Cell cycle distribution of cord blood-derived ... - Wiley Online Library

British Journal of Haematology, 2000, 108, 621±628

Cell cycle distribution of cord blood-derived haematopoietic progenitor cells and their recruitment into the S-phase of the cell cycle C. L U C O T T I , 1 L. M A L ABA R BA , 1 V. R O S TI , 2 G. B E RG A M A S C H I , 1 M. DAN OVA , 1 R. I N V E R N I Z Z I , 1 A. P E C C I , 1 I. R A M AJ O L I , 1 C. P E RO TTI , 3 L. T O R R E T TA , 3 M. D E A M I C I , 4 L. S A LVAN E S C H I 3 A ND M. C A Z Z O L A 1 1Department of Internal Medicine and Medical Therapy, Section of Internal Medicine and Medical Oncology, 2Research Laboratory of Organ Transplantation, Clinical Immunology Unit, 3Department of Immunohaematology and Blood Transfusion, and 4Department of Paediatrics, IRCCS Policlinico San Matteo and University of Pavia School of Medicine, Pavia, Italy Received 18 August 1999; accepted for publication 12 November 1999

Summary. The objective of this study was to evaluate the cycling status of cord blood (CB)-derived colony-forming cells (CFC) and long-term culture-initiating cells (LTC-IC), and their recruitment into the S-phase of the cell cycle. By using the cytosine arabinoside (Ara-C) suicide approach, we found that only small proportions of both CFC and LTC-IC were in the S-phase of the cell cycle. These estimates were con®rmed by ¯ow cytometric DNA analysis, which showed that 96 6 2% of CB-derived CD34 cells were in G0/G1 and only 1´6 6 0´4% in the S-phase. Staining of CD34 cells with an antistatin monoclonal antibody, a marker of the G0 phase, indicated that among CD34 cells with a ¯ow cytometric DNA content typical of the G0/G1 phase 68 6 7% of cells were in the G0 phase of the cell cycle. Incubation (24 h) with interleukin 3 (IL-3), recombinant human stem cell factor (SCF) and granulocyte colony-stimulating factor

(G-CSF) signi®cantly increased the proportion of cells in the S-phase for both CFC and LTC-IC without inducing any loss in numbers. Flow cytometric DNA analysis also showed an increase in CD34 cells in the S-phase upon continuous exposure to these cytokines. Our ®ndings indicate that: (i) very few CB-derived CFC or LTC-IC were in the S-phase of the cell cycle; (ii) a substantial amount of CD34 cells with a ¯ow cytometric DNA content typical of the G0/G1 fraction was cycling, as found in the G1 phase of the cell cycle; and (iii) 24-h incubation with IL-3, SCF and G-CSF could drive a proportion of progenitor cells into the S-phase without reducing their number. These data might be useful for gene transfer protocols and the ex vivo expansion of CB-derived progenitor cells.

The importance of umbilical cord blood (CB) as a potential source of haematopoietic progenitor cells for bone marrow transplantation has been recently highlighted (Gluckman, 1996). However, because of the low numbers of progenitor cells (Broxmeyer et al, 1992), the use of cord blood for bone marrow transplantation has been limited so far to predominantly paediatric patients (Gluckman et al, 1989; Kurtzberg et al, 1996). Over the last few years, many studies have been carried out with the aim of establishing a combination of cytokines that would allow ex vivo expansion of the more primitive haematopoietic progenitor cells (Piacibello et al, 1997;

Zandstra et al, 1998), but, despite this concerted effort, no de®nitive agreement on a common expansion protocol has so far been reached. In contrast to the large body of studies on the expansion of CB progenitor cells, less information is available on the cycling status of CB-derived haematopoietic progenitor cells of both committed (colony-forming cells, CFCs) and early (long-term culture-initiating cells, LTC-ICs) type. The de®nition of the cycling status of these progenitor cells is of interest for different reasons. For instance, retroviral gene transfer requires the target cells to be cycling: in this regard, CB-derived progenitor cells could be an appealing source of cells for gene transfer protocols (Kohn et al, 1995), provided they are in the S-phase of the cell cycle. Should these cells be in a quiescent status, they would need to be driven into the cell cycle in order to achieve good ef®ciency in gene transfer. Moreover, in recent years, the

Correspondence: Dr Mario Cazzola, Division of Haematology, IRCCS Policlinico San Matteo, 2, P.zzle Golgi, 27100 Pavia, Italy. e-mail: [email protected]. q 2000 Blackwell Science Ltd

Keywords: CD34, cell cycle, CFC, LTC-IC, statin.

