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Stem Cells, Vol. 14, No. 5, 566-576, September 1996
© 1996 AlphaMed Press

ORIGINAL PAPER

Extensive Amplification of Single Cells from CD34+ Subpopulations in Umbilical Cord Blood and Identification of Long-Term Culture-Initiating Cells Present in Two Subsets

E.A. de Wynter, G. Nadali, L.H. Coutinho, N.G. Testa

CRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Withington, Manchester, United Kingdom

Key Words. CD34+ subsets • Amplification • Long-term culture-initiating cells • Cord blood • Colony-forming cells • Primitive cells

Correspondence: Dr. E. A. de Wynter, CRC Department of Experimental Haematology, Paterson Institute for Cancer Research,Withington, Manchester, M20 9BX, United Kingdom.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
CD34+ cord blood cells were isolated with immunomagnetic beads and fractionated by fluorescence-activated cell sorting (FACS) into three subpopulations: CD34+38+DR+, CD34+38–DR+ and CD34+38–DR–, using antibodies specific for these cell surface markers. Cells from each of the three subsets were plated as single cells in serum-free medium supplemented with a combination of growth factor and individual cells were monitored for proliferation and the capacity to form colony-forming cells. Single cells from the CD34+38+DR+ subset showed the lowest expansion capacity, generating up to 1.1 x 106 cells at five weeks, while individual cells from both the CD34+38–DR+ and CD34+38–DR– subsets could be expanded up to 1.8 x 106 and 9.2 x 106 cells, respectively, over a period of six weeks. The different subpopulations also generated colony-forming cells which gave rise to erythroid, myeloid and erythroid/myeloid colonies. CD34+38–DR+ cells generated large numbers of colonies within two weeks in liquid culture, but this rapidly declined. Generation of lineage-committed colony-forming cells was better sustained in the CD34+38–DR– population and continued for up to six weeks in culture.

Overall, the generation of colony-forming cells declined with time in culture, although the cell numbers continued to expand. However, when the same populations were plated on irradiated bone marrow stroma, both the CD34+38–DR+ and the CD34+38–DR– cells were capable of producing granulocyte-macrophage colony-forming cells (GM-CFCs) for 10 to 12 weeks. As hemopoiesis was sustained for almost three months, it appears that these populations were significantly enriched in long-term culture-initiating cells (LTC-ICs). Although both populations generated GM-CFCs, the CD34+38–DR– cells sustained production of higher numbers of colony-forming cells than the CD34+38–DR+ population. These results demonstrate that cells from cord blood can be efficiently monitored at the single-cell level for proliferation, expansion and colony-forming capacity. Furthermore, at least two populations of LTC-ICs can be distinguished in cord blood CD34+38– cells by the differential expression of the HLA-DR antigen.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Most mature blood cells have a short life span and must be produced continuously from primitive cells in the bone marrow. These primitive cells have the ability to proliferate and differentiate into mature blood cells, and possess the characteristic feature of bearing the CD34 antigen on their cell surface. If enriched populations of these CD34+ cells are used in transplantation, hemopoiesis can be fully reconstituted, indicating that pluripotent stem cells are present in the CD34+ population [1].

Human umbilical cord blood is a particularly rich source of these transplantable cells and has been used successfully in the clinic to transplant children with various hemopoietic disorders [2-4]. Although the numbers of primitive cells vary in cord blood samples, there is a possibility that a single collection of cord blood may contain sufficient cells to engraft and repopulate the hemopoietic system of an adult recipient [5, 6]. This has stimulated considerable interest in the biology of cord blood cells and generated a number of reports on the capacity of these progenitor cells to proliferate and differentiate in various ex vivo culture systems in the presence or absence of serum. Clearly, these CD34+ cells are suitable for transplantation, but as the isolated cells represent a heterogeneous population, it is not possible to determine the contribution of different CD34+ subpopulations to the overall expansion.

