Key Words. AC133 antigen CD34 antigen Cord blood Peripheral blood 2003;21:

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1 Stem Cells Original Article Differences Between Peripheral Blood and Cord Blood in the Kinetics of Lineage-Restricted Hematopoietic Cells: Implications for Delayed Platelet Recovery Following Cord Blood Transplantation KAZUTA YASUI, KAYOKO MATSUMOTO, FUMIYA HIRAYAMA, YOSHIHIKO TANI, TORU NAKANO Osaka Red Cross Blood Center, Osaka, Japan; Department of Molecular Cell Biology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Key Words. AC133 antigen CD34 antigen Cord blood Peripheral blood ABSTRACT Cord blood (CB) cells are a useful source of hematopoietic cells for transplantation. The hematopoietic activities of CB cells are different from those of bone marrow and peripheral blood (PB) cells. Platelet recovery is significantly slower after transplantation with CB cells than with cells from other sources. However, the cellular mechanisms underlying these differences have not been elucidated. We compared the surface marker expression profiles of PB and CB hematopoietic cells. We focused on two surface markers of hematopoietic cell immaturity, i.e., CD34 and AC133. In addition to differences in surface marker expression, the PB and CB cells showed nonidentical differentiation pathways from AC133 + CD34 + (immature) hematopoietic cells to terminally differentiated cells. The majority of the AC133 + CD34 + PB cells initially lost AC133 expression and eventually became AC133 CD34 cells. In contrast, the AC133 + CD34 + CB cells did not go through the intermediate AC133 CD34 + stage and lost both markers simultaneously. Meanwhile, the vast majority of megakaryocyte progenitors were of the AC133 CD34 + phenotype. We conclude that the delayed recovery of platelets after CB transplantation is due to both subpopulation distribution and the process of differentiation from AC133 + CD34 + cells. Stem Cells 2003;21: INTRODUCTION As an adjunct to existing techniques for bone marrow (BM) and peripheral blood (PB) cell transplantation, cord blood (CB) cell transplantation recently has been developed [1-4]. Quantitative and qualitative differences among hematopoietic progenitor cells from these three sources have been demonstrated [5-9]. One significant difference between CB cells and PB and BM cells is the rate of platelet recovery after transplantation. Although the number of platelets can be maintained once the cells are grafted, the rate of platelet recovery is delayed after CB transplantation [5-7]. The rate of granulocyte and platelet recovery following PB stem cell transplantation correlates with the number of transplanted CD34 + cells. However, a similar correlation has not been found with CB transplantation. Empirically, the number of nucleated cells present after transplantation has been regarded as the primary indicator of successful engraftment [10]. Correspondence: Kayoko Matsumoto, Ph.D., Osaka Red Cross Blood Center, Morinomiya , Johtoh-ku, Osaka , Japan. Telephone: ; Fax: ; kayokoma@a1.mbn.or.jp Received July 22, 2002; accepted for publication September 9, AlphaMed Press /2003/$5.00/0 STEM CELLS 2003;21:

2 144 Differentiation of CB and PB Cells The process of differentiation from immature hematopoietic cells to the various hematopoietic lineages is defined by the expression patterns of surface markers. Monoclonal antibodies against cell-surface antigens are utilized not only for the identification of differentiated blood cells, but also for the isolation of immature hematopoietic stem/progenitor cell subsets [11-13]. Antibodies to CD34 are commonly used in the isolation of human hematopoietic stem cells for transplantation [14, 15]. However, the utility of CD34 as a marker has been challenged by studies using the murine transplantation model [16-18]. In addition, several lines of evidence suggest the existence of human CD34 hematopoietic stem cells and that such cells are less mature than CD34 + stem cells [19-21]. Therefore, the newly discovered stem cell marker AC133 represents a strong candidate for the isolation of human-transplantable hematopoietic stem cells. The AC133 antigen is a five-transmembrane-spanning receptor that is mainly expressed on CD34 + cells, and is a marker of primitive human hematopoietic cells [22-25]. AC133 + cells are expressed on hematopoietic stem cells and have repopulating ability, since only AC133 + CD34 + cells repopulate nonobese diabetic/severe combined immunodeficient mice [26] and AC133 + cells are capable of establishing human-sheep hematopoietic chimeras following xenotransplantation [25]. Furthermore, precursor endothelial cells are