• ontogeny;
  • cord blood;
  • expansion;
  • stem cells;
  • cytokines


  1. Top of page
  2. Abstract
  6. Acknowledgements
  7. References

Time course studies revealed that the combination of Flt-3 ligand (FL), Steel factor (SF) and interleukin-3 (IL-3) did not elicit as large an amplification of the long-term culture-initiating cell (LTC-IC) population in serum-free cultures of CD34+CD38 cord blood (CB) cells as was obtained in similar cultures of adult human CD34+CD38 bone marrow (BM) cells (4- v 90-fold maximum increases), even though both total and colony-forming cell (CFC) numbers initially increased more rapidly in CB cultures. Multifactorial analysis of the short-term (10 d) effects of different cytokines identified FL and IL-6 in combination with the soluble IL-6 receptor (sIL-6R) as most important for expanding the CB LTC-IC population. In contrast, their counterparts in adult BM were most effectively stimulated by FL, SF and IL-3. For rapid generation of increased numbers of CFC, SF with either FL or IL-6/sIL-6R were found to be the most important contributors in cultures of CD34+CD38 CB cells, whereas, in analogous BM cultures, IL-6/sIL-6R and TPO (in addition to FL, SF and IL-3) were required. These findings reinforce the principle of altered cytokine responsiveness as a hallmark of early haemopoietic cell differentiation and demonstrate how cytokine requirements may change during human ontogeny. Identification of conditions for optimizing the expansion of different subsets of primitive CB cells has additional important implications for clinical transplantation and gene transfer.

It has been known for many years that the haemopoietic cell differentiation process changes during ontogeny. This involves changes not only in the expression of a variety of lineage-specific genes, e.g. in developing erythroid cells ( Fantoni et al, 1981 ), T cells ( Ikuta et al, 1990 ) and B cells ( Hardy & Hayakawa, 1991; Li et al, 1993 ), but also in the surface markers expressed on these cells and on their precursors ( Terstappen et al, 1991 ; Huang & Terstappen, 1994; Traycoff et al, 1994 ; Morrison et al, 1995 ; Rebel et al, 1996b ). Subtle differences between the responses of some fetal and adult progenitor cell types to cytokines have also been reported ( Rich & Kubanek, 1980; Migliaccio & Migliaccio, 1988; Mayani et al, 1995 ; Gardner et al, 1990 ; Deutsch et al, 1995 ; Hogge et al, 1996 ; van de Ven et al, 1995 ) and evidence of an intrinsically determined, greater proliferative potential of haemopoietic stem cells (HSC) and progenitors from fetal (or newborn) sources by comparison with adult bone marrow (BM) has been documented ( Micklem et al, 1972 ; Harrison, 1983; Lansdorp et al, 1993 ; Lu et al, 1993 ; Rebel et al, 1996a ).

In a previous study we identified high concentrations of Flt-3 ligand (FL), Steel factor (SF) and interleukin-3 (IL-3) combined in a 5:5:1 (wt) ratio as necessary and sufficient (amongst several cytokines tested) to stimulate > 50-fold increases in human long-term culture-initiating cell (LTC-IC) numbers in short-term (10 d) suspension cultures initiated with CD34+CD38 normal adult BM cells ( Petzer et al, 1996b ; Zandstra et al, 1997a ). In the same cultures, production of colony-forming cells (CFC) could be further increased if IL-6, granulocyte colony-stimulating factor (G-CSF), or nerve growth factor-β was also present. In addition to the different numbers and types of cytokines required to optimize LTC-IC and CFC amplification, these two responses were found to be differently affected by changes in the relative and absolute concentrations of the cytokines in the medium. Interestingly, in a subsequent series of experiments in which CD34+CD38 cord blood (CB) cells were cultured under similar conditions, the LTC-IC population was expanded only slightly ( Kogler et al, 1996 ; Conneally et al, 1997 ; Bhatia et al, 1997 ), in spite of a large amplification of the CFC population ( Migliaccio et al, 1992 ; Lansdorp et al, 1993 ). This marked and selective difference in the extent of LTC-IC expansion observed in cultures of similarly stimulated CD34+CD38- CB and adult BM cells prompted us to undertake a more detailed investigation of their respective cytokine requirements. The results demonstrated FL and IL-6 plus the soluble IL-6 receptor (sIL-6R) to be the most important factors for stimulating LTC-IC amplification in serum-free cultures of human CD34+CD38 CB cells, in contrast to adult BM CD34+CD38 cells which required FL, SF and IL-3. Differences in the cytokine requirements for amplifying different subpopulations of CFC from these two ontologically distinct sources of input cells were also identified.


