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Cobblestone Area-Forming Cells in Human Cord Blood Are Heterogeneous and Differ from Long-Term Culture-Initiating Cells

^a University of Bristol, Division of Transplantation Sciences, Bristol, United Kingdom;
^b presently with the Department of Surgery, Prince of Songkla University, Pattani, Thailand

Key Words. CD34⁺ • CD34^- • Cobblestone-area-forming cells • Cord blood • Bone marrow stroma

Patricia Denning-Kendall, Ph.D., Paul O’Gorman Lifeline Centre, Southmead Hospital, Bristol, BS10 5NB, United Kingdom. Telephone: 44-117-959-6237; Fax: 44-117-959-5342; e-mail: P.A.Denning-Kendall{at}bristol.ac.uk

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion References

 
The long-term culture-initiating cell (LTC-IC) assay is a physiological approach to the quantitation of primitive human hematopoietic cells. The readout using identification of cobblestone area-forming cells (CAFC) has gained popularity over the LTC-IC readout where cells are subcultured in a colony-forming cell assay. However, comparing the two assays, cord blood (CB) mononuclear cell (MNC) samples were found to contain a higher frequency of CAFC than LTC-IC (126 ± 83 versus 40 ± 31 per 10⁵ cells, p = 0.0001). Overall, 60% of week-5 cobblestones produced by CB MNC were not functional LTC-IC and were classified as "false." Separation of CB MNC using immunomagnetic columns showed that false cobblestones were CD34^-/lineage⁺. Purified CD34⁺ cells, as expected, gave very similar readouts in the two assays, with 4,084 and 3,468/10⁵ cells being CAFC and LTC-IC, respectively. CD34^-/lineage^- cells did not form cobblestones or become CD34⁺ on stroma or in cytokine culture. Human CB MNC contain a population of mature lineage⁺ cells, possibly mature T or B cells, which, although producing cobblestone areas (CA), are not functional LTC-IC. The CAFC readout by this method, therefore, is unreliable for estimation of primitive hematopoietic cells by limiting dilution analysis in whole human CB or MNC and also may not detect CD34^- CA stem cells.

	INTRODUCTION

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The characterization and quantitation of primitive human hematopoietic cells is essential for understanding the developmental sequence of hematopoiesis, stem cell manipulation, and transplantation. Assay of primitive hematopoietic cells is difficult because of their rarity and expression of surface markers such as CD38 and Thy-1 [1], with different subsets representing stages of differentiation. The most primitive hematopoietic cells can ultimately only be recognized by their ability to repopulate marrow of a myeloablated recipient of the same species. However, a physiological in vitro approach is to measure long-term culture-initiating cells (LTC-IC) [2] in a modified Dexter culture system [3] using the fact that after 5 or more weeks of culture, LTC-IC will produce de novo colony-forming cells (CFC).

Alternatively, the cobblestone area-forming cell (CAFC) readout exploits a visual end point for the assay, eliminating trypsinization and the CFC assay. Wells are scored as being positive or negative for the presence of cobblestone areas (CA, tightly knit group of phase-dark, angular cells in the stroma) and can be scored at more than one time point, e.g., after 5 and 8 weeks. In the murine model, CAFCs seen between days 28–45 of long-term culture are directly related to bone marrow repopulating ability [4].

Both LTC-IC and CAFC frequencies have been reported in the study of stem cell storage [5], expansion [6, 7], or mobilization [8]. In the clinical setting these assays have been used to assess stem cell defects (e.g., in aplastic anemia [9] and Diamond Blackfan anemia [10]) and to investigate bone marrow reserve after stem cell transplantation [11, 12] or in patients with autoimmune cytopenias [13].

In the present work, we set out to measure the number of primitive cells in cord blood (CB) that had been processed for clinical banking. We chose limiting dilution on cryopreserved human bone marrow stroma feeder layers [14] with the visual CAFC readout. It soon became apparent that further validation of the assay was required. We have therefore compared the frequency of CAFC with LTC-IC in CD34-enriched and CD34-depleted fractions of CB.

	MATERIALS AND METHODS

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CB Collection
Umbilical CB samples were from normal full-term deliveries collected by gravity into sterile 50-ml tubes containing 1,000 IU heparin after the umbilical cord had been clamped and cut by the midwife. All collections were performed with written informed consent from the mothers and with local hospital ethics committee approval.