present in the AC133 + cell population [27]. Recent studies have shown that AC133 expression is not limited to primitive blood cells, but is also present on unique cell populations with stem cell activities in nonhematopoietic tissues [27, 28]. Accordingly, AC133 can be regarded as a useful marker for immature human cells. In a previous study [29], we reported that the ratio of AC133-expressing cells to CD34 + cells was much higher in CB than in PB. This subpopulation diversity might have been due to the different biological characteristics of these cell types. In the present study, we speculated that the biological characteristics of the AC133 + CD34 + PB and CB cells might differ. We found that the differentiation of AC133 + CD34 + CB cells was quite different from that of AC133 + CD34 + PB cells in terms of surface marker expression, colony-forming ability, and ploidy level of the megakaryocytes (MKs). The delayed recovery of platelets after CB transplantation could be due to intrinsic differences in the AC133 + CD34 + (immature) hematopoietic cell population. MATERIALS AND METHODS Cell Sources Cord blood was collected following normal pregnancies and deliveries, and with the informed consent of the parents. Buffy coat PB cells were obtained from volunteer blood donors. All samples were processed within 24 hours of collection. Cell Purification by Magnetic-Activated Cell Sorting (MACS) Mononuclear cells (MNCs) were isolated from PB and CB and were subjected to immunomagnetic separation using a MACS AC133 Cell Isolation Kit or a MACS CD34 Progenitor Cell Isolation Kit (Miltenyi Biotech; Auburn, CA; as described previously [29]. Briefly, MNCs were incubated for 30 minutes at 6ºC with an FcR-blocking reagent (human IgG) and AC133 or CD34 MicroBeads, i.e., microbeads conjugated with monoclonal mouse anti-human antibodies that were directed against either AC133/1 or CD34. After washing with phosphate buffered saline (PBS)/0.5% bovine serum antigen/5 µm EDTA, the labeled cells were filtered through a 30-µm nylon mesh and loaded onto a column that was installed in a magnetic field. Trapped cells were eluted after the column was removed from the magnet. The collected cells were applied to a second column and the purification step was repeated. For AC133 CD34 + selection, the MNCs were first subjected to immunomagnetic separation using the MACS AC133 Cell Isolation Kit. Cells in the flow-through fraction were collected and applied to a second column after reincubation with AC133 MicroBeads. Cells in the second flowthrough fraction (AC133 cells) were collected and subjected to immunomagnetic separation using the MACS CD34 Progenitor Cell Isolation Kit. The cells that were trapped in the second MACS CD34 column were designated as the AC133 CD34 + population. Cell Sorting and Flow Cytometric Analysis For AC133 + CD34 + and AC133 CD34 + isolation from noncultured CB and PB cells, MACS-purified CD34 cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-cd34 (HPCA-2-FITC; Becton Dickinson; San Jose, CA; and phycoerythrin (PE)-conjugated anti-ac133/2 (Miltenyi Biotech) antibodies for 30 minutes on ice in the dark. The cells were then washed once in PBS. Cell sorting was performed using a fluorescence-activated cell sorter (FACS) Vantage machine (Becton Dickinson) that was equipped with a 488-nm argon laser. For the isolation of AC133 + CD34 +, AC133 CD34 +, AC133 Low/+ CD34 Low/, and AC133 CD34 cells, the AC133 + cells from CB or PB were cultured for 6 days, stained with FITC-labeled anti-cd34 and PE-labeled anti-ac133/2 antibodies, and sorted as described above. The labeled cells were analyzed on a FACSCalibur flow cytometer (Becton Dickinson) using the CellQuest software (Becton Dickinson). The expression of AC133 antigen on CD34 + cells from PB or CB was investigated by

3 Yasui, Matsumoto, Hirayama et al. 145 dual-color immunolabeling with FITC-labeled anti-cd34 and PE-labeled anti-ac133/2 antibodies. The kinetics of AC133 and CD34 expression on CD61 and CD13 cells were investigated by triple-color immunolabeling with peridinin chlorophyll-protein-labeled anti-cd34, PE-labeled anti- AC133/2, and FITC-labeled anti-cd61 (Becton Dickinson) or anti-cd13 (Becton Dickinson) antibodies. Colony-Forming Cell Assay For BFU-E, myeloid colony-forming units-granulocytemacrophage (CFU-GM), and multilineage CFU-granulocyte-erythroid-macrophage analyses, cells were suspended at a concentration of cells/ml in Iscove s modified Dulbecco s medium (IMDM) that contained 2% fetal bovine serum. The cell suspensions were mixed with 10 volumes of methylcellulose-based semisolid culture medium (Metho- Cult GF H4434V; StemCell Technologies