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cycling status of both CFCs and LTC-ICs derived from different sources (bone marrow, peripheral blood and apheretic products obtained after mobilization protocols with growth factors and/or chemotherapy) has been de®ned (Ponchio et al, 1995, 1997; Danova & Aglietta, 1997; Lemoli et al, 1997), whereas it has not yet been de®nitely clari®ed for CB-derived haematopoietic progenitor cells. In this work, we evaluated the cycling status of both CFCs and LTC-ICs derived from umbilical CB using the cytosine arabinoside (Ara-C) suicide technique, which is based on the capacity of Ara-C to kill selectively cells that are in the Sphase of the cell cycle (Dresch et al, 1983). We found that a small number of CB-derived haematopoietic progenitor cells were in the S-phase of the cell cycle. We have also con®rmed these results by ¯ow cytometric analysis of the DNA content of CD34 cells and determined the proportion of these cells in the different phases of the cell cycle (including the G0 by means of an antistatin monoclonal antibody). Finally, we have found that the combination of interleukin 3 (IL-3), recombinant human stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) can trigger most CFCs and LTC-ICs into the S-phase of the cell cycle within 24 h of incubation. MATERIALS AND METHODS Cells and cell separation procedures. After informed consent, cord blood was obtained from normal full-term deliveries. Mononuclear light-density cord blood cells (LDCBCs) were obtained by centrifugation on a Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) gradient (1077 g/ml) for 30 min at 400 g. Interface cells were washed three times and resuspended at 1 ´ 106/ml in Iscove's modi®ed Dulbecco's medium (IMDM; Irvine Scienti®c, Santa Ana, CA, USA). The selection of CD34 cells was carried out using the MiniMacs magnetic cell-sorting device (Miltenyi Biotec, Germany), according to the manufacturer's speci®cations. Brie¯y, 1 ´ 108 LDCBCs were pelleted and resuspended in 300 ml PBS/0´5% FCS/5 mM EDTA, pH 7´2 (`the buffer'). Human IgG (100 ml) and 100 ml of a mouse IgG1 anti-CD34 antibody (clone QBEND/10) were added to the cells and gently but thoroughly mixed; the suspension was then incubated for 15 min at 48C. After washing, the cells were resuspended in 400 ml fresh buffer. Colloidal superparamagnetic MACS Microbeads (100 ml), recognizing the anti-CD34 antibody, were then added and the cell suspension was incubated for 15 min at 6±128C. After incubation, the cells were washed, resuspended in 400 ml fresh buffer and added to a MiniMacs column pre®lled with buffer and placed on the magnet. CD34 cells were then recovered by removing the column from the magnet and eluting them with 1 ml buffer (this last step was repeated twice to recover as many positive cells as possible). The collected CD34 cells were then washed once, counted and resuspended in the appropriate medium (see below). The purity of the selected CD34 cells was on average > 95% and the recovery on average < 60%. Growth factors. Highly puri®ed recombinant human interleukin 3 (IL-3) and recombinant human granulocyte± macrophage colony-stimulating factor (GM-CSF) were kindly