In attempts to characterize these subpopulations, CD34+ cells from cord blood have been separated into various populations based on the expression of antigens such as CD45RA, CD71, CD38, HLA-DR, Thy1 and c-kit. Some studies have assigned a particular phenotype to the "true" stem cell [7-9]. However, quantitation of the numbers of CD34+ cells with a "stem cell" phenotype is not particularly informative because it does not define the proliferative potential of these cells. To characterize the functional properties of the cell subpopulations isolated in this study, especially in terms of their expansion potential and ability to sustain hemopoiesis in vitro, we have separated subsets of CD34+ cells according to their expression of CD38 and HLA-DR antigens using three-color immunofluorescence and fluor-escence-activated cell sorting (FACS). Single cells were expanded in a serum-free medium supplemented with a combination of growth factors for 14 days, then subcultured every seven days, and the progeny assessed weekly for their ability to proliferate and generate colony-forming cells over six weeks in liquid culture. The subpopulations were also examined for enrichment in long-term culture-initiating cells by assessing their ability to sustain long-term hemopoiesis in vitro on irradiated bone marrow stroma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Growth Factors
Purified recombinant human granulocyte colony stimulating factor (rHuG-CSF) and recombinant human stem cell factor (rHu-SCF) were obtained from Amgen, Inc., (Thousand Oaks, CA). Recombinant interleukin 3 (IL-3) and interleukin 6 (IL-6) were obtained from Sandoz (Basel, Switzerland). Insulin-like growth factor 1 (IGF-1) was purchased from Collaborative Biomedical Products (Becton Dickinson; Oxford, England) and basic fibroblast growth factor (bFGF) was a generous gift from Dr. G. Molineux (Amgen). Recombinant erythropoietin (rHu-Epo) was obtained from Boehringer (Mannheim; Germany).

Cells
Human umbilical cord blood samples were collected with informed consent from full-term normal deliveries. Samples were collected in sterile tubes containing preservative-free heparin. Before separation, the samples were diluted 1:1 with phosphate buffered saline (PBS), and the mononuclear cells (MNCs) isolated by centrifugation on Ficoll-Hypaque (Lymphoprep, 1.077 g/ml, Nycomed; Birmingham, UK) at 400 g for 25 minutes. The MNCs at the interface were collected and washed in PBS containing 0.5% bovine serum albumin (BSA).

Isolation of CD34+ Cells
After the MNCs were washed and counted, the CD34+ fraction was isolated using MiniMACS columns and a CD34 Isolation Kit, as previously described (Miltenyi Biotec; Bergisch Gladbach, Germany) [10]. Briefly, MNCs were labeled with a blocking agent and a CD34 antibody (QBEND/10) from the Isolation Kit for 15 minutes at 4° to 8°C. Cells were then washed once in a cold solution of PBE (PBS, 0.5% BSA and 5 mM EDTA). The cell pellet was then resuspended in a small volume of PBE and incubated with microbeads conjugated to an anti-mouse antibody. Target cells were isolated by passing the entire cell suspension through a MiniMACS column in a magnetic field where the CD34 microbead-labeled cells were retained on the column. The CD34+ fraction was recovered by releasing the magnetic field and flushing the target cells from the column. In general, the purity of cells isolated was greater than 80%, as determined by flow cytometry analysis.

FACS Sorting of CD34+ Cells
To select cells with the appropriate phenotype, the CD34+ cells were labeled with directly conjugated antibodies, CD34-FITC (HPCA-2), CD38-PE (Becton Dickinson; San Jose, CA) and a third antibody HLA-DR-TC from Caltag Laboratories (San Francisco, CA). Isotype-matched FITC- (fluorescein isothiocyanate), PE- (phycoerythrin), and TC- (TriColor) conjugated mouse monoclonals served as controls. Labeled cells were analyzed on a FACS Vantage flow cytometer equipped with an Argon-Ion laser tuned at 488 nm. Forward- and side-light scatter and three fluorescence signals were determined for each cell, and the data were acquired in list mode. Data list mode files were analyzed with PC-LYSYS II (BDIS). Single-cell sorting was performed on a FACS Vantage using the Automatic Cell Deposition Unit (ACDU). To eliminate doublets and cell aggregates, a pulse processor module was used on the forward- and side-scatter width parameters. After deposition, wells were examined by microscopy to verify 1 cell/well. Although some wells had no cells, there were no wells with more than one cell. On average, the sorting efficiency, defined here as the number of wells containing a single cell, ranged from 72% to 87%.

