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Comparison of Hematopoietic Activities of Human Bone Marrow and Umbilical Cord Blood CD34 Positive and Negative Cells

Department of Immunology, Institute of Basic Medical Sciences, Center for TARA, University of Tsukuba and CREST (JST), Tsukuba Science-City, Ibaraki, Japan
^* These two authors contributed equally to this work.

Key Words. Hematopoietic stem cells • NOD/SCID mouse • Cord blood • SCID-repopulating cell • CD34⁺ cells

Correspondence: Dr. Hiromitsu Nakauchi, Institute of Basic Medical Sciences and Center for TARA, University of Tsukuba, Tsukuba Science-city, Ibaraki 305, Japan.

	Abstract

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Although the hematopoietic activities of human CD34⁺ bone marrow (BM) and cord blood (CB) cells have been well characterized, the phenotype of nonobese-diabetic severe combined immunodeficient (NOD/SCID) mice repopulating cells (SRCs) in CB and BM has not yet been fully examined. To address this issue, various hematopoietic activities were compared in terms of total and CD34⁺ CB and BM cells. Clonal culture of fluorescence-activated cell sorter (FACS) CD34⁺ CB and BM cells revealed a higher incidence of colony-forming cells with greater proliferation capacity in CB over BM CD34⁺ cells. CB CD34⁺ cells also demonstrated higher secondary plating efficiency over BM cells. In addition, we demonstrated that mice transplanted with CB mononuclear cells (MNCs) showed significantly higher levels of chimerism than those transplanted with BM MNCs. However, recipients of FACS-sorted CD34⁺ CB cells showed significantly lower levels of chimerism than those that received total CB MNCs, suggesting a role of facilitating cells in the CD34^– cell population. To further analyze the role of CD34^– cells, the NOD/SCID repopulating ability of FACS-sorted CB CD34^–c-kit⁺Lin^– and CD34^–c-kit-Lin^– cells were examined. However, SRCs were not detected in those cells. Taken together, these data suggest that CB is a better source of hematopoietic stem cells and that there are cells in the CD34^– fraction that facilitate repopulation of hematopoiesis in the NOD/SCID environment.

	Introduction

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Umbilical cord blood (CB) has been utilized for human hematopoietic stem cell (HSC) transplantation as an alternative source to bone marrow (BM) [1-4]. HSCs in CB as well as in BM are also considered to be an ideal target for gene therapy. Purification and characterization of HSCs are, therefore, important not only for studies of the biological properties of HSCs but also for clinical application.

CD34 antigen has been used as a marker of human hematopoietic stem/progenitor cells [5-7]. In fact, most colony-forming cells are found in the CD34⁺ cell fraction [8-10]. Several lines of evidence have indicated that although CD34⁺ cells are less frequent in CB than in BM, CB contains a significantly larger number of colony-forming cells than BM [11-13]. These data suggest that hematopoietic stem/progenitor cells are enriched in CB CD34⁺ cells more than in BM CD34⁺ cells. However, in vitro colony-forming cells are a heterogeneous population including both lineage-uncommitted and -committed hematopoietic progenitors. Furthermore, these colony-forming cells appear to be biologically distinct from more primitive HSCs, with long-term marrow-repopulating ability as indicated in mouse studies [14-17]. Although enriched human BM as well as CB CD34⁺ cells have already been used for clinical transplantation to marrow-ablated patients, it has not yet been concluded whether CD34⁺ cells in CB and BM are equally capable of long-term marrow reconstitution. Thus, it is crucial to compare not only in vitro but in vivo hematopoietic activities of the CB and BM CD34⁺ cells.

In contrast to murine HSCs, however, reconstitution assays for human HSCs require xenotransplantation models. Recently, various animal models, such as fetal lambs, dogs, severe combined immunodeficient (SCID) mice and nonobese-diabetic-SCID (NOD/SCID) mice [18-21] have been tried for in vivo functional assays of human HSCs. Among these, Dick et al. have recently described a successful engraftment of human HSCs in CB and BM mononuclear cells (MNCs) in NOD/SCID mice, showing that CB MNCs are enriched for SCID-repopulating cells (SRCs) compared with BM MNCs or mobilized PB MNCs [22]. However, the phenotype of SRCs was not elucidated in this study. More recently, using the same NOD/SCID xenograft system, they demonstrated that SRCs are also present in the CD34^– fraction of human CB cells [23].

In this report, using in vitro colony assays as well as an in vivo reconstitution assay in NOD/SCID mice, we attempted to elucidate differences between CB- and BM-derived fluorescence-activated cell sorter (FACS)-purified CD34⁺ cells. We have also examined the reconstitution ability of CD34^–Lin- cells in comparison with CD34⁺ cells in CB. We show here that SRCs are enriched in CB CD34⁺ cells more than in BM CD34⁺ cells and that there are cells in the CD34^– fraction in CB that facilitate repopulation of hematopoiesis in the NOD/SCID environment.

