-leukemia activity of cord blood. Bone Marrow Transplant 1995; 15: Dan K, An E, Futaki M, Inokuchi K, Gomi S, Yamada T, Ogata K, Tanabe Y, Ohki I, Shinohara T, Nomura T: Megakaryocyte, erythroid and granulocyte-macrophage colony formation in myelodysplastic syndromes. Acta Hematol 1993; 89: Smith C, Casparetto C, Collins N, Gillio A, Muench MO, O'Reilly RJ, Moore MA: Purification and partial characterization of a human hematopoietic precursor population. Blood 1991; 77: Andrews RG, Bryant EM, Bartelmez SH, Muirhead DY, Knitter GH, Benginger W, Strong DM, Bernstein ID: CD 34+ marrow cells, devoid of T and B lymphocytes, reconstitute stable lymphopoiesis and myelopoiesis in lethally irradiated allogeneic baboons. Blood 1992; 80: Tracoff CM, Abboud MR, Laver J, Brandt JE, Hoffman R, Law P, Ishizawa L, Srour E: Evaluation of the in vitro behavior of phenotypically deifined populations of umbilical cord blood hematopoietic progenitor cells. Exp Hematol 1994; 22: Bender JG, Unverzagt K, Walker DE, Lee W, Smith S, Williams S, Van Epps ED: Phenotypic analysis and characterization of CD 34+ cells from normal human bone marrow, cord blood, peripheral blood, and mobilized peripheral blood from patients undergoing autologous stem cell transplantation. Clin Immunol Immunopathol 1994; 70: Craig W, Kay R, Cutler RL, Lansdorp PM: Expression of Thy-I on human hematopoietic progenitor cells. J Exp Med 1991; 177: Terstappen LW, Huang S, Safford M, Lansdorp PM, Loken MR: Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD 34+ CD 38- progenitor cells. Blood 1991; 77: Cicuttni FM, Welch K, Boyd AW: Characterrization of CD 34+ HLA-DR-CD 38- and CD 34+ HLA-DR- CD 38- progenitor cells from human umbilical cord

7 (313)27 blood. Growth Factors 1994; 10: Hao QL, Shah AJ, Thiemann FT, Smogorzewska EM, Crooks GM: A functional comparison of CD 34+ CD 38- cells in cord blood and bone marrow. Blood 1995; 86: Reems JA, Storb BT: Cell cycle and functional differences between CD 34+/CD 38 hi and CD 34+/ 34 lo human marrow cells after in vitro cytokine exposure. Blood 1995; 85: Laporte JP, Gorin NC, Rubinstein P, Lesage S, Portnoi MF, Barbu V, Lopez M, Douay L, Najman A: Cord-blood transplantation from an unrelated donor in an adult with chronic myelogenous leukemia. New Eng J Med 1996; 335: (Received, December 13, 1996) (Accepted for publication, January 9, 1997)