Single-Cell Expansion
Purified single cells were deposited in 96-well U-bottom plates. Each well contained 100 ml of serum-free medium (X-Vivo 10 medium [Bio-Whittaker; UK], and in one experiment, serum-free medium, a kind gift from Dr. I. L. Ponting [11]) containing 100 ng/ml SCF, 5 x 104 U/ml G-CSF, 10 ng/ml IL-3, 200 U/ml IL-6, 2 U/ml Epo, 2.5 ng/ml bFGF and 10 ng/ml IGF-1. All cultures were incubated in 5% CO2, 5% O2 in air at 37°C in a fully humidified incubator for 14 days before replating. Replating of expanded clones was performed by dispersing the cells from one well into three or four new wells on a 96-well plate containing fresh serum-free medium and the growth factors. Plates were incubated under identical conditions as outlined above. At seven-day intervals after replating, the wells containing expanded clones were examined; one was replated further into new wells with fresh growth mixture, the contents of a second well were assayed in the CFC-MIX assay, while a third was counted and the morphology of the expanded cells determined by staining with May-Grünwald/Giemsa. This procedure was repeated for up to six weeks after the initial single-cell plating or until there was no further expansion.

CFC-MIX Assays
Five hundred to one thousand sorted cells from each of the selected populations were plated in a CFC-MIX assay as previously described [12]. Briefly, cells were added to a 1 ml mixture of 30% fetal calf serum (FCS), 10% deionized BSA, 10% 5637-conditioned medium (pre-tested medium from the 5637 EJ bladder carcinoma cell line), 2 units Epo and 1.35% methylcellulose. After thorough mixing, cells were plated in triplicate and incubated for 14 days at 37°C in 5% CO2 and 5% O2 in air. The expanded clones from single cells were assayed in a similar manner. Colonies of granulocyte-macrophage colony-forming cells (GM-CFCs) and BFU-Es were assessed according to established methods after 14 days' incubation [13, 14].

Two-Stage Long-Term Cultures (LTCs)
Bone marrow cultures were established and maintained as previously described [12]. Primary cultures were initiated with 107 nucleated cells in LTC medium containing Iscove's modified Dulbecco's medium (IMDM) at 350 mOsM/kg, 10% horse serum, 10% FCS and 5 x 10–7 M hydrocortisone (Sigma; Aldrich, UK) in 12.5 cm2 tissue culture flasks. Cultures were maintained at 33°C and 5% CO2 in air with weekly feeding. Confluent marrow stroma developed within three to four weeks and was then irradiated at 15 Gy. These confluent layers were recharged with cells from the three CD34+ subpopulations. Due to the low incidence of the CD34+38–DR– population, cell numbers had to be adjusted in the inoculum in accordance with the yield of sorted cells from the different cord blood samples. The number of clonogenic cells generated from each culture over the next 10 to 12 weeks was determined by removing aliquots of the total cellular content of each flask and culturing these cells in the CFC-MIX assay.

Statistical Analysis
Growth and maintenance of CFCs from the three CD34+ subsets were analyzed using methods appropriate for binomial data. Significance levels in the LTC experiments were determined by calculation of areas under the curve and determination of systematic ordering of profiles using a non-parametric test (Friedman's Test).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Isolation and Analysis of CD34+ Subpopulations
On average, 1% of MNCs were recovered using the MiniMACS columns with a purity for CD34+ cells of more than 80%. A representative experiment is shown in Figure 1Go. The forward- and side-scatter display shows a compact population of cells after CD34 selection by MiniMACS (Fig. 1AGo). The expression of CD34, CD38 and HLA-DR antigens on the selected cells was analyzed using three-color immunofluorescence, and Figures 1B through 1DGo illustrate a typical flow cytometric dot-plot where CD34-FITC, CD38-PE and HLA-DR-TC are combined. For sorting, gates were set to achieve > 99% control cells in the negative population. Four populations could be identified, and two CD34+38– subsets based on HLA-DR expression clearly distinguished. By far the majority of CD34+ cells was positive for both CD38 and HLA-DR, with less than 1% of cells lacking both markers. Cells with high distribution of these antigens were sorted into the CD34+38+DR+ population. The population of CD34+38– cells was divided into two subsets—the HLA-DR+ and HLA-DR– fractions—and these were collected separately. The percentages of CD34+ cells in all four populations from 15 samples are detailed in Table 1Go. It is clear that the CD34+38–DR– cells were the least abundant in all experiments, with figures ranging from 0.02% to 0.73%, which translates to 2 x 102 to 7 x 103 cells per experiment. As the CD34+38+DR– cells had already acquired the CD38 activation marker and had in pilot experiments behaved in a similar manner to the CD34+38+DR+ cells, they were not included in the present study.