Inc.; Vancouver, Canada; Aliquots (1.1 ml) were plated in duplicate in 35-mm dishes (Falcon 1008) and incubated for 14 days in a humidified atmosphere with 5% CO 2 at 37ºC. The clonogenic potential of MK progenitors was assayed using the serum-free collagen-based system, MegaCult-C (StemCell Technologies), according to the manufacturer s instructions. Cell cultures were supplemented with 50 ng/ml thrombopoietin (TPO), 10 ng/ml interleukin-6 (IL-6), and 10 ng/ml IL-3. The test cells were suspended in PBS. Aliquots (0.1 ml) of the cell suspensions were mixed with 2.0 ml of serum-free medium that contained the cytokines. After adding 1.2 ml of cold collagen solution, the final culture mixtures were dispensed in 0.75-ml aliquots into the two wells of a chamber slide. After incubation for days in double-chamber slides, the MK colonies were distinguished by immunostaining for CD well plates that contained 1 ml of StemPro-34 serumfree medium (GIBCO BRL; Grand Island, NY; invitrogen.com), which was supplemented with 2 mm L- glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, 50 ng/ml Flt3 ligand (FL; Diaclone Research; Besancon, France; 10 ng/ml TPO (Pepro- Tech Inc.; Rocky Hill, NJ; and 50 ng/ml stem cell factor (SCF; PeproTech). Cultures were maintained in a humidified atmosphere at 37 C and 5% CO 2. The three cytokines were added twice a week, and half the medium that contained growth factors was changed on day 6 or 7. In order to stimulate MK formation, the cells were cultured in nontreated 24-well plates in 1 ml of IMDM that contained 20% human serum and 10 ng/ml TPO, according to the slightly modified method of Mazur et al. [30]. TPO was added twice a week, and half the medium containing growth factors was changed on day 6. Ploidy was tested using the CycleTEST PLUS DNA Reagent kit (Becton Dickinson), according to the manufacturer s recommendations. RESULTS Purification and Colony-Forming Activities of AC133 + CD34 + and AC133-CD34 + Cells in Human Steady-State PB and CB Cells The expressions of AC133 on PB and CB CD34 + cells are shown in Figure 1. More than 80% of the CD34 + CB cells expressed AC133, whereas only about 40% of the CD34 + PB cells expressed AC133. To analyze the differentiation Liquid Cultures Cell cultures were usually initiated with cells in nontreated Figure 1. Expression of AC133 + cells in the CD34 + population. Representative FACS profiles (A) and percentages of AC133 + cells in the CD34 + populations (B) of PB and CB are shown. The percentages of AC133 + cells in PB and CB were 81.0 ± 5.7 and 43.2 ± 6.9 (mean ± standard error; n = 5), respectively. Statistical analysis of the data by the Student s t-test gives p <

4 146 Differentiation of CB and PB Cells AC133 CD34 + subpopulations were isolated from PB and CB using a cell sorter (Fig. 2). These cell populations were subjected to colony-forming analysis in methylcellulose or collagen-based cultures, and the BFU-E, CFU-GM, CFU- Mix (units of mixed colonies), and CFU-Meg (units of MKs) values were measured. The CFU-Mix values are not shown because the results were inconclusive. The colony formation values for PB and CB cells are shown in Figure 3. The BFU-E value for the AC133 + CD34 + PB cells was about one-third of that obtained for the AC133 CD34 + cells. In contrast, AC133 CD34 + PB cells produced very few CFU-GM colonies. As for the CFU-Meg values, there was no significant difference between the AC133 + CD34 + and AC133 CD34 + PB cell populations. CB cells gave essentially the same BFU-E and CFU-GM results. However, the CFU-Meg value for the AC133 + CD34 + CB cell population was significantly lower than that for the AC133 CD34 + population (p < 0.05; Student s t-test). Figure 2. Expressions of AC133 and CD34 on FACS-purified cells from PB and CB. AC133 and CD34 expressions after purification of AC133 + CD34 + and AC133 CD34 + populations from PB and CB. In both cases, the purity was >98%. abilities of subpopulations that were classified according to their AC133 and CD34 antigens, the AC133 + CD34 + and Kinetics of AC133 and CD34 Expression and Colony Formation in Suspension Cultures of AC133 + PB and CB Cells When MACS-purified AC133 + PB and CB cells (>99% of the cells expressed CD34 antigen) were cultured in serumfree liquid medium, which contained TPO, Flt3 ligand, and SCF, the cells differentiated and lost surface expression of CD34 and AC133. The kinetics of surface marker loss from AC133 + PB and CB cells were analyzed (Fig. 4). After 3 days Figure 3. Colony formation in AC133 + CD34 + and AC133 CD34 + populations from PB and CB. BFU-E and CFU-GM values for cells and CFU-MK values for cells are shown (n = 4).