  1. Top of page
  2. Abstract
  6. Acknowledgements
  7. References


CB from normal, full-term infants delivered by caesarean section were collected in tubes containing heparin according to protocols approved by the University of British Columbia Clinical Screening Committee for Research Involving Human Subjects. A CD34+ cell-enriched fraction (∼ 40% CD34+ cells) was isolated using a StemSepTM column (StemCell Technologies, Vancouver, BC) as previously described ( Conneally et al, 1997 ). Light-density normal human BM cells (< 1.077 g/cm3) were isolated using Ficoll-Paque (Pharmacia, Uppsala, Sweden) from previously frozen harvests obtained from cadaveric organ donors (Northwest Tissue Center, Seattle, Wash.). The CD34+CD38 subpopulation (geqslant R: gt-or-equal, slanted 99% pure) was then isolated from both sources of cells by fluorescence-activated cell sorting (FACS) as described ( Lansdorp & Dragowska, 1992; Petzer et al, 1996b ). Briefly, cells were washed in phosphate-buffered saline (PBS) containing 2% fetal calf serum (FCS, StemCell), resuspended in Hank's hepes-buffered salt solution containing 2% FCS and 0.1% sodium azide (HFN), incubated at leqslant R: less-than-or-eq, slant 107 cells/ml for 30 min at 4°C with a combination of anti-CD34 (8G12 FITC) (kindly provided by Dr P. Lansdorp, Terry Fox Laboratory) and anti-CD38 (Leu 17-PE, Becton Dickinson, San Jose, Calif.) and then washed twice in HFN. 2 μg/ml propidium iodide (PI, Sigma Chemicals, St Louis, Mo.) was added to the final wash. The cells were then resuspended in HF (no azide) for sorting on a FACStar Plus® (Becton Dickinson) equipped with a 5 W argon laser and a 30 mW helium laser. Viable (PI) cells with low-medium forward light scattering properties, low side scattering characteristics and a CD34+CD38 phenotype were collected in Eppendorf tubes containing serum-free Iscove's medium (described below). Positive staining was defined as the emission of a level of fluorescence that exceeded levels obtained by geqslant R: gt-or-equal, slanted 99% of the cells from the same starting population when these were stained with control antibodies labelled with the corresponding fluorochrome.

Stroma-free liquid suspension cultures

CD34+CD38 cells were incubated at 500 CB or 2000 BM cells/ml in Iscove's medium containing no serum but supplemented with 20 ng/ml of bovine serum albumin, 10 μg/ml of human insulin and 200 μg/ml of human transferrin (BIT, StemCell) plus 40 μg/ml low density lipoproteins (Sigma), 10−4 M 2-mercaptoethanol (2-ME) and various growth factors as previously described ( Petzer et al, 1996b ). For the time-course experiments, 2.5 ml cultures were maintained in 35 mm petri dishes and subcultured weekly by transferring either 1/4 (week 1) or 1/10th (weeks 2–7) of the cells present into new petri dishes after dilution of the cells into the original volume of fresh medium and cytokines. For the multifactorial experiments, 100 μl cultures were incubated unperturbed in round-bottom 96-well plates for 10 d at 37°C. At the end of 10 d (for the multifactorial experiments) or weekly (for the kinetic experiments), aliquots of the cells were assayed for LTC-IC and CFC. All cytokines evaluated were purified recombinant human proteins obtained from the following sources: Flt-3 ligand (FL, Immunex, Seattle, Wash.) SF and G-CSF (Amgen, Thousand Oaks, Calif.), IL-1 (Biogen, Cambridge, Mass.) and IL-3 (Novartis, Basel, Switzerland), IL-6 (Cangene, Missassauga, Ont.), soluble IL-6 receptor (sIL-6R, R & D Systems, Minneapolis, Min.), and thrombopoietin (TPO, ZymoGenetics, Seattle, Wash.).

Colony assays

Cell suspensions to be assayed for granulopoietic (CFU-GM), erythropoietic (CFU-E and BFU-E) and multi-lineage (CFU-GEMM) CFC were plated at suitable cell concentrations (to give < 100 colonies per 1.1 ml culture) in Iscove's medium containing 0.9% methylcellulose, 30% FCS, 1% bovine serum albumin, 10−4 M 2-ME (Methocult H4430, StemCell), 20 ng/ml each of IL-3, GM-CSF (Novartis), IL-6 and G-CSF, and 50 ng/ml SF. These assay cultures were then incubated at 37°C for 2–3 weeks prior to scoring all colonies using criteria adopted in our laboratory for many years.

LTC-IC assays

LTC-IC frequencies were determined by placing aliquots of the cells to be tested in 35 mm tissue culture dishes already containing semi-confluent feeder layers of irradiated (8000 cGy) mouse M2-10B4 and Sl/Sl fibroblasts previously engineered to express 4 ng/ml of human IL-3, 190 ng/ml of human G-CSF and 4 ng/ml of human SF ( Hogge et al, 1996 ) plus 2.5 ml of human myeloid long-term culture medium (Myelocult, StemCell). This medium contains 12.5% horse serum, 12.5% FCS and 10−4M 2-ME. 10−6 M hydrocortisone sodium hemisuccinate (Sigma) was added just prior to use. LTC-IC assays were incubated for 6 weeks at 37°C with weekly removal of half of the nonadherent cells and replacement of half of the medium ( Hogge et al, 1996 ). After 6 weeks, all of the remaining nonadherent cells from each assay culture were removed, added to the corresponding trypsinized adherent cells, and the combined pool was then washed and assayed for CFC as described above. The colony counts obtained were used to calculate the total yield of CFC (CFU-GM plus BFU-E plus CFU-GEMM) at the end of 6 weeks from each input innoculum tested. This value provides a relative but, nevertheless, quantitative measure of the number of LTC-IC initially present ( Sutherland et al, 1990 ). Previous work has shown that the number of colonies generated per LTC-IC remains unchanged after expansion of the LTC-IC in growth factor-supplemented serum-free media ( Sutherland et al, 1991 ; Petzer et al, 1996a ; Zandstra et al, 1997a ).