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  		Figure 1. Flow cytometric analysis of isolated CD34+ cord blood cells from a representative experiment; three-color immunofluorescence staining with FITC-conjugated anti-CD34, PE-conjugated anti-CD38 and TC-conjugated anti-HLA-DR monoclonal antibodies. Control cells were stained with conjugated irrelevant mouse monoclonals as described in Material and Methods. Sorting gates are defined as shown where R1 = lymphocyte gate; R2 = CD34+38–DR–; R3 = CD34+38–DR+ and R4 = CD34+38+DR+.

 

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  		Table 1. Percentage of CD34+ subpopulations expressing the CD38 and HLA-DR antigens
 
Colony-Forming Capacity
In three experiments, cells from each of the three separated populations were assayed for their content of CFCs. Results are shown in Table 2Go for each of the three subsets. The majority of GM-CFCs and BFU-Es were confined to the CD34+38+DR+ fraction, although 14% and 3% of GM-CFCs were detected in the CD34+38–DR+ and CD34+38–DR– populations, respectively. There was a far greater depletion of erythroid progenitors in the CD34+38–DR+ and CD34+38–DR– populations compared with the CD34+38+DR+ cells. These results confirm that this latter subset consists primarily of committed progenitors.


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  		Table 2. CFCs generated from each CD34+ subpopulation
 
Expansion of Single Cells
To investigate the expansion potential of each of the selected populations, single cells from each subset were individually sorted into serum-free medium containing growth factors, and their proliferation followed for at least six weeks. In four experiments, a total of 720 wells were examined. The percentage of wells containing proliferating cells, (assayed as >5 cells per well), at around day 14 for each subpopulation averaged 77% for CD34+38+DR+, 34% for CD34+38–DR+ and 38% for CD34+38–DR– and reflected the rate of expansion in the early stages of culture (Fig. 2Go). There was significant difference in growth between the CD34+38+DR+ phenotype compared with the CD34+38–DR+ and CD34+38–DR– groups (p < 0.0001). However, there was no significant difference in growth between the CD34+38–DR+ and CD34+38–DR– subsets at this time point (p = 0.52). As shown in Table 3Go, the range in cell numbers per well varied, as the CD34+38–DR+ subset had cell numbers ranging from 103 to 4.6 x 104 at day 14, while similar numbers per well were obtained in the CD34+38–DR– population about one week later. However, median values for both subpopulations were identical at day 14.



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  		Figure 2. Percentage of wells containing proliferating cells at day 14 after initial sorting of single cells into serum-free media supplemented with growth factors. Cultures were scored for proliferation (>5 cells) and results are an average of six experiments. Standard errors were less than ten percent. The asterisk (*) indicates that the percentage of growth is significantly higher in this CD34+ subpopulation (p <= 0.0001).

 

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  		Table 3. Growth and expansion of single cells in serum-free medium supplemented with a combination of growth factors
 
To determine the total cell expansion of individual cells, the number of cells per well and the number of wells used were recorded at each replating (Fig. 3Go). Single cells from cord blood have a remarkable potential for expansion, particularly among the CD34+38–DR– population where an individual cell could generate between 2.7 x 105 and 9.2 x 106 progeny after six weeks in liquid culture, as illustrated in Table 3Go. In some experiments, the cells from this subset continued to expand for >50 days. Cells from the CD34+38–DR+ fraction were less active, with a yield of between 1.2 to 1.8 x 106 progeny from a single cell over the same time period. There was a reduced potential among the CD34+38+DR+ fraction for expansion as the cell numbers peaked at week 5 but declined to 0.96 to 2.5 x 105 by week 6 of culture. Even among this population, however, individual cells could be expanded up to 106 cells after five weeks. There was significant difference in expansion between the three populations at weeks 4, 5 and 6 (p = 0.001, 0.05 and 0.03, respectively). In all of the subsets, the cells obtained were predominantly macrophages by six weeks.