Gelatin Sedimentation
CB samples were red cell depleted by sedimentation with 3% gelatin at 30°C as previously described [15].

Preparation of CB Mononuclear Cells and Purification of CD34⁺ Cells
CB mononuclear cells (MNCs) were prepared from 40–50 ml CB using Ficoll-Hypaque (Lymphoprep [d = 1.077 g/ml], Nycomed; Birmingham, UK; http://www.amersham.co.uk) followed by treatment with ammonium chloride lysing solution to remove red cells. For CD34⁺ enrichment, MNC were applied to a MiniMACS (magnetic cell sorting) column (Miltenyi Biotec Ltd.; Camberley, UK; http://www.miltenyibiotec.com) as described previously [6]. Cells were eluted to produce a CD34-enriched fraction (MACS⁺) and a CD34-depleted fraction (MACS^-).

Preparation of Lin⁺ and Lin^- Cells
The MACS^- fraction was further separated into lineage-positive (lin⁺) and lineage-negative (lin^-) fractions. Cells were stained with a cocktail of phycoerythrin (PE)-conjugated antihuman CD2 (MT910), CD3 (UCHT1), CD4 (MT310), CD14 (TUK4), CD16 (DJ130c), CD19 (HD37), CD20 (B-Ly1), CD33 (WM-54), CD41 (5B12), CD56 (NKH-1), glycophorin A (JC159 (10 µl/10⁷ cells), and fluorescein isothiocyanate (FITC)-conjugated CD45 in 50 µl phosphate-buffered saline. Lin⁺ stained cells were conjugated to anti-PE microbeads and were recovered after passage through two LS positive selection MACS columns (purity 98.2% ± 1.0%). The first fraction of unlabeled cells (10⁶ cells from 50 ml CB) eluted from the LS column contained approximately 84% lin^- cells and was further purified (purity 96.9% ± 3.0%, yield 10⁵ cells) by passage through an LD MACS depletion column according to manufacturer’s instructions (Miltenyi Biotec Ltd.).

Flow Cytometry
Cells were assessed for CD34⁺ content by dual labeling with FITC-conjugated anti-CD45 (clone T29/33, DAKO; Carpenteria, CA; http://www.dako.com) and PE-conjugated anti-CD34 (clone HPCA-2, Becton Dickinson; San Jose, CA; http://www.bd.com). Samples were examined on an Epics XL flow cytometer and data were analyzed using the Epics Listmode software (both from Coulter; Fullerton, CA; http://www.beckman.com). Only CD34⁺, CD45⁺ events with low side scatter were counted as CD34⁺ progenitor cells. Cells in the MACS^- fraction (CD34^- cells) and lin^- cells after culture were phenotyped using the panel of PE-conjugated antibodies described for the preparation of lin⁺ cells together with CD34-PE.

Assay for Hematopoietic CFCs
Hematopoietic CFCs were assayed as described previously [15] using 1.3% methylcellulose in Iscove’s modified Dulbecco’s medium (IMDM) containing 10% 5637 conditioned medium, 10% bovine serum albumin (HCC-9300), 30% fetal calf serum (FCS; HCC-6150, both from Stem Cell Technologies; Vancouver, BC; http://www.stemcell.com), and 2 U/ml erythropoietin ("Eprex;" Janssen-Cilag Ltd.; High Wycombe, UK; http://www.janssen-cilag.co.uk).

Assessment of CAFC and LTC-IC
The frequency of week-5 CAFC and LTC-IC was determined using a limiting dilution assay as previously described [14]. Briefly, cells of interest were cultured on cryopreserved irradiated bone marrow feeder cells in 96-well plates at six different concentrations with 24 replicates per dilution. For assessment of CAFC after 5 weeks of culture, all wells were scored microscopically for the presence of 12 or more closely associated, small embedded cells (Fig. 1).

View larger version (155K): [in this window] [in a new window]   Figure 1. Cobblestone area (CA) formed in normal bone marrow stroma. Figure shows a large CA consisting of angular phase-dark cells associated with a fat cell (F) together with phase-bright supernatant cells (S) after coculture of irradiated (15 Gy) normal bone marrow stroma 5 weeks after seeding with cord blood MNCs. M = macrophage.