Multifactorial design experiments

The same type of multilevel orthogonal factorial analysis ( Box et al, 1978 ) was used to determine the relative roles of various cytokines in stimulating an expansion of each of the progenitor populations evaluated (i.e. LTC-IC, CFU-GM and BFU-E/CFU-GEMM) here as was used previously to examine the cytokine responses of primitive human BM cells ( Petzer et al, 1996b ; Zandstra et al, 1997a ). In the present CB experiments the six cytokines evaluated were FL (300 ng/ml), SF (300 ng/ml), IL-3 (60 ng/ml), IL-6 plus sIL-6R (considered as a pair at 60 and 1000 ng/ml, respectively), TPO (1000 U/ml) and IL-1 (36 U/ml). All 64 possible combinations of each of these were tested in two subexperiments. Each of the two subexperiments (32 combinations per subexperiment) was performed with a pool of CD34+CD38 cells isolated from three different CB specimens. Each of the two subexperiments also included four replicate cultures containing all six cytokines at one third of the concentrations used in the other combinations. Such a design enabled the effects of individual, paired and triplet cytokine combinations to be assessed in the 32, 16 and eight possible combinations of each (i.e. the numbers of combinations used for the determination of significance). In a second pair of experiments the same six cytokines were similarly tested at the same concentrations for their ability to amplify LTC-IC and CFC numbers in 10 d cultures initiated with CD34+CD38 cells isolated from different samples of normal adult human BM. In these experiments the cytokines assessed were FL, SF and IL-3 (as one group), IL-6 and sIL-6R (as a second group), and TPO and IL-1 (separately). This resulted in the evaluation of 16 possible combinations of these cytokine groups plus four replicate cultures containing all six cytokines at one third of the concentrations used in the test groups.

The net effect of each cytokine (or group of cytokines) on the amplification of each progenitor population monitored was calculated as described by Box et al (1978 ), from the sum of the differences between all of the values obtained in the presence versus the absence of the particular cytokine (or group of cytokines) being considered. Calculations were performed using the Jass Software program (Joiner Associates, Madison, Wis.). To enable the data from the different subexperiments to be combined for this analysis (the CB and BM experiments were analysed separately), the logarithm (log) of each of the population changes measured in a given subexperiment was first determined, the mean of all of the log expansions measured in that subexperiment was then subtracted from it and the difference thus obtained was then divided by the standard deviation of all of the individual log expansion values. The results of the multifactorial analyses of these normalized data sets reveal statistically significant effects (in this case on the amplification of CFC and LTC-IC from CB or adult BM CD34+CD38 cells) as those lying outside the range defined by the mean ± 2 SD for the distribution of values from a given source of cells and endpoints.


  1. Top of page
  2. Abstract
  6. Acknowledgements
  7. References

Different kinetics of progenitor expansion in cultures of CB and adult BM cells stimulated by FL, SF and IL-3

Highly purified CD34+CD38 cells were cultured in serum-free medium supplemented with 300 ng/ml FL and SF and 60 ng/ml IL-3 with weekly transfer of an aliquot of the cells to new wells containing fresh medium (to maintain the cell density at < 2.5 × 106 /ml) as described in Methods. Fig 1 shows the projected accumulated (fold) change in the total, CFC and LTC-IC populations based on measurements of these performed over the entire 7-week course of the experiments. Weekly assessment of the cytokine and glucose concentrations in these cultures (by ELISA and colourimetric glucose oxidase-based assays, respectively) showed that these constituents remained consistently > 50% of their initial values (data not shown), suggesting that none of the cellular expansions observed was limited at any time by excessive cell densities or medium component depletion ( Zandstra et al, 1997b ). The rate of total cell expansion in the CB cell cultures was significantly higher than in analogous cultures of BM cells during the first 3 weeks (mean increase ± SEM =  67 000 ± 9, 600-fold v 950 ± 370-fold, P < 0.01). These values imply maximum average cell generation times of 31 and 50 h for the expanding CB and BM cells, respectively. Initial expansion of the CFC population was also significantly faster in the CB cultures than in the BM cultures (to reach values that were 8000 ± 2500-fold v 1400 ± 50-fold above input, respectively, by the end of the first 3 weeks, P < 0.05). During the fourth and fifth week the total cell numbers in the CB cultures were maintained but did not increase further and the CFC population began to decline. In contrast, in the BM cultures both the total number of cells and the number of CFC continued to expand at an undiminished rate. The LTC-IC population in the BM cultures also expanded rapidly and continuously to achieve a peak value of 91 ± 42-fold above input by the end of the first 3 weeks. Thereafter, the number of LTC-IC in the BM cultures declined, but slowly, so that even after another 4 weeks they were still 8 ± 4-fold higher than the input value. In the CB cultures LTC-IC numbers also increased initially, but less rapidly and for a shorter period of time. As a result, maximum values of only 4 ± 2-fold above input were reached within the first 2 weeks.


Figure 1. 00 CD34+CD38 adult BM cells. Results are the mean ± SEM of data from three experiments.

Download figure to PowerPoint

It should be noted that in the first week the number of CFC in both CB and BM cultures increased more rapidly than the total number of cells. This indicated that, during this early period, acquisition of in vitro colony-forming ability (in semi-solid medium) by primitive cells that lack this function was occurring faster than the concomitant rate at which this property was being lost.

Identification of specific cytokine requirements for generating different types of primitive haemopoietic progenitors in 10 d cultures of CD34+CD38 CB cells

The experimental results presented in Fig 1 suggested that CD34+CD38 CB cells were not as responsive to FL, SF and IL-3 as their adult BM counterparts in terms of their ability to produce expanded populations of LTC-IC. To determine whether this apparent defect of CB cells might be corrected by stimulating them with other cytokines, CD34+CD38 cells were isolated from two different pools of CB cells, and aliquots of 50 of these cells were then incubated for 10 d in 100 μl suspension cultures containing different combinations of FL, SF, IL-3, IL-6/sIL-6R, TPO and IL-1. These cytokines were chosen because of their previously reported effects on very primitive CB and/or BM progenitors ( Migliaccio et al, 1992 ; Brandt et al, 1992 ; Lansdorp & Dragowska, 1992; Tsujino et al, 1993 ; Sui et al, 1995 ; Moore & Hoskins, 1994; Petzer et al, 1996b ). In this analysis, IL-6 and the sIL-6R were studied as a pair because it had been previously reported that on these cells the sIL-6R has no stimulatory effect on its own ( Sui et al, 1995 ).