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  		Figure 3. Diagram showing the scheme adopted for the expansion of a single cell. Details as described in Materials and Methods.

 
Of the single sorted cells, about 7% CD34+38+DR+, 33% CD34+38–DR+ and 33% CD34+38–DR– failed to grow in our culture conditions, although the cells were alive for at least 21 days as assessed by trypan blue exclusion.

The capacity of the different cell subsets to generate colony-forming cells in liquid culture is illustrated in Figure 4Go. Colonies formed from the expanded clones of these individual cells were erythroid, myeloid or erythroid/myeloid, although in general, erythroid colonies predominated. The CD34+38+DR+ cells rapidly lost the ability to generate CFCs by the third week in culture, whereas CD34+38–DR– cells continued to generate significant numbers of CFCs up to week 6. In the CD34+38–DR+ subset, large numbers of CFCs were produced within two to three weeks, but this did not extend beyond week 4. By six weeks, colonies could only be detected in the CD34+38–DR– subset, and the percentage of clones giving rise to CFCs at the different time points is summarized in Table 4Go. Again, there were significant differences between the three subpopulations throughout the term of culture, particularly by week 6.



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  		Figure 4. Cumulative production of CFCs generated from a single cell with time. CD34+38+DR+ (white), CD34+38–DR+ (grey), CD34+38–DR– (black).

 

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  		Table 4. Percentage of each CD34+ subpopulation giving rise to CFCs at different time points in liquid culture
 
CD34+ Subpopulations in LTC on Irradiated Stroma
To further compare the capacity of the three subpopulations to initiate and sustain hemopoiesis in LTCs, the three different subsets were seeded on pre-established irradiated bone marrow stroma. The cell numbers in the inoculum were adjusted in accordance with the yield from each individual experiment. Nonadherent cells in the supernatant were harvested weekly and examined for their content of CFCs, and the colonies obtained expressed as numbers of GM-CFCs per 1,000 cells plated in the initial inoculum (Fig. 5Go).



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  		Figure 5. Generation of GM-CFCs per 1,000 cells seeded onto irradiated bone marrow stroma. Results illustrate four individual experiments (1-4) where numbers of cells in the inoculum varied between 5 x 103-104 for CD34+38+DR+; 103-5 x 103 for CD34+38–DR+; 40-5 x 102 for CD34+38–DR– subsets. CD34+38+DR+ = solid line (—); CD34+38–DR+ = small dash (--); and CD34+38–DR– = broken line (– –).

 
The CD34+38–DR– cells generated high numbers of CFCs which gradually increased and reached a maximum around weeks 4 to 5. At this time, there was a significant difference between the numbers of GM-CFCs generated by the different subsets (p = 0.05) with a systematic ordering of profiles throughout the period of culture (p = 0.01). The numbers of CFCs from the CD34+38–DR+ cultures were generally lower than those observed in LTCs of CD34+38–DR– cells. In contrast, there was a rapid decline in the GM-CFC content of LTCs initiated with CD34+38+DR+ cells between weeks 1 and 2 with colony numbers approximately 10- to 100-fold lower than those from the CD34+38–DR+ and CD34+38–DR– LTCs, respectively.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
Cord blood progenitor cells (CFC) are present at higher frequencies and give rise to hemopoietic colonies containing cells with high replating efficiency compared to bone marrow progenitor cells [15-18]. In the present study we used a rapid, immunomagnetic technique to enrich for CD34+ cells and fractionated this population by FACS sorting based on the expression of CD38 and HLA-DR antigens. Flow cytometry studies have indicated that expression of these two antigens varied significantly among adult bone marrow, fetal bone marrow, mobilized peripheral blood and cord blood [8]. The HLA-DR marker is commonly linked with the identity of hemopoietic stem cells in bone marrow, but this does not apply to cord blood cells [19-21]. In bone marrow, cells with a low level of HLA-DR antigen are enriched for primitive CFU-blasts, high proliferative potential CFCs (HPP-CFCs) and LTC initiating cells (LTC-ICs). The reverse has been reported in cord blood, where Traycoff et al. [22] showed that the HLA-DR+ subpopulation of CD34+ cells was more primitive than the CD34+DR– population. Other studies imply that primitive progenitor cells may be fractionated on the basis of lack of expression of the CD38 antigen alone, but it is now clear that functional differences exist between CD38– and DR– subfractions of CD34+ bone marrow cells [23]. In our study, we separated cord-blood-derived CD34+ cells lacking the CD38 antigen into two populations based on HLA-DR expression and distinguished two primitive cell populations. To our knowledge, no other studies have investigated the functional character of cells with this phenotype in cord blood.