5 Yasui, Matsumoto, Hirayama et al. 147 Figure 4. Kinetics of AC133 and CD34 expression in liquid cultures of AC133 + cells from PB and CB. AC133 + cells were cultured under serum-free conditions in the presence of Flt-3 ligand, SCF, and TPO. The AC133 and CD34 expression profiles are shown. of culture, most of the PB AC133 + CD34 + cells retained CD34 expression (95.1% ± 0.8%; mean ± standard deviation of three samples), whereas approximately half of these cells had lost AC133 expression. CD34 expression decreased progressively between days 3 and 6. By day 9, more than 80% of the cells expressed neither CD34 nor AC133. Thus, AC133 + PB cells in culture lost AC133 antigen expression first, and later lost CD34 antigen expression. The order of antigen loss in AC133 + CD34 + CB cell cultures was different from that in the AC133 + CD34 + PB cell cultures. The vast majority of the CB AC133 + cells (91.1% ± 2.1%; mean ± standard deviation of three samples) lost both CD34 and AC133 expression simultaneously, with the absence of an intermediate AC133 CD34 + population (Fig. 4). After 6 days in liquid culture, the differentiated PB and CB AC133 + cells were fractionated into four subpopulations: AC133 + CD34 + ; AC133 CD34 + ; AC133 Low/+ CD34 Low/ ; and AC133 CD34 (Fig. 5A), and the colony-formation activity of each subpopulation was analyzed. Most of the BFU-E (80.2% ± 5.8%; mean ± standard deviation of four samples) were due to the AC133 CD34 + PB cells. Although the BFU-E value for the AC133 CD34 + CB cells was twofold greater than that for the AC133 + CD34 + CB cells, a significant proportion of the BFU-E was attributable to the AC133 CD34 + subpopulation. The AC133 + CD34 + and AC133 Low/+ CD34 Low/ populations contained more than 90% of the CFU-GM, and the plating efficiency of AC133 Low/+ CD34 Low/ cells was a little higher than that of the AC133 + CD34 + cells, in both PB and CB cultures. Almost all the CFU-Meg was attributable to the AC133 CD34 + subpopulation in both CB and PB cell cultures. In addition, the CFU-Meg values for the PB culture were 10- fold higher than the values obtained for the CB cell cultures. As shown in Figure 4, the proportion of AC133 CD34 + CB cells was very small. Therefore, the absolute CFU-Meg values were much lower in CB cultures than in PB cultures. The expression levels of the AC133 and CD34 antigens on CD13 + GM lineage cells and on CD61 + MK lineage cells were examined in liquid cultures of AC133 + CD34 + PB and CB cells. We cultured MACS-purified AC133 + cells from CB and PB for 9 days. Approximately 8% of the CD61 + PB cells initially expressed both AC133 and CD34. During differentiation, the cells became AC133 CD34 mainly via an AC133 CD34 + population (Fig. 6A). On the other hand, when AC133 + CB cells were examined, there were virtually no CD61 + cells at the start of culture. By day 3 of culturing, AC133 + CD34 +, AC133 CD34 +, and AC133 CD34 cell populations were found among the CD61 + cells. Finally, CD61 + cells expressed neither AC133 nor CD34 on day 6 (Fig. 6B). Thus, the AC133 CD34 + population was not a major path in the CB culture. A similar experiment was carried out using an anti-cd13 monoclonal antibody in place of the anti-cd61 antibody, to examine the expressions of AC133 and CD34 on myeloid