The CFC and LTC-IC contents of each of the two input CD34+CD38 cell suspensions, and of the cells harvested from each of the 80 different 10-d-old liquid suspension cultures (36 different combinations of growth factors plus four replicates in each of two subexperiments) were obtained by comparing the total output of each of these functionally defined cell types from a fixed number of input CD34+CD38 cells. The validity of this approach to assess changes in LTC-IC numbers (rather than by comparing numbers obtained from limiting dilution analyses of each test cell suspension) is based on previous evidence that the numbers and types of CFC generated from individual LTC-IC, although highly variable, remained on average the same when sufficiently large numbers of freshly isolated and cultured LTC-IC were compared ( Sutherland et al, 1991 ; Petzer et al, 1996a ; Zandstra et al, 1997a ).

For the various conditions evaluated using CB input cells, changes in BFU-E numbers (relative to the start value) ranged from a > 100-fold decrease to a 1100-fold increase, and for CFU-GEMM from a > 100-fold decrease to a 700-fold increase. Multiparameter analysis of the variance of the normalized data from both CB subexperiments was used (as described in Methods) to identify individual or interactive effects of single cytokines, or particular combinations of cytokines, as those lying significantly outside a range defined by the mean ± 2 SD of the normal probability distribution of the effects measured for all cytokine combinations tested ( Box et al, 1978 ; Zandstra et al, 1997a ). A comparison of the standard deviation of the two sets of replicate centre points (i.e. data from the CB cultures containing all factors at one third of the concentrations used in all other groups) established that the interexperimental variability was not statistically significant in this analysis (P < 0.05, data not shown).

Fig 2 shows the outcome of this analysis for the 10 d production of different types of CFC from CD34+CD38 CB cells. In the case of BFU-E and CFU-GEMM, only the effects of SF (P < 0.001 for both) and IL-6/sIL-6R (P < 0.01 for BFU-E and P < 0.05 for CFU-GEMM) were significant whereas, for the generation of CFU-GM in the same cultures, only the effects of SF (P < 0.001) and FL (P = 0.07) were significant. These findings indicated that different cytokines regulated the generation from CB cells of progenitors committed to different lineages.


Figure 2. different combinations of cytokines, performed in two runs, each with a different source of pooled CB (see text).

Download figure to PowerPoint

Corresponding changes in LTC-IC numbers varied from a >100-fold decrease to a 380-fold increase over input LTC-IC values, and FL (P < 0.01) and IL-6 plus the sIL-6R (P < 0.01) were the only cytokines identified by multiparameter analysis of variance to be significant contributors to LTC-IC expansion in these cultures (Fig 3). Thus, the cytokines that maximize LTC-IC amplification from CB cells are different from those that maximize the concomitant expansion of CFC committed to either the erythroid or the granulopoietic pathways. Interestingly, in this analysis, TPO was not identified as a significant contributor to LTC-IC amplification from CD34+CD38 CB cells.


Figure 3. Fig 3. Linearized normal probability distribution of the normalized effect of different combinations of cytokines on LTC-IC expansion from the same cultures shown in Fig 2. Analysis was performed in the same manner as for Fig 2.

Download figure to PowerPoint

The superiority of the FL plus IL-6/sIL-6R combination on CB LTC-IC expansion by comparison to the combination of FL, SF, IL-6, IL-3 and G-CSF or the combination of FL plus TPO, was further confirmed in additional experiments ( 1 Table I). In contrast, as also predicted by the factorial design experiments, the expansion of CB CFC remained greatest in the SF containing cocktail.

Table 1. Table I. Effects of selected culture conditions on the expansion of the CFC and LTC-IC populations in 10 d serum-free cultures of CD34+CD38 CB cells. The number of CFC per 100 CD34+CD38 input cells was 36, 13, 16 and 13 for experiments 1, 2, 3 and 4, respectively. The number of CFC per 100 CD34+CD38 input cells from week 6 harvests of LTC-IC assays in the same experiments was 2500, 380, 1500 and 26, respectively. nd: not determined.Thumbnail image of

Different cytokine requirements for generating CFC and LTC-IC in 10 d cultures of CD34+CD38 adult BM cells

The same combinations of cytokines tested in the CB experiments were then investigated for their effects on CD34+CD38 BM cells using a similar protocol. However, in this case, FL, SF and IL-3 were considered as a single group, IL-6/sIL-6R as a second group, and only TPO and IL-1 were evaluated individually. These groupings were chosen based on our previous results with adult BM cells which had shown that TPO, IL-1, IL-3, SF and FL, but not IL-6, all had independent positive effects on LTC-IC expansion, although neither TPO nor IL-1 enhanced LTC-IC expansion beyond that achieved with FL plus SF plus IL-3 (plus IL-6 plus G-CSF) ( Petzer et al, 1996b ). A total of 40 liquid suspension cultures were thus evaluated (16 different combinations of cytokines plus four replicates in each of two independent experiments). Changes in BFU-E numbers ranged from a > 50-fold decrease (relative to input) to a 1200-fold increase according to the cytokine combination present, and changes in CFU-GM ranged from a > 50-fold decrease to a 64-fold increase. For LTC-IC, values ranged from an 8-fold decrease to an 88-fold increase. (CFU-GEMM numbers, both before and after the 10 d culture period, were too low to permit meaningful analysis.) Comparison of the standard deviation of the two sets of replicate centre points (i.e. data from cultures containing all cytokines at one third of the concentrations used in all other groups) established that the interexperimental variability was again not statistically significant (data not shown).