In general, as cells progress toward lineage commitment and maturity, they acquire lineage and activation antigens. We confirmed that CD34+38+DR+ cells were enriched for both myeloid and erythroid CFCs, and that committed progenitors are associated with this phenotype. By contrast, both the CD34+38–DR+ and CD34+38–DR– populations were significantly reduced in their content of GM-CFCs and BFU-Es, implying that these populations were initially enriched for the more immature cells.

Proliferation rates varied between the different subpopulations, with CD34+38+DR+ cells proliferating faster over the first 14 days. This is in agreement with work by Migliaccio et al. [24], who expanded unfractionated CD34+ cells in serum-free conditions and noted various growth phases, suggesting that different populations of cells may be triggered to proliferate at different times. The initial delay in expansion of the CD34+38–DR+ and CD34+38–DR– subsets is in keeping with these populations being enriched for more primitive cells which require more time to enter the cell cycle, as many of these primitive cells are in G0 [25]. However, we also observed that cells within these primitive subpopulations varied in the rate of expansion (Fig. 2Go), so that this parameter alone cannot be taken as an indication of the degree of immaturity of a cell.

The expansion potential of the three cell subpopulations was monitored in serum-free conditions. Single cells from both the CD34+38–DR+ and CD34+38–DR– populations could generate between 106 and 9 x 106 over five to six weeks. This scale of expansion from a single cell indicates that both subsets contain cells with HPP. It is difficult to evaluate any direct comparison with previous studies, as both the starting populations of cells and the culture conditions vary widely [26-29]. Many studies have demonstrated the expansion of total cell numbers ranging from 20- to >1,000-fold. From the results here, it is clear that at the single-cell level, the potential for expansion from some individual cells is much greater (nearly 107 cells, Table 3Go).

A recent report examining a subpopulation of CD34+ cells (CD34+CD45RAlowCD71low) at the single-cell level agrees with the large expansion capacity of these cord blood cells with the generation of CD34+ progeny after 18 to 29 days of culture in serum-free medium [30]. However, in that study, continuous generation of CFCs was not assessed. From these data we infer that here, at least in the initial stages of proliferation, a fraction of the progeny from single cells was CD34+, as cells giving rise to colonies in clonogenic assays express this antigen [31]. As only the progeny of the CD34+38–DR+ and CD34+38–DR– cells, not the parental cell (compare Table 1Go), formed colonies in the semi-solid assays, the initial single cells were likely to be pre-CFCs.

We conclude that both the CD34+38–DR+ and CD34+38–DR– subsets contained very primitive hemopoietic progenitors enriched for pre-CFCs which do not form colonies in semi-solid assays. A proportion of these cells have extensive proliferation capacity, give rise to clonogenic progeny and can be induced to proliferate and differentiate in serum-free conditions. It is not clear whether self-renewal occurred, as those cells involved in self-renewal may divide more slowly and would be obscured by the extensive proliferation of the maturing cells. Some cells were not induced to proliferate at all in this system and remained quiescent. Although most of these were alive, they did not respond to the growth conditions used, suggesting that either these conditions are not optimal or this fraction of cells may be too primitive to be activated in vitro in a liquid culture system.