	Materials and Methods

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Monoclonal Antibodies and Cytokines
Fluorescent isothiocyanate (FITC)-conjugated mouse monoclonal anti-human CD34 and CD45 monoclonal antibodies (mAbs) were purchased from Pharmingen (San Diego, CA). Phycoerythrin (PE)-conjugated mouse anti-human CD3, CD11b, CD19, CD31, CD34, CD33, CD41, CD56, CD117 (c-kit) and Glycophorin-A (GP-A) mAbs were purchased from DAKO (Glostrup, Denmark).

Recombinant human erythropoietin (Epo) was a gift from Chugai Pharmaceutical Co. (Tokyo, Japan). Recombinant human stem cell factor (SCF) and GM-CSF were provided by Kirin Brewery Co. (Tokyo, Japan). Recombinant human interleukin 1ß (IL-1ß), IL-3, and IL-6 were purchased from Genzyme (Cambridge, MA).

Samples and Cell Separation
CB samples were obtained from umbilical veins of normal, full-term infants. BM was obtained from adult volunteers under informed consent. These samples were diluted 1:2 in phosphate-buffered saline before separation over Ficoll/Hypaque. Cells were then stained with FITC-conjugated anti-CD34, PE-conjugated anti-CD117 (c-kit), and biotin-conjugated lineage markers (CD 3, CD19, CD56, and GP-A), followed by the antigen-allophycocyanin cell (APC)-conjugated streptavidin. The CD34⁺Lin^–, CD34^–c-kit⁺, and CD34^–c-kit^– cells were purified by sorting with flow cytometry (FACS vantage, Becton Dickinson; San Jose, CA). For transplantation of CD34⁺Lin^– cells into NOD/SCID mice, CD34⁺ cells were enriched using anti-FITC microbeads with application in magnetic column (Miltenyi Biotec; Bergisch Gladbach, Germany) before sorting.

Colony Forming Unit (CFU) Assay
For CFU-culture (CFU-C) assay, CD34⁺ cells were clone-sorted into 96-well flat-bottomed microtiter plates (Costar; Cambridge, MA) with 0.1 ml Iscove's modified Dulbecco's medium ([IMDM], GIBCO; Grand Island, NY), containing 1.2% methylcellulose (Aldrich Chemical; Milwaukee, WI), 30% fetal bovine serum, 1% deionized bovine serum albumin (BSA) (Sigma; St. Louis, MO), 5 x 10^–5 M 2-mercaptoethanol (2-ME) (Sigma), 2 u/ml Epo, and 5% phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM). After 14 days of culture at 37°C under 5% CO₂, colony formation was counted under inverted microscopy. For the high-proliferating potential colony-forming cells (HPP-CFC) assay, recombinant human SCF (rhSCF) (50 ng/ml), rhGM-CSF (100 ng/ml), IL-1ß (10 ng/ml), IL-3 (100 U/ml), and IL-6 (100 U/ml) were added in the medium described above instead of Epo and PHA-LCM, and colony formation was counted after 28 days of culture.

For the replating CFU-C and HPP-CFC assay, CD34⁺ cells were clone-sorted in 96-well U-bottomed microtiter plates (Costar) and primarily cultured for seven days with 0.06 ml IMDM, containing 30% fetal calf serum, 1% deionized BSA, 5 x 10^–5 M 2-ME, rhSCF (50 ng/ml), rhIL-1ß (10 ng/ml), and rhIL-6 (100 U/ml). After the culture, all the cells in each well were harvested and dispersed in 48-well plates (Costar) with the medium for CFU-C and HPP-CFC assays as described. The cells were cultured for the next 14 days, and colony formation was counted.

Transplantation in NOD/SCID Mice
MNCs or CD34⁺ cells purified from BM and CB were transplanted by tail-vein injection into sublethally irradiated (325 cGy using 137Cs -irradiator) eight-week-old NOD/Shi-Scid Jic (NOD/SCID) mice (Clea Japan; Tokyo, Japan) according to our standard protocol.