Statistics
All results are reported as mean ± standard deviation (SD). Statistical analysis on groups was performed using a two-tailed t-test. Correlation coefficients were calculated using linear regression analysis.

	RESULTS

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Frequency of CAFC in Red-Cell-Depleted CB
The mean frequency of CAFC in red-cell-depleted CB was 72 ± 65/10⁵ nucleated cells (n = 7). Flow cytometric analysis showed that, on average, 0.29 ± 0.11% (290/10⁵) of nucleated cells were CD34⁺, and therefore over 25% of CD34⁺ cells were apparently forming cobblestones on bone marrow stroma. Results for individual samples are shown in Table 1. This frequency was unexpectedly high and prompted investigation of the CAFC method compared with the LTC-IC readout.

View larger version (17K): [in this window] [in a new window]   Figure 2. Frequency of CAFC and LTC-IC in CB MNCs. CB MNCs were seeded onto preformed, irradiated normal bone marrow feeder layers in 96-well plates at 6 different dilutions, with 24 replicates per dilution. After culture for 6 weeks as described in Materials and Methods, individual wells were scored as positive or negative for the presence of a CA consisting of 12 or more tightly packed phase-dark stromal embedded cells. After calculation of the percentage of negative wells at each dilution, limiting dilution analysis was used to estimate the frequency of CAFC/105 MNCs. To estimate the frequency of functional LTC-IC, the entire contents of culture wells were placed in individual enriched methylcellulose for CFC assay. After 14 days incubation, wells were then scored as positive or negative for the presence of CFC, and the frequency of LTC-IC was estimated by limiting dilution analysis.

Distribution of CAFC and LTC-IC after MiniMACS Fractionation
Five CB samples containing 1.4–2.0 x 10⁸ MNCs were processed on the MiniMACS column for CD34⁺ enrichment. Cells in the MACS⁺ fraction contained 93% of the eluted CD34⁺ cells and 99.7% of the eluted colony-forming unit (CFU)-granulocyte macrophage (GM). Few CD34⁺ cells were found in the negative fraction and these produced mainly small BFU-E (15 ± 6 BFU-E/10⁵ cells) and accounted for less than 1% of the eluted CFU.

The results of CAFC and LTC-IC estimation on MNCs, MACS^-, and MACS⁺ fractions are shown in Table 2. As expected, MNCs contained more CAFC than LTC-IC. Although the CD34⁺ cells in the MACS⁺ fraction accounted for only 1.2% of the eluted cells, they accounted for all of the LTC-IC activity of CB MNCs. The percentage of CD34⁺ cells that were LTC-IC was almost identical between the MNCs fraction (4.8%) and the MACS⁺ fraction (4.3%). In addition, for CD34⁺ cells, CAFC results correlated very closely with LTC-IC results. This correlation also held for cytokine-cultured CD34⁺ cells over a wide range of frequencies (range, 58–2,128 CAFC and 68–1,666 LTC-IC/10⁵ cells). These data suggest that cobblestones produced by CD34⁺ cells correlate with LTC-IC and that false cobblestones are not generated by expansion culture of CD34⁺ cells under myeloid conditions.

These data suggest that the false cobblestones were in the MACS^- fraction.

Are False CAFC Plastic Adherent?
Some centers routinely adherent cell deplete MNCs before seeding feeder layers for LTC-IC assays, a method adopted for LTC-IC analysis of bone marrow samples to deplete any third-party stromal cells. Petengell et al. [19] compared CAFC and LTC-IC frequencies in CB adherent cell-depleted MNCs and found that although most had a lower LTC-IC than CAFC frequency, there was no instance of the dramatic differences reported for our CB samples. It was possible therefore that the majority of false CAFC were removed by adherence.

To test this hypothesis, both MNCs and the MACS^- fraction from CB were assayed for CAFC and LTC-IC before and after adherent cell depletion. Adherent cells were removed from CB by overnight incubation of 20 x 10⁶ MNC at 37°C in a plastic flask containing 10 ml of IMDM supplemented with 15% FCS. As expected, the MNC samples contained more (2.6–11.0-fold) CAFC than LTC-IC. When adherent cells were removed, the frequency of CAFC decreased on average by 17% (Table 3), but results were still much higher than the MNC LTC-IC frequencies. Additionally, in three of four samples, adherent cell depletion removed up to 60% of the functional LTC-IC. Adherent cell depletion therefore does not specifically remove false cobblestones but removes a significant proportion of LTC-IC.