Multiparameter analysis of variance of the experimental data revealed that, as previously reported ( Petzer et al, 1996b ), the addition of one or more cytokines to the combination of SF, FL and IL-3 (P < 0.001 for both BFU-E and CFU-GM) was required to obtain maximal expansion of either of these populations. For BFU-E, these were TPO (P < 0.01) and IL-6/sIL-6R (P < 0.05), whereas for the CFU-GM, only IL-6/sIL-6R (P < 0.05) contributed an additional significant effect (Fig 4). IL-1 did not have any significant effect on the generation of either type of CFC. Thus, the initial generation of different types of CFC from CD34+CD38 adult BM cells is also regulated by specific cytokines. In the same cultures, only the combination of FL, SF and IL-3 (P > 0.001) had a significant effect on LTC-IC expansion (Fig 4), confirming our previous results for this population ( Petzer et al, 1996b ).


Figure 4. +CD38 adult BM cells. Analysis was performed in the same manner as for Fig 2. Effects were obtained from an analysis of 16 different combinations of cytokines, performed in duplicate, each with a different source of BM.

Download figure to PowerPoint


  1. Top of page
  2. Abstract
  6. Acknowledgements
  7. References

We showed recently that a significant net increase in LTC-IC (up to 60-fold within 10 d) can be achieved in cytokine-stimulated cultures of CD34+CD38 adult BM cells. Moreover, this occurs without compromising the concomitant expansion of CFC which, in the same cultures, may increase several hundred-fold during the same period of time ( Zandstra et al, 1997a ). Additional studies of such cultures have led to several other important findings. First, optimization of the initial amplification of LTC-IC and CFC from a very primitive subset of adult BM cells (i.e. highly purified CD34+CD38 cells) requires different types and concentrations (both absolute and relative) of cytokines. Second, both the numbers and types of progeny produced from individual CD34+CD38 cells exposed to the same cytokine combinations in such cultures varies widely ( Petzer et al, 1996a ), although deterministic effects of certain cytokines on these outcomes are also demonstrable ( Zandstra et al, 1997a ). Third, conditions optimized for the amplification of BM LTC-IC are not as effective in stimulating their counterparts in cultures of CB cells ( Kogler et al, 1996 ; Conneally et al, 1997 ). This suggested that the cytokines able to regulate primitive haemopoietic cell proliferation and differentiation may change during ontogeny as well as during the differentiation process itself.

The present study was designed to formally test this latter possibility by defining the cytokine requirements for expanding LTC-IC and CFC numbers in short-term cultures of CD34+CD38 CB cells. The roles of six cytokines in contributing to each of these outcomes was examined through the use of a multifactorial experimental design and analysis of variance of the results obtained. The six cytokines tested were: FL, SF, IL-1, IL-3, IL-6/sIL-6R and TPO. These cytokines were chosen because they had all been found to have a stimulatory effect (either alone or in combination) on adult BM LTC-IC or CFC cultured under similar conditions ( Petzer et al, 1996b ). For expansion of adult BM LTC-IC, FL plus SF plus IL-3 was confirmed as the minimal combination of cytokines able to elicit a maximal response. In contrast, for CB cells, FL and IL-6/sIL-6R were the only cytokines of the six tested that had a significant effect in promoting LTC-IC amplification. An equivalent role for SF only became apparent at the later stage of CFC expansion where it was effective on multipotent, erythropoietic and granulopoietic CFC alike. However, expansion of CB CFC also required a second cytokine; FL in the case of the granulopoietic CFC, and IL-6/sIL-6R in the case of the erythroid and multipotent CFC. Thus, as summarized schematically in Fig 5, the cytokines required to generate the earliest types of CFC from CB cells reflect a partitioning of the cytokine requirements for expanding LTC-IC from the same source of cells plus the acquisition of a new requirement for SF stimulation. Expansion of analogous adult BM CFC populations was also promoted by a combination of cytokines that included some that stimulated LTC-IC and others, like IL-6/sIL-6R, that did not. In addition, expansion of BM CFC included significant responses to IL-3 and TPO which were not shared by their CB counterparts.


Figure 5. Fig 5. Schematic comparing the different cytokine effects on functionally similar progenitor cells in CB and adult BM.

Download figure to PowerPoint

At first glance these latter results might appear to differ from those Piacibello et al (1997 ) who recently reported an ability of TPO plus FL to stimulate a marked expansion of LTC-IC and CFC in cultures of CB cells. However, in their study, large progenitor expansions were not seen until after a much longer period of culture (> 5 weeks) and the cells were kept throughout the duration of the experiment in the original culture vessels. We have observed the occasional development of an adherent layer when CD34+CD38 CB cells were cultured for prolonged periods of time (4–6 weeks) in the same culture dish, in which case the type of calculations used by Piacibello et al (1997 ) to derive expansion values would become invalid and probably overestimate the true numbers attainable. On the other hand, our results do bear out the recent findings of Tajima et al (1996 ) who showed that for CD34+ human CB cells the IL-6R-negative (IL-6R) population included the LTC-IC, CFU-GEMM, BFU-E and megakaryopoietic CFC whereas most CB CFU-GM were IL-6R+. This distribution of IL-6R and IL-6R+progenitor phenotypes exactly matches the populations whose expansion in vitro we have shown to be stimulated by IL-6 in combination with the sIL-6R. Our findings thus support the concept that provision of a high concentration of sIL-6R in combination with IL-6 can bypass the need for constitutive IL-6R expression to allow IL-6 to stimulate a maximal gp 130-mediated response.