6 148 Differentiation of CB and PB Cells Figure 5. Colony-forming ability of cells obtained from the PB and CB AC133 + populations at day 6. A) FACS profiles and fractions of the AC133 + CD34 + (fraction 1), AC133 CD34 + (fraction 2), AC133 Low/+ CD34 Low/ (fraction 3), and AC133 CD34 (fraction 4) cell populations are shown. B) BFU-E and CFU-GM values for cells and CFU-MK values for cells in each individual fraction (fractions 1-4 in A) are shown (n = 3). lineage cells. The expression patterns were essentially identical in PB and CB cell cultures (Figs. 6C and 6D), except in the case of the AC133 + CD34 population at day 6. The percentage of AC133 + CD34 cells in the CB cell culture was significantly higher than in the PB cell culture. Time Course of CD61 Expression and Ploidy Level We examined the expression of CD61 antigen and the ploidy level under conditions of preferential MK differentiation (Fig. 7), as described in the Materials and Methods section. CD61 expression in both PB and CB cell cultures occurred earlier in AC133 CD34 + cells than in AC133 + cells. Furthermore, there were no differences between PB and CB cells in terms of the emergence of CD61 expression. In the PB cell culture, AC133 CD34 + cells gave rise to MKs that contained >16N DNA content earlier than AC133 + cells. MKs containing >16N DNA content were barely detectable when CB AC133 CD34 + or AC133 + cells were cultured. DISCUSSION The AC133 + CD34 + and AC133 CD34 + cells were purified from CB and PB, and the colony-formation activities of these populations were examined. The AC133 + CD34 + cells produced GM, erythroid, and MK colonies. In contrast, the AC133 CD34 + cells gave rise to erythroid and MK colonies, but produced very few GM colonies. This result was confirmed by the liquid culture expansion of single-cell-sorted AC133 + CD34 + and AC133 CD34 + populations (data not shown). These data clearly show that the differentiation of AC133 CD34 + cells is quite different from that of AC133 + CD34 + cells, and suggest that the low percentage of AC133 CD34 + cells in CB is one of the reasons for the slow recovery of MK lineages after transplantation [5-7]. To further analyze this point, CB and PB AC133 + cells were cultured for 6 days in liquid medium that contained cytokines. We then examined the clonogenic capacities of the AC133 + CD34 +, AC133 Low/+ CD34 Low/, AC133 CD34 +, and AC133 CD34 populations. The clonogenicity of cells in liquid culture was similar to that obtained from colonyformation analyses of primary cells. Erythroid-MK colonyforming cells mainly occurred in the AC133 CD34 + population. However, almost no CFU-GM were found in that population. Instead, CFU-GM were detected in the AC133 + CD34 + and AC133 Low/+ CD34 Low/ populations.

7 Yasui, Matsumoto, Hirayama et al. 149 Figure 6. AC133 and CD34 expressions in CD61 + and CD13 + cells from AC133 + PB and CB cell cultures. The mean ± standard error (n = 3) values of individual fractions (AC133 + CD34 +, AC133 CD34 +, AC133 + CD34, and AC133 CD34 ) are shown. Recently, common lymphoid and myeloid progenitor cells, which have the ability to differentiate into various lymphoid and myeloid lineages, respectively, were isolated using a murine system and a sophisticated combination of monoclonal antibodies [31, 32]. In addition, the common myeloid progenitors have been further delineated into either MK/erythrocyte or GM lineages, both of which were identified by surface marker expression profiling in mutually exclusive manners [31]. However, the generation and isolation of these progenitor cells from human hematopoietic organs have not been previously described. We have shown that the AC133 and CD34 expression patterns may be used for the classification of lineage-restricted colonies. In order to analyze the expressions of AC133 and CD34 during lineage-specific differentiation, we examined the expressions of two lineage-specific markers during Figure 7. Kinetics of CD61 expression and polyploidy in liquid culture under conditions of megakaryocyte induction. AC133 + CD34 + cells (dotted lines) and AC133 CD34 + cells (solid lines) from CB (open circles) and PB (closed circles) were cultured in the presence of human plasma and TPO. CD61 expression (A) and polyploidy (B) of cultured cells are shown. Cells with >16N DNA content were scored as polyploid cells. A representative example of three independent experiments is shown.