provided by Sandoz International (Basel, Switzerland); recombinant human granulocyte colony-stimulating factor (G-CSF) and recombinant human stem cell factor (SCF) by Amgen (Thousand Oaks, CA, USA). Recombinant human erythropoietin (Epo) was obtained from Boehringer Mannheim (Mannheim, Germany). Cytosine arabinoside (Ara-C) suicide assay. LDCBCs were incubated in IMDM with 20% fetal bovine serum (FBS; HyClone, Logan, UT, USA) or in IMDM containing 5 ´ 10 5 M b-mercaptoethanol (Sigma Chemicals, Milan, Italy), 10 mg/ml human insulin (Sigma), 200 mg/ml iron-saturated human transferrin (ICN Pharmaceuticals, Costa Mesa, CA, USA), 20 mg/ml deionized bovine serum albumin (StemCell Technologies, Vancouver, BC, Canada), with or without 100 ng/ml SCF (Amgen), 20 ng/ml IL-3 (Sandoz) and 20 ng/ ml G-CSF (Amgen). Equal volumes of cell suspension were incubated at 378C under 5% CO2 in air for 24 h, in the presence or absence of 10 6 M Ara-C. This concentration of Ara-C was chosen according to preliminary experiments carried out in our laboratory and according to published data (Preisler & Epstein, 1981; Lemoli et al, 1997), showing that a plateau of speci®c killing (with a minimal aspeci®c toxic effect) was reached at a concentration ranging between 1 and 2 ´ 10 6 M. The cells were then transferred into a tube, washed twice with fresh medium and resuspended in IMDM, and appropriate quantities from each sample were assayed for LTC-ICs and CFCs. The proportions of CFCs and LTC-ICs killed by Ara-C (i.e. the proportion of progenitor cells in the S-phase) were then calculated. Clonogenic assay. Clonogenic assays were performed as described previously (Carlo Stella et al, 1988), with minor modi®cations. Brie¯y, 2 ´ 104 LDCBCs were plated in 35-mm Petri dishes in 1-ml aliquots of IMDM containing 30% FBS (HyClone), 5 ´ 10 5 M b-mercaptoethanol, 0´9% (w/v) methylcellulose, GM-CSF, IL-3 (10 ng/ml each factor), SCF (50 ng/ ml) and 3 IU/ml erythropoietin. After 14 d incubation at 378C under 5% CO2, the number of colonies was scored using an inverted microscope. LTC-IC assay. For assay of LTC-IC in CB cell suspensions, 3 ´ 106 LDCBCs were resuspended in 2´5 ml of myeloid longterm culture (LTC) medium (StemCell Technologies) supplemented just before use with 10 6 M freshly dissolved hydrocortisone sodium hemisuccinate (Sigma) and plated in 35-mm Petri culture dishes onto a preprepared feeder layer of 3 ´ 105 irradiated (8000 cGy) M210B4 ®broblasts (Lemoine et al, 1988) (kindly provided by Dr C. J. Eaves) The cultures were maintained at 378C under 5% CO2 for 5 weeks with weekly replacement of half of the medium and nonadherent cells with fresh LTC medium. At the end of the 5 weeks, all of the non-adherent cells were removed and combined with the cells harvested from the adherent fraction by trypsinization (Sutherland & Eaves, 1994). These cells were then washed and aliquots were assayed for their CFC content in a clonogenic assay as described above. The number of LTC-IC present in the initial sample (per 106 mononuclear cells) was calculated as follows: LTC-IC frequency

A 1 1 ´R´ ´ B C 4

q 2000 Blackwell Science Ltd, British Journal of Haematology 108: 621±628

Cell Cycle of Cord Blood-derived Haematopoietic Progenitors where A is the number of CFCs derived from the plating of the harvested cells, B is the number of cells plated for secondary CFCs, C is the number of cells used to start the LTC, R is the cell recovery at the end of the LTC, and 4 represents the average CFC output per single LTC-IC. The last value was calculated in limiting dilution assays performed in our laboratory, as described previously (Sutherland et al, 1990), by determining the cell dilution that resulted in < 37% negative wells, equivalent to single-hit kinetics (one LTC-IC per well) according to the Poisson distribution (Taswell, 1981). Immunocytochemical detection of statin. Statin was detected on cytocentrifuge preparations of freshly harvested CD34 cells by an immunoalkaline-phosphatase method (streptavidin± biotin complex, LSAB2 kit; Dakopatts). Brie¯y, cells were ®xed in 70% ethanol at 208C for 20 min and rehydrated in PBS. After permeabilization of the cells with PBS/Tween/BSA solution for 10 min, slides were incubated in a moist chamber at room temperature with the antistatin monoclonal antibody S-44 (kindly provided by Dr E. Wang), diluted 1:200 for 12 h. After washing with PBS, they were incubated with a biotinylated anti-mouse rabbit Ig and then with the phosphatase alkaline/streptavidin complex. After washing, slides were stained with the following medium: naphthol-AS-BI phosphate (50 mg), dimethylformamide (0´6 ml), tris-HCl, pH 8´2, 0´05 mM (100 ml), levamisole 1 M, sodium nitrite 4% (0´5 ml) and New Fuchsin 5% (0´2 ml) for 15 min. Slides were ®nally washed and counterstained with Mayer's Hemalum for 5 min. Incubation of CD34 cells with growth factors. For the assessment of the progression through the cell cycle, 1 ´ 105/ ml CD34 cells were incubated in Iscove's medium containing 5 ´ 10 5 M b-mercaptoethanol, 10 mg/ml human insulin, 200 mg/ml iron-saturated human transferrin, 20 mg/ml deionized bovine serum albumin in the presence of 100 ng/ml SCF, 20 ng/ml IL-3 and 20 ng/ml G-CSF. After 6, 12 and 24 h, aliquots of cells were analysed for their DNA content as described below.