Analysis of Human Cell Engraftment in NOD/SCID Mice
To determine engraftment of human cells in NOD/SCID mice, MNCs of peripheral blood (PB), BM, spleen, or liver prepared by lysis of red blood cells with lysis buffer containing 0.83% ammonium chloride and 0.1% sodium bicarbonate (pH 7.0), were stained with FITC- or PE-conjugated mAbs against human panleukocyte marker CD45, lineage-specific markers CD3 (T cell), CD11b (myelomonocyte), CD19 (B cell), CD31 (platelet), CD33 (myelomonocyte), CD41 (platelet and megakaryocyte), CD56 (natural killer [NK] cell), glycophorin-A (erythroid cell), and hematopoietic progenitor cell markers CD34 and CD117 (c-kit). The cells were washed twice with staining medium (SM) and then resuspended in SM containing propidium iodide (2 ug/ml) to gate out dead cells. Cells were analyzed by flow cytometry (FACS vantage), as described previously [14].

	Results

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FACS Analysis of BM and CB Cells
BM and CB MNCs were analyzed by flow cytometry for the expression of CD34, c-kit, and lineage markers, including CD3, CD19, CD56, GP-A,. As shown in Figure 1, the frequencies of CD34⁺Lin^– cells and CD34^–c-kit⁺Lin^– cells in BM MNCs were 1.7% ± 0.1% and 4.2% ± 1.2%, respectively, and those of CB cells were 0.6% ± 0.2% and 0.2% ± 0.1%, respectively.

View larger version (29K): [in this window] [in a new window]   Figure 1. FACS analysis of BM and CB cells. BM(A) and CB(B) MNCs were stained with FITC-conjugated anti-CD34, PE-conjugated anti-CD117 (c-kit), and biotin-conjugated lineage markers (CD 3, CD19, CD56, GP-A), followed by the APC-conjugated streptavidin. Sorting gates are indicated. Frequencies of each fraction in BM and CB MNC are indicated. Data are representative in several independent experiments.

Engraftment of Human CB or BM Cells in NOD/SCID Mice
The results of in vitro studies on the colony-forming abilities of CB and BM CD34⁺ cells led us to examine whether CB shows higher reconstitution ability than BM in NOD/SCID mice. We first compared efficiencies of engraftment of human CB and BM cells in NOD/SCID mice.

To determine the frequency of SRCs in CB cells, a limited number of CB MNCs were intravenously injected into sublethally irradiated mice. Four to eight weeks after transplantation, the PB of the recipient mice was examined for chimerisms using human CD45 expression as a marker of human hematopoietic cell engraftment by flow cytometry. As shown in Table 2, all of the mice (6/6) transplanted with 10 million CB MNCs showed 0.1% or higher chimerisms in the PB, which was a threshold level reproducibly detectable by our flow cytometry analysis. In contrast, only one mouse (1/6) transplanted with 0.1 million CB cells showed over 0.1% chimerism. Based on the limiting dilution assay, the frequency of SRCs in CB was calculated as described previously [25-27] and was estimated to be 1/8.6 x 10⁵ (Fig. 2).

View larger version (14K): [in this window] [in a new window]   Figure 2. The frequency of SCID repopulating cells in total CB by limiting dilution analysis. The number of CB cells transplanted to a NOD/SCID mouse (X axis) was plotted versus the logarithm of the percentage of mice with unsuccessful engraftment. The frequency of SRCs was shown to be 1/8.6 x 105 at 37% of graft failure.

View larger version (49K): [in this window] [in a new window]   Figure 3. Phenotypic analysis of the human hematopoietic cells in BM of NOD/SCID mice nine weeks after transplantation with CB MNC. BM cells were stained with the FITC-conjugated anti-human CD45 mAbs and PE-conjugated human hematopoietic lineage-specific antibodies indicated and then analyzed with flow cytometry. Data are representative in several independent experiments.

Engraftment of CD34^– Cells from Human CB Cells in NOD/SCID Mice
We have previously reported that in adult mouse bone marrow, long-term marrow repopulating cells are enriched in the CD34^lo/– fraction, whereas progenitors are enriched in the CD34⁺ cell fraction of the c-kit⁺, Sca-1⁺, Lin^– cells [14]. To test whether SRCs in human CB are also present in the CD34^–, c-kit⁺ cell fraction, we purified CD34^–, c-kit⁺, Lin^– cells by flow cytometry (Fig. 1). Given that 10 million total CB MNCs are sufficient for 100% engraftment in NOD/SCID mice (Table 2), 0.3 million of the FACS-purified CD34^–, c-kit⁺, Lin^– cells, which corresponds to 15 million (i.e., more than 10 million) of total CB cells calculated with the frequency of the CD34^–, c-kit⁺, Lin^– cell fraction in total CB cells were injected per NOD/SCID mouse. However, as shown in Table 3, none of the six recipient mice injected with CD34^–c-kit⁺Lin^– cells showed over 0.1% chimerism with human CD45⁺ hematopoietic cells in the PB. Similarly, 1 to 5 million CD34^–, c-kit^–, Lin^– cells, nearly equivalent to 10 million total CB MNCs, were injected to test their reconstitution ability in NOD/SCID mice, but none of them showed over 0.1% chimerism by human CD45⁺ hematopoietic cells in the recipients' PB.