8 150 Differentiation of CB and PB Cells AC133 + CD34 + cell culture. CD61 and CD13 antigens were utilized as markers of MK and GM lineages, respectively. The vast majority of the CD61 + MK cells differentiated via AC133 CD34 + cells into AC133 CD34 cells. This tendency was also found in a population of cells that were positive for glycophorin A, which is an erythroid marker (data not shown). However, the expressions of both AC133 and CD34 were lost almost simultaneously by the CD13 + GM cells. This finding is consistent with the data that only AC133 CD34 + cells could give rise to erythroid-mk colonies. We also analyzed the relationship between AC133 + CD34 + cells and AC133 CD34 + cells. Since AC133 is a marker of immature hematopoietic cells, it is likely that AC133 CD34 + cells are the descendants of AC133 + CD34 + cells. We purified AC133 + CD34 + cells from CB and PB, and subsequently examined the AC133 and CD34 expression patterns in cell culture. Unexpectedly, the pattern of loss of AC133 was different in CB cells than it was in PB cells. The vast majority of the AC133 + CD34 + cells in the CB AC133 + cell culture had lost both AC133 and CD34. In contrast, only 50% of the PB AC133 + CD34 + cells lost these two antigens simultaneously; the other 50% lost AC133 first and then CD34. This implies that immature AC133 + CD34 + CB cells do not give rise to AC133 CD34 + cells, which include erythro-mk progenitors. It has been reported that CD61 expression is delayed in CB cultures [5]. However, our results show that the CD61 expression patterns of AC133 + CD34 + and AC133 CD34 + cells from CB and PB are indistinguishable. Since CD61 expression occurs later in AC133 + CD34 + cells than in AC133 CD34 + cells, the apparent delay in CB cells is probably due to the abundance of AC133 + CD34 + cells in CB. In contrast, and as reported previously [5, 7], impaired polyploidization of CB cells was observed even after AC133 separation. Therefore, the insufficient polyploidization of CB cells is not due to subpopulation differences between CB and PB. Taken together, delayed platelet recovery after CB transplantation can be divided into two mechanistic categories. The first mechanism relates to differences in the subpopulations of hematopoietic cells in CB and PB. The percentage of AC133 CD34 + cells, which includes most erythro-mk progenitors, is very low in CB. The second mechanism pertains to the different characteristics of AC133 + CD34 + and AC133 CD34 + cells in PB and CB. A combination of these two mechanisms mediates weak platelet production immediately after CB transplantation. Although long-term platelet production is driven from self-renewing hematopoietic stem cells, the early phase of platelet production is mediated by multipotential and/or erythro-mk progenitors. From this point of view, we propose that the expression of AC133 could be used as an index of the rate of platelet recovery in the early stages after CB transplantation. ACKNOWLEDGMENTS We would like to thank K. Nakamura (Osaka University) for operating the FACS and Y. Horie and N. Yamashita for technical assistance. REFERENCES 1 Kohli-Kumar M, Shahidi NT, Broxmeyer HE et al. Haemopoietic stem/progenitor cell transplant in Fanconi anaemia using HLA-matched sibling umbilical cord blood cells. Br J Haematol 1993;85: Vowels MR, Tang RL, Berdoukas V et al. Brief report: correction of X-linked lymphoproliferative disease by transplantation of cord-blood stem cells. N Engl J Med 1993;329: Wagner JE, Broxmeyer HE, Byrd RL et al. Transplantation of umbilical cord blood after myeloablative therapy: analysis of engraftment. Blood 1992;79: Gluckman E, Broxmeyer HA, Auerbach AD et al. 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