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Immuno¯uorescent detection of statin, propidium iodide DNA staining and ¯ow cytometry. For ¯ow cytometric analysis, CD34 cells were ®xed in 70% cold ethanol at 48C for at least 30 min. Fixed cells in suspension were rehydrated in PBS, treated for 20 min with 5% normal goat serum (NGS) in PBS and permeabilized with PBS/Tween/BSA solution for 10 min at room temperature in a moist chamber with a 1:200 dilution in PBS of the antistatin monoclonal antibody S-44 (Wang, 1985; Pellicciari et al, 1995), washed for 10 min in PBS and then incubated with a 1:50 dilution of a phycoerythrin (PE)conjugated goat anti-mouse IgG (Sigma Chemical) in PBS/ Tween/BSA solution. For DNA staining, a previously described single-step procedure on a separate sample of ethanol-®xed cells was used (Rosti et al, 1995). Flow cytometric determinations, for both statin positivity and DNA content, were made with a Becton Dickinson FACStar ¯ow cytometer, using conditions previously described (Mangiarotti et al, 1998). Statistical Methods. Results are expressed as means 6 standard deviation (SD). The Student t-test for paired data was used to test the probability of signi®cant differences between samples; all data were analysed using the statistical package STATVIEW 4´02 (BrainPower, Calabasas, CA, USA) run on a Macintosh LCII personal computer (Apple Computer, Cupertino, CA, USA). RESULTS Cell cycle status of CFCs CB-derived mononuclear cells (n 6) were incubated for 24 h in a liquid culture containing IMDM and 20% FBS with or without 10 6 M Ara-C and then plated in semisolid medium for the evaluation of the growth of the CFCs. As shown in Table I, the proportion of CFCs in the S-phase (i.e. the proportion of CFCs killed by Ara-C during the 24 h of incubation) was 17 6 8%, suggesting that the great majority of CB-derived CFCs did not enter the S-phase within the 24-h incubation. With respect to the single subtypes of CFCs, the number of CFU-GM and BFU-E grown after 24-h incubation

Table I. Number of clonogenic (CFC) and early (LTC-IC) haematopoietic progenitors in steady state (24-h incubation with FBS) and their kinetics of activation into the S-phase after exposure to IL-3, G-CSF and SCF (GF). The proportion of CFC and LTC-IC in the S-phase of the cell cycle is also shown.

BFU-E* CFU-GM* CFC* LTC-IC*

24 h FCS

24 h FCS Ara-C

S-phase cells (%)³

24 h SF GF

24 h SF GF Ara-C

S-phase cells after GF incubation (%)³

20 6 6 12 6 2 35 6 9 23 6 16

15 6 6² 11 6 2² 29 6 8² 19 6 7²

25 6 2 261 17 6 8 17 6 9

24 6 11 11 6 5 37 6 14 22 6 9

8 6 7§ 3 6 2§ 11 6 6§ 4 6 3§

64 6 8 68 6 3 70 6 8 81 6 7

* Progenitor cell numbers are expressed per 2 ´ 104 CB mononuclear cells or per 1 ´ 106 CB mononuclear cells for CFCs (n 6) (and their subtypes) and LTC-ICs (n 4) respectively. Values shown are means 6 SD. ² Compared with 24-h FCS incubation, P > 0´05 (Student t-test for paired value). ³ The percentage of CFCs and LTC-ICs in the S-phase is derived from the ratio of the number of CFC or LTC-IC grown after incubation with AraC to the number of CFCs and LTC-ICs grown in control cultures (without Ara-C), according the formula reported in the MATERIAL AND METHODS section). § Compared with 24-h SF GF incubation, P < 0´05 (Student t-test for paired value). q 2000 Blackwell Science Ltd, British Journal of Haematology 108: 621±628

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C. Lucotti et al TGFb1 or by other similar inhibitory components present in the FBS and con®rming that most of CB-derived CFCs were not in the S-phase of the cell cycle.

Fig 1. Kinetics of recruitment of CB-derived CFCs and LTC-ICs into the S-phase. Solid symbols indicate the percentage of CFCs (circles) and LTC-ICs (squares) in the S-phase of the cell cycle upon continuous liquid culture in the presence of 20% FBS. Open symbols indicate the percentage of CFCs (circles) and LTC-ICs (squares) upon continuous liquid culture in a serum-free medium in the presence of IL-3, SCF and G-CSF. Each point represents the mean 6 SD of three different experiments.