The patterns of changing cytokine responses observed here indicate a striking association between the transition of cells from the LTC-IC to the CFC compartment and the particular cytokines that stimulate these cells to expand their numbers. Notably, the factor requirements for expansion of LTC-IC in cultures of CB and adult BM cells were different, but simpler than the factor requirements for CFC expansion from cells of either source. The latter include the cytokines required by LTC-IC plus at least one new factor, its identity being dependent upon the ontological origin of the CFC being produced and their lineage assignment (Fig 5). These findings thus provide examples where similar biological outcomes may be obtained by the activation of different cytokine receptors. Further comparisons of the intracellular events activated may provide new clues about common intracellular events that mediate the proliferation and differentiation of functionally similar, primitive haemopoietic cells.

Cord blood has recently attracted interest as a source of haemopoietic stem cells both for BM transplantation and gene therapy applications. The use of CB cells as a source of marrow repopulating cells has been demonstrated both in related and unrelated transplant recipients ( Wagner et al, 1992 , 1996; Kurtzberg et al, 1996 ). However, the majority of such transplants have been carried out in paediatric populations and the feasibility of extending this approach routinely to adult (larger) recipients has not yet been addressed. Many centres have embarked on studies to try to expand CB cell progenitors with the aim of producing transplants that would achieve rapid and permanent engraftment of adults. Various cocktails of cytokines including IL-1, IL-3, IL-6, G-CSF, GM-CSF, SF, FL and TPO have been shown to stimulate the rapid proliferation in vitro of CD34+ cells isolated from adult BM, mobilized blood and CB, resulting in significant increases in total cell numbers as well as in various types of lineage-restricted and multi-potent CFC ( Haylock et al, 1992 ; Koller et al, 1993 ; Sato et al, 1993 ; Shapiro et al, 1994 ; Alcorn et al, 1996 ). The present identification of the different cytokine combinations required to optimize the rapid production of more primitive haemopoietic progenitors from CB and adult BM cells is therefore likely to be of considerable practical importance. It is, of course, still critical to establish whether the results obtained here for CB and adult BM LTC-IC will prove to be predictive for human stem cells with in vivo repopulating potential. However, our recent results with cytokine-stimulated CB cells assayed for their ability to repopulate NOD/SCID mice both before and after retroviral infection are consistent with this prediction ( Conneally et al, 1997 , 1998).


  1. Top of page
  2. Abstract
  6. Acknowledgements
  7. References

The authors thank Dianne Reid and Gayle Thornbury for excellent technical assistance, Bernadine Fox for manuscript preparation, and Dr Peter Lansdorp, StemCell Technologies, Novartis, Immunex, Amgen, Biogen, Cangene and Zymogenetics for gifts of reagents. This work was supported by operating grants from the National Cancer Institute of Canada (NCIC) with funds from the Terry Fox Run, the British Columbia (BC) Science Council, and Novartis. P. Zandstra held studentships from the Natural Sciences and Engineering Research Council of Canada and the BC Science Council, E. Conneally an NCIC Terry Fox Physician-Scientist Fellowship, and C. Eaves a Terry Fox Cancer Research Scientist award from the NCIC.