	Discussion

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Following pioneering studies by Leary et al. and Broxmeyer et al. [28-30], evidence has accumulated indicating that CB is an abundant source of stem and progenitor cells and that CB cells can be used for transplantation [24, 31-34]. We first tried to confirm those results using the clonal cell culture system. To exclude the possibility that the hematopoietic activity of CB MNCs is enhanced by coexisting CB-derived stromal cells that produce various cytokines, CB and BM MNCs were separated into various fractions with mAbs such as CD34, c-kit, and lineage markers (Lin) and their hematopoietic activity compared in vitro by a clonal culture utilizing a FACS clone-sorting system [35]. Analysis of colony-forming activity by sorted CB and BM CD34⁺ cells revealed that, although the frequency of CD34⁺ cells is smaller in CB than in BM (Fig. 1), CB CD34⁺ cells contain a significantly higher number of CFCs, e.g., three times more CFU-Cs, seven times more HPP-CFCs, and four times more secondary CFCs. Since these experiments were carried out by a clonal culture, the effect of coexisting stromal cells has been excluded. On the other hand, colony-forming activity was not at all detectable in the CD34^–Lin^– fraction, which was equivalent to 1.8 million total CB MNCs (data not shown).

Study of human hematopoietic stem cells has been difficult due to the absence of a suitable in vivo assay system. In the present study, we have also compared in vivo stem cell activity of human umbilical CB and adult BM cells using a NOD/SCID xenotransplantation model. The levels of chimerism in mice transplanted with CB MNCs were significantly higher than those in mice transplanted with BM MNCs. These results suggest that CB MNCs contain a larger number of cells with higher hematopoietic activity than BM MNCs. The frequency of SRC in CB estimated by the limiting dilution assay in our study was 1/8.6 x 10⁵, which was almost comparable to the 1/9.3 x 10⁵ total CB MNCs described by Dick and colleagues [22].

One of the most intriguing findings in the present study is that the level of chimerism in the mice transplanted with total CB cells was significantly higher than that of those transplanted with purified CB CD34⁺Lin^– cells despite the fact that a corresponding number of CD34⁺Lin^– CB cells were transplanted. This discrepancy may be explained by the presence of facilitating cells in the CD34^–Lin⁺ fraction that is believed to promote generation of human hematopoietic cells from CD34⁺ cells in NOD/SCID mice [36-37]. It could also be due to the presence of immune cells that eliminate mouse NK cells and facilitate engraftment of SRCs in the NOD/SCID environment. Alternatively, there may be cells in the CD34^– fraction of human CB or BM that do not form colonies but repopulate hematopoietic cells in NOD/SCID mice.

We have previously reported that long-term hematopoietic repopulating cells in mice are enriched in the CD34^lo/–c-kit⁺Sca-1⁺Lin^– fraction but not in CD34⁺ cells [14]. Recent reports have also suggested that human CD34^– cells in CB and BM cells contain in vivo hematopoietic repopulating cells in NOD/SCID mice [23] and in sheep [38]. These observations led us to study whether the fractions of CD34^– cells in CB may contain SRCs. Since transplantation of 10⁷ total CB cells successfully established chimerism at significant levels in all the NOD/SCID mice examined, we transplanted cells from the CD34^–c-kit⁺Lin^– or CD34^–c-kit^–Lin^– fraction nearly equivalent to 10⁷ total CB cells, as determined by the estimation of frequency of each fraction in CB. However, neither CD34^–c-kit⁺Lin^– cells nor CD34^–c-kit^–Lin^– cells transplanted generated any detectable level of chimerisms in NOD/SCID mice (0/6 and 0/3, respectively). Thus, in the CD34^–Lin^– population, SRCs appear to be present at a frequency less than 1 in 10⁷ cells. These results do not necessarily rule out the possibility of CD34^– stem cells, because there may be cells present in the CD34^–Lin^– or in CD34⁺Lin^– fraction that can become SRCs only when accessory and/or feeder cells are present as indicated in a recent report by Bhatia et al. [23].

	Acknowledgments

 
We would like to thank T. Morita for FACS operation and M. Ito for secretarial help. We would also like to thank Dr. Mihiro Shiina for providing umbilical cord blood and Dr. Toshiro Nagasawa for providing bone marrow samples. This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture in Japan, the Science and Technology Agency of the Japanese Government, and the Japan Society for the Promotion of Science (JSPS-RFTF96I00202).

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