View larger version (54K): [in this window] [in a new window]   Figure 3. Separation of cord blood MACS- (CD34-) cells into lin+ and lin- fractions. Top plots show MACS- cells after incubation with a cocktail of PE-conjugated lineage antibodies (10 µl/107 cells) together with FITC-conjugated CD45 before separation. Two lin- populations (A and B) have separate side-scatter properties. Lin+ cells are mainly CD45+. Those that are CD45- stain for glycophorin A. For application to MACS separation columns, cells were incubated with anti-PE microbeads. Lin+ cells were recovered after passage through two LS-positive selection MACS columns (shown bottom left). The fraction of unlabeled cells eluted from the first LS column contained lin- cells and was further purified using a depletion column. The resultant lin- fraction clearly shows CD45hi and CD45lo populations that have different morphology on cytospins.

Culture of MACS^-/Lin^- Cells
Although these cells have no 5-week LTC-IC activity, it was possible that they could expand and express CD34 in cytokine culture. Cells were placed in serum-free medium (StemSpan SFEM, StemCell Technologies) with stem cell factor, Flt3, and thrombopoietin at 100 ng/ml, and interleukin (IL)-3 and IL-6 at 20 ng/ml at 37°C for 14 days. No CFU-GM were found in fresh cells or after 14 days of culture; CD34, CD13, or CD33 were not expressed and the cell number did not increase. Cells remained CD45⁺.

	DISCUSSION

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This study describes measurement of primitive human hematopoietic cells in CB by limiting dilution on normal bone marrow stroma. The CAFC frequency of CB was on average fourfold higher than the LTC-IC frequency. Cell separation experiments showed that CB contains a population of nonadherent, CD34^-/lin⁺ cells that can form CA on irradiated human bone marrow stroma. These are not functional LTC-IC, and therefore interfere with the CAFC scoring method. They account for 86/10⁵ (range, 0–230/10⁵) MNC compared with a range of 10–100/10⁵ MNC that are functional LTC-IC producing CFC after 5 weeks of culture on stroma feeder layers. In some CB samples, the CAFC readout is 11-fold higher than the LTC-IC readout (Fig. 2).

These cells could possibly be mature T or B cells. It is unlikely that the false cobblestones are produced by stem cells of different origin, such as mesenchymal stem cells (MSCs), as false cobblestones were present in the fraction enriched for hematopoietic lin⁺ cells. Secondly, no cobblestones are ever seen in cultures of the bone marrow stromal feeder layer even though it is grown from whole marrow buffy coat that includes MSCs. Also, when second passage MSCs (>90% pure) are placed in long-term hematopoietic culture medium, fibroblast and fat cell types differentiate but no cobblestones are seen.

When MACS^- cells were separated into lin⁺ and lin^- fractions, we used a method similar to that of Nakamura et al. [20] except that we removed CD34⁺ cells first. Our cell preparations produced almost identical flow cytometric plots with the MACS^-/lin^- fraction containing a CD45^hi and CD45^lo population. We have analyzed these populations further and found that a high proportion of the CD45^hi population had blast cell morphology, were CD38^-, and expressed CD7 and CD11b, consistent with them being lymphoid progenitors [21]. A few cells expressed CD133, an antigen associated with CD34⁺ long-term repopulating cells [22] and an extremely rare subset of CD34^- cells [23]. A few expressed CD90, another hematopoietic stem cell marker [1], and a significant number of cells in this population expressed ABCG2 transporter, a gene associated with uncommitted progenitors [24]. The CD45^lo population was considered to be contaminating neutrophil precursors.