  1. Top of page
  2. Abstract
  6. Acknowledgements
  7. References
  • 1
    Alcorn, M.J., Holyoake, T.L., Richmond, L., Pearson, C., Farrell, E., Kyle, B., Dunlop, D.J., Fitzsimons, E., Steward, W.P., Pragnell, I.B., Franklin, I.M. (1996) CD34-positive cells isolated from cryopreserved peripheral-blood progenitor cells can be expanded ex vivo and used for transplantation with little or no toxicity. Journal of Clinical Oncology, 14, 1839 1847.
  • 2
    Bhatia, M., Bonnet, D., Kapp, U., Wang, J.C.Y., Murdoch, B., Dick, J.E. (1997) Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture. Journal of Experimental Medicine, 186, 619 624.
  • 3
    Box, G.E.P., Hunter, W.G., Hunter, J.S. (1978) Statistics for Experimenters: an Introduction to Design, Data Analysis, and Model Building. John Wiley & Sons, Toronto.
  • 4
    Brandt, J., Briddell, R.A., Srour, E.F., Leemhuis, T.B., Hoffman, R. (1992) Role of c-kit ligand in the expansion of human hematopoietic progenitor cells. Blood, 79, 634 641.
  • 5
    Conneally, E., Cashman, J., Petzer, A., Eaves, C. (1997) Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice. Proceedings of the National Academy of Sciences of the United States of America, 94, 9836 9841.
  • 6
    Conneally, E., Eaves, C.J., Humphries, R.K. (1998) Efficient retroviral-mediated gene transfer to human cord blood stem cells with in vivo repopulating potential. Blood, 91, 3487 3493.
  • 7
    Deutsch, V.R., Olson, T.A., Nagler, A., Slavin, S., Levine, R.F. (1995) The response of cord blood megakaryocyte progenitors to IL-3, IL-6 and aplastic canine serum varies with gestational age. British Journal of Haematology, 89, 8 16.
  • 8
    Fantoni, A., Farace, M.G., Gambari, R. (1981) Embryonic hemoglobins in man and other mammals. Blood, 57, 623 633.
  • 9
    Gardner, J.D., Liechty, K.W., Christensen, R.D. (1990) Effects of interleukin-6 on fetal hematopoietic progenitors. Blood, 75, 2150 2155.
  • 10
    Hardy, R.R. & Hayakawa, K. (1991) A developmental switch in B lymphopoiesis. Proceedings of the National Academy of Sciences of the United States of America, 88, 11550 11554.
  • 11
    Harrison, D.E. (1983) Long-term erythropoietic repopulating ability of old, young, and fetal stem cells. Journal of Experimental Medicine, 157, 1496 1504.
  • 12
    Haylock, D.N., To, L.B., Dowse, T.L., Juttner, C.A., Simmons, P.J. (1992) Ex vivo expansion and maturation of peripheral blood CD34+ cells into the myeloid lineage. Blood, 80, 1405 1412.
  • 13
    Hogge, D.E., Lansdorp, P.M., Reid, D., Gerhard, B., Eaves, C.J. (1996) Enhanced detection, maintenance and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human Steel factor, interleukin-3 and granulocyte colony-stimulating factor. Blood, 88, 3765 3773.
  • 14
    Huang, S. & Terstappen, L.W.M.M. (1994) Lymphoid and myeloid differentiation of single human CD34+, HLA-DR+, CD38 hematopoietic stem cells. Blood, 83, 1515 1526.
  • 15
    Ikuta, K., Kina, T., MacNeil, I., Uchida, N., Peault, B., Chien, Y.H., Weissman, I.L. (1990) A developmental switch in thymic lymphocyte maturation potential occurs at the level of hematopoietic stem cells. Cell, 62, 863 874.
  • 16
    Kogler, G., Callejas, J., Hakenberg, P., Enczmann, J., Adams, O., Daubener, W., Krempe, C., Gobel, U., Somville, T., Wernet, P. (1996) Hematopoietic transplant potential of unrelated cord blood: critical issues. Journal of Haematotherapy, 5, 105 116.
  • 17
    Koller, M.R., Emerson, S.G., Palsson, B.O. (1993) Large-scale expansion of human stem and progenitor cells from bone marrow mononuclear cells in continuous perfusion cultures. Blood, 82, 378 384.
  • 18
    Kurtzberg, J., Laughlin, M., Graham, M.L., Smith, C., Olson, J.F., Halperin, E.C., Ciocci, G., Carrier, C., Stevens, C.E., Rubinstein, P. (1996) Placental blood as a source of hematopoietic stem cells for transplantation into unrelated recipients. New England Journal of Medicine, 335, 157 166.
  • 19
    Lansdorp, P.M. & Dragowska, W. (1992) Long-term erythropoiesis from constant numbers of CD34+ cells in serum-free cultures initiated with highly purified progenitor cells from human bone marrow. Journal of Experimental Medicine, 175, 1501 1509.
  • 20
    Lansdorp, P.M., Dragowska, W., Mayani, H. (1993) Ontogeny-related changes in proliferative potential of human hematopoietic cells. Journal of Experimental Medicine, 178, 787 791.
  • 21
    Li, Y.S., Hayakawa, K., Hardy, R.R. (1993) The regulated expression of B lineage associated genes during B cell differentiation in bone marrow and fetal liver. Journal of Experimental Medicine, 178, 951 960.
  • 22
    Lu, L., Xiao, M., Shen, R-N., Grigsby, S., Broxmeyer, H.E. (1993) Enrichment, characterization and responsiveness of single primitive CD34+++ human umbilical cord blood hematopoietic progenitors with high proliferative and replating potential. Blood, 81, 41 48.
  • 23
    Mayani, H., Little, M.T., Dragowska, W., Thornbury, G., Lansdorp, P.M. (1995) Differential effects of the hematopoietic inhibitors MIP-1α, TGFβ and TNFα on cytokine-induced proliferation of subpopulations of CD34+ cells purified from cord blood and fetal liver. Experimental Hematology, 23, 422 427.
  • 24
    Micklem, H.S., Ford, C.E., Evans, E.P., Ogden, D.A., Papworth, D.S. (1972) Competitive in vivo proliferation of foetal and adult haematopoietic cells in lethally irradiated mice. Journal of Cell Physiology, 79, 293 298.
  • 25
    Migliaccio, A.R. & Migliaccio, G. (1988) Human embryonic hemopoiesis: control mechanisms underlying progenitor differentiation in vitro. Developmental Biology, 125, 127 134.
  • 26
    Migliaccio, G., Migliaccio, A.R., Druzin, M.L., Giardina, P.J.V., Zsebo, K.M., Adamson, J.W. (1992) Long-term generation of colony-forming cells in liquid culture of CD34+ cord blood cells in the presence of recombinant human stem cell factor. Blood, 79, 2620 2627.