However, when the two populations, CD34+38–DR+ and CD34+38–DR–, were cultured on irradiated bone marrow stroma, GM-CFCs were generated for up to 12 weeks, indicating that these populations were enriched for LTC-ICs and that their expansion capacity is not fully expressed in liquid cultures. Interestingly, there was a significant increase in the GM-CFC numbers generated from CD34+38–DR– compared with CD34+38–DR+ throughout the term of culture. This difference could reflect LTC-ICs with different capacities for both self-renewal and generation of progenitor cells. In limiting dilution assays, the LTC-ICs are detected by their capacity to generate CFCs after five to eight weeks [32], and both the CD34+38–DR+ and CD34+38–DR– populations maintained their ability to produce GM-CFC beyond five weeks.

Another possibility is that the continued generation of GM-CFCs after week 5 may be derived from a fraction of the CD34+38–DR– cells with extensive self-renewal capacity, defined as the capacity to generate further LTC-ICs. We noted that a small fraction of cells within this latter subset continued to generate CFCs in liquid culture. In any case, both the CD34+38–DR+ and CD34+38–DR– populations were better supported by irradiated stroma than in liquid culture and appeared to be highly enriched for immature hemopoietic cells. The data indicate that although immature cells are known to reside within the CD34+38– compartment, there may be at least two types of LTC-ICs capable of initiating and sustaining long-term cultures but differing by as much as 10-fold in their capacity to generate myeloid cells in LTCs. These cells can be distinguished based on the expression of the HLA-DR antigen. It is tempting to speculate that they may be related to the cells mainly responsible for short-term and long-term engraftment. Clearly, the hemopoietic stem cell compartment consists of a hierarchy of cells which displays one or more of the characteristics which have been used to define them [33]. The populations with the more immature phenotypes examined in this study fulfill some of the criteria for the hemopoietic stem cell, as they express the CD34 antigen, lack maturation and activation antigens, require a combination of growth factors for stimulation and proliferation and can generate and sustain long-term hemopoiesis in a stromal environment in vitro.

We are now examining the same CD34+ subsets in our culture conditions to determine if the LTC-ICs within these populations can be expanded. It is possible that the LTC-ICs may have already expanded in the current serum-free conditions at an early stage of culture. Experiments at the single-cell level would clarify how each individual LTC-IC contributes to proliferation, and this would have important clinical implications where grafts are manipulated for transplantation.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
This work was supported by the Cancer Research Campaign, U.K. Dr. G. Nadali was supported by Policlinico Borgo Roma, Azienda Ospediale di Verona, Italy, and Dr. L. H. Coutinho was supported by a grant from the North West Regional Health Authority. The authors would also like to thank Professor J. Hows for supplying umbilical cord blood samples, Mr. M. Hughes and Mr. J. Barry for technical assistance with flow cytometry and Mr. D. Ryder for statistical analysis of the data.


   Footnotes
 
Provisionally accepted April 8, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Berenson RJ, Bensinger WI, Hill RS et al. Engraftment after infusion of CD34+ marrow cells in patients with breast cancer or neuroblastoma. Blood 1991;77:1717-1722.[Abstract/Free Full Text]

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  3. Pahwa RN, Fleischer A, Than S et al. Successful hematopoietic reconstitution with transplantation of erythroid-depleted allogeneic human umbilical cord blood cells in a child with leukemia. Proc Natl Acad Sci USA 1994;91:4485-4488.[Abstract/Free Full Text]

  4. Gluckman E, Broxmeyer HE, Auerbach AD et al. Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical cord blood from an HLA identical sibling. N Engl J Med 1989;321:1174-1178.[Medline]

  5. Broxmeyer HE, Hangoc G, Cooper S et al. Growth characteristics and expansion of human umbilical cord blood and estimation of its potential for transplantation in adults. Proc Natl Acad Sci USA 1992;89:4109-4113.[Abstract/Free Full Text]

  6. Cardoso AA, Li ML, Bartard P et al. Release from quiescence of CD34+38– human umbilical cord blood cells reveals their potentiality to engraft adults. Proc Natl Acad Sci USA 1993;90:8707-8711.[Abstract/Free Full Text]

  7. Huang S, Terstappen LWMM. Lymphoid and myeloid differentiation of single human CD34+, HLA-DR+, CD38– hematopoietic stem cells. Blood 1994;83:1515-1526.[Abstract/Free Full Text]

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Received March 7, 1996; accepted for publication June 10, 1996.




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