It is generally considered that fresh CD34^-/lin^- cells are devoid of repopulating ability [20, 23, 25], but cytokine stimulation can rapidly induce expression of CD34, together with repopulating activity on a small percentage of these cells [20, 23, 26]. Nakamura et al. [20] demonstrated generation of CD34⁺ cells from the high-side-scatter/CD45^lo population of cells, which we have shown to be predominantly mature myeloid cells and unlikely to contain any CD34^- stem cells. More recently, cell sorting has defined the CD34-inducible population as rare AC133⁺/CD7^-/CD38^- cells [23]. Although our CD45^hi low-side-scatter population contained CD133⁺ cells, we did not observe any expression of CD34 after culture. This may be due to the exhaustive removal of CD34⁺/LTC-IC from the MNC preparation before separation into lin^- and lin⁺ fractions. In addition, our culture conditions were not designed for the support of primitive lymphoid cells, so it is unlikely that the large proportion of CD7⁺ cells would survive and become CD34⁺. This prediction is validated by Storms et al. [21], who demonstrated that CD7⁺/CD34^- lymphoid progenitors needed stroma-based culture in the presence of natural killer cell-stimulating cytokines to survive.

Our estimates of the proportion of LTC-IC in CB using the LTC-IC readout suggest that, on average, 32/10⁵ MNC or 43/10³ CD34⁺ cells in CB are LTC-IC. These compare favorably with reported frequencies of 59/10⁵ MNCs [27], 23/10⁵ MNCs [28], and 71/10³ CB CD34⁺ cells [29] using the LTC-IC readout after culture on normal bone marrow stroma feeder layers. The CAFC readout, although popular, seems to be fraught with problems. There are a few reports showing similar CAFC and LTC-IC readouts on human bone marrow [19, 28, 30]. Petengell et al. [19] showed that CAFC and LTC-IC frequencies were very similar (r = 0.73) with no examples of the large discrepancies we reported; however, the median week-5 CAFC frequency reported for CB MNCs was unusually low at 1/12,506 (8/10⁵ MNCs). This may be due to different culture conditions producing a less supportive stroma where the presence of CD34^-CAFC may be less obvious.

Similar problems have also been encountered with other feeder cell layers. Mobilized peripheral blood CAFC frequencies using the M2–10B4 cell line were 3.1 times higher than the LTC-IC frequencies for all samples, although the identity of the false CAFC was not investigated [30]. Overall, the LTC-IC readout for the frequency of primitive human hematopoietic cells seems more reliable and has the advantage of being a functional assay. Interpretation of CAFC frequencies should be approached with appropriate caution. Many other cell lines, such as murine MS5 and FBDM-1, are also used for limiting dilution assays, and these may give variable support to CAFC because they do not always contain a complete complement of supportive cells and may be unsuitable for use with purified CD34⁺ cells [31].

It should also be noted that there may be an increased detection of LTC-IC when feeder layers are genetically engineered and transfected with human growth factor genes. Genetically engineered M2–10B4 gave a mean LTC-IC frequency for CB of 31/10⁵ MNCs, which is very similar to our value of 32/10⁵, whereas parent M2–10B4 feeder layers gave a frequency of 7/10⁵ for the same samples [32]. Similarly, if the plating density of test cells is too high, overcrowding probably leads to depletion of nutrients and stromal damage and results in a falsely low result [33]. In general, however, cells are plated at low densities to ensure high proportions of negative wells, and we would not envisage this to be a problem. Results from all LTC-IC assays must be interpreted with great care, taking into account the type of feeder layer and the readout.

The results described here refer to human CAFC seeded on human bone marrow stroma feeder layers. In the murine model initially developed for the CAFC assay by Ploemacher et al. [4], there is a direct correlation between the number of nucleated cells required to produce long-term donor chimerism and the frequency of CAFC, suggesting that the false cobblestone phenomenon may not occur in this case.

We conclude that the CAFC readout is unreliable in the human system and may overestimate the frequency of primitive hematopoietic cells in human CB. Under certain conditions with purified CD34⁺ cells, the CAFC and LTC-IC readout produces very similar results, but if the CAFC assay is to be used, it must first be validated against the LTC-IC readout for each type of sample and feeder layer.

	ACKNOWLEDGMENT

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The authors wish to thank the midwives in the delivery suite at Southmead Hospital and all mothers who donated cord blood for the project. We also wish to thank the Bud Flanagan Leukaemia Fund, which donated laboratory equipment. This work was supported in part by grants from the Leukaemia Research Fund, London, UK; Children with Leukaemia; Southmead Hospital Stem Cell; and University of Bristol Cancer Research Funds.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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