  • 27
    Moore, M.A.S. & Hoskins, I. (1994) Ex vivo expansion of cord blood-derived stem cells and progenitors. Blood Cells, 20, 468 481.
  • 28
    Morrison, S.J., Hemmati, H.D., Wandycz, A.M., Weissman, I.L. (1995) The purification and characterization of fetal liver hematopoietic stem cells. Proceedings of the National Academy of Sciences of the United States of America, 92, 10302 10306.
  • 29
    Petzer, A.L., Hogge, D.E., Lansdorp, P.M., Reid, D.S., Eaves, C.J. (1996a) Self-renewal of primitive human hematopoietic cells (long-term-culture-initiating cells) in vitro and their expansion in defined medium. Proceedings of the National Academy of Sciences of the United States of America, 93, 1470 1474.
  • 30
    Petzer, A.L., Zandstra, P.W., Piret, J.M., Eaves, C.J. (1996b) Differential cytokine effects on primitive (CD34+CD38) human hematopoietic cells: novel responses to flt3-ligand and thrombopoietin . Journal of Experimental Medicine, 183, 2551 2558.
  • 31
    Piacibello, W., Sanavio, F., Garetto, L., Severino, A., Bergandi, D., Ferrario, J., Fagioli, F., Berger, M., Aglietta, M. (1997) Extensive amplification and self-renewal of human primitive hematopoietic stem cells from cord blood. Blood, 89, 2644 2653.
  • 32
    Rebel, V.I., Miller, C.L., Eaves, C.J., Lansdorp, P.M. (1996a) The repopulation potential of fetal liver hematopoietic stem cells in mice exceeds that of their adult bone marrow counterparts. Blood, 87, 3500 3507.
  • 33
    Rebel, V.I., Miller, C.L., Thornbury, G.R., Dragowska, W.H., Eaves, C.J., Lansdorp, P.M. (1996b) A comparison of long-term repopulating hematopoietic stem cells in fetal liver and adult bone marrow from the mouse. Experimental Hematology, 24, 638 648.
  • 34
    Rich, I.N. & Kubanek, B. (1980) The ontogeny of erythropoiesis in the mouse detected by the erythroid colony-forming technique. Journal of Embryology and Experimental Morphology, 58, 143 155.
  • 35
    Sato, N., Sawada, K., Koizumi, K., Tarumi, T., Leko, M., Yasukouchi, T., Yamaguchi, M., Takahashi, T.A., Sekiguchi, S., Koike, T. (1993) In vitro expansion of human peripheral blood CD34+ cells. Blood, 82, 3600 3609.
  • 36
    Shapiro, F., Yao, T., Raptis, G., Reich, L., Norton, L., Moore, M.A.S. (1994) Optimization of conditions for ex vivo expansion of CD34+ cells from patients with stage IV breast cancer. Blood, 84, 3567 3574.
  • 37
    Sui, X., Tsuji, K., Tanaka, R., Tajima, S., Muraoka, K., Ebihara, Y., Ikebuchi, K., Yasukawa, K., Taga, T., Kishimoto, T., Nakahata, T. (1995) gp130 and c-kit signalings synergize for ex vivo expansion of human primitive hemopoietic progenitor cells. Proceedings of the National Academy of Sciences of the United States of America, 92, 2859 2863.
  • 38
    Sutherland, H.J., Eaves, C.J., Lansdorp, P.M., Thacker, J.D., Hogge, D.E. (1991) Differential regulation of primitive human hematopoietic cells in long-term cultures maintained on genetically engineered murine stromal cells. Blood, 78, 666 672.
  • 39
    Sutherland, H.J., Lansdorp, P.M., Henkelman, D.H., Eaves, A.C., Eaves, C.J. (1990) Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proceedings of the National Academy of Sciences of the United States of America, 87, 3584 3588.
  • 40
    Tajima, S., Tsuji, K., Ebihara, Y., Sui, X., Tanaka, R., Muraoka, K., Yoshida, M., Yamada, K., Yasukawa, K., Taga, T., Kishimoto, T., Nakahata, T. (1996) Analysis of interleukin 6 receptor and gp130 expressions and proliferative capability of human CD34+ cells. Journal of Experimental Medicine, 184, 1357 1364.
  • 41
    Terstappen, L.W.M.M., Huang, S., Safford, M., Lansdorp, P.M., Loken, M.R. (1991) Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+CD38 progenitor cells. Blood, 77, 1218 1227.
  • 42
    Traycoff, C.M., Abboud, M.R., Laver, J., Brandt, J.E., Hoffman, R., Law, P., Ishizawa, L., Srour, E.F. (1994) Evaluation of the in vitro behavior of phenotypically defined populations of umbilical cord blood hematopoietic progenitor cells. Experimental Hematology, 22, 215 222.
  • 43
    Tsujino, Y., Wada, H., Misawa, M., Kai, S., Hara, H. (1993) Effects of mast cell growth factor, interleukin-3, and interleukin-6 on human primitive hematopoietic progenitors from bone marrow and cord blood. Experimental Hematology, 21, 1379 1386.
  • 44
    Van De Ven, C., Ishizawa, L., Law, P., Cairo, M.S. (1995) IL-11 in combination with SLF and G-CSF or GM-CSF significantly increases expansion of isolated CD34+ cell population from cord blood vs. adult bone marrow. Experimental Hematology, 23, 1289 1295.
  • 45
    Wagner, J.E., Broxmeyer, H.E., Byrd, R.L., Zehnbauer, B., Schmeckpeper, B., Shah, N., Griffin, C., Emanuel, P.D., Zuckerman, K.S., Cooper, S., Carow, C., Bias, W., Santos, G.W. (1992) Transplantation of umbilical cord blood after myeloablative therapy: analysis of engraftment. Blood, 79, 1874 1881.
  • 46
    Wagner, J.E., Rosenthal, J., Sweetman, R., Shu, X.O., Davies, S.M., Ramsay, N.K.C., McGlave, P.B., Sender, L., Cairo, M.S. (1996) Successful transplantation of HLA-matched and HLA-mismatched umbilical cord blood from unrelated donors: analysis of engraftment and acute graft-versus-host disease. Blood, 88, 795 802.
  • 47
    Zandstra, P.W., Conneally, E., Petzer, A.L., Piret, J.M., Eaves, C.J. (1997a) Cytokine manipulation of primitive human hematopoietic cell self-renewal. Proceedings of the National Academy of Sciences of the United States of America, 94, 4698 4703.
  • 48
    Zandstra, P.W., Petzer, A.L., Eaves, C.J., Piret, J.M. (1997b) Cellular determinants affecting the rate of cytokine depletion in cultures of human hematopoietic cells. Biotechnology and Bioengineering, 54, 58 66.