with Ara-C was not statistically different from the number of CFU-GM and BFU-E grown after 24-h incubation without Ara-C (Table I), indicating that also at the level of the single subtypes CB-derived CFCs were not in the S-phase of the cell cycle. The yield of CFU-GEMM was very low, so that statistical comparison between control cultures and Ara-C treated cultures was not possible (not shown). As FBS can contain a signi®cant amount of transforming growth factor b1 (TGFb1), which is known to inhibit the entry of haematopoietic progenitor cells into the S-phase (Cashman et al, 1990; Sitnicka et al, 1995), we also performed a 24-h Ara-C incubation in serum-free culture. The killing of CFCs by Ara-C was not statistically different between serum-free cultures and FBS-containing cultures (data not shown), indicating that no effect on the cell cycle was exerted by

Kinetics of activation of CFCs into the S-phase To study the kinetics of recruitment of CB-derived CFCs into S-phase, we performed two different types of experiments. First, we incubated CB-derived mononuclear cells under the same conditions (IMDM 20% FCS with or without Ara-C) used for the 24-h Ara-C suicide, but for a longer period of time (up to 36 h), and then we assessed the number of surviving CFCs after plating in methylcellulose. We found that 41 6 11% of CFCs were killed by Ara-C after 36 h of incubation, indicating that a substantial proportion of CFCs entered the S-phase after the ®rst 24 h, during which no signi®cant killing was observed (Fig 1). In a second set of experiments, mononuclear cells were incubated for up to 36 h in a serum-free medium containing SCF, IL-3 and G-CSF with or without Ara-C. This approach was chosen because in previous experiments (Ponchio et al, 1995) this combination of cytokines was shown to trigger most of the quiescent population of CFCs and LTC-ICs derived from the peripheral blood into the S-phase without any signi®cant loss of their number. Under these conditions, we observed that after 24 h 69% of CFCs had entered the S-phase, and that within 36 h > 90% of the CFCs were recruited into the S-phase (Table I; Fig 1). We also found that after at least 24 h of incubation with this combination of cytokines (and in the absence of Ara-C) there was no signi®cant loss of the number of CFCs compared with their input number, suggesting that, as for their peripheral blood counterpart, IL-3, SCF and G-CSF can trigger cell proliferation for CB-derived CFCs without inducing a substantial amount of differentiation (Table II). Cell cycle status of LTC-ICs and kinetics of activation into the S-phase We also examined the cycling status of CB-derived LTC-ICs (n 4) using the same strategy as that for the CFCs. The long-term cultures were incubated at 378C because in preliminary experiments we found a higher number of LTC-ICs growing at this temperature than at 338C. In four different experiments in which mononuclear cells were

Table II. Changes in the number of CFCs and LTC-ICs during 24 h of incubation with different types of media.

Progenitor cells

% of input cells after 24-h incubation with 20% FCS (without Ara-C)*

% of input cells after 24-h incubation in SF with GF (without Ara-C)*

BFU-E CFU-GM CFC (total) LTC-IC

84 6 9 81 6 16 82 6 12 91 6 12

106 6 27 72 6 12 86 6 4 92 6 18

Progenitor cells numbers are expressed as a percentage (6 SD) of the number detected at the beginning of the incubation (input cell). Increase or decrease of the cell numbers (and their percentage) were not statistically signi®cant (Student t-test P > 0´05). * Results are expressed as a mean 6 SD of six (for CFCs) and four (for LTC-ICs) different experiments. q 2000 Blackwell Science Ltd, British Journal of Haematology 108: 621±628

Cell Cycle of Cord Blood-derived Haematopoietic Progenitors incubated for 24 h in liquid cultures containing IMDM and 20% FCS with or without Ara-C, we observed 17 6 9% more killing than in control cultures (Table I); a similar result was obtained when incubation with Ara-C was performed in serum-free conditions. Thus, these data clearly indicated that CB-derived LTC-ICs were not in the S-phase, similar to their committed counterpart. The same combination of cytokines and the same time-course experiments used for CFCs were also used for studying the kinetics of progression of the LTC-ICs into the S-phase. Table I shows that after 24 h of incubation with IL-3, SCF and G-CSF > 80% of LTC-ICs were killed by Ara-C and that within 36 h > 95% of LTC-ICs had entered the S-phase of the cell cycle (Fig 1). As for CFCs, we found that this combination was able to sustain, at least for 24 h, the number of LTC-ICs at the input level (Table II), while recruiting most of the LTC-ICs in the S-phase of the cell cycle. Flow cytometric analysis of cell cycle phase distribution and statin expression of CB-derived CD34 cultured cells With DNA ¯ow cytometry, the majority of freshly harvested CB-derived CD34 cells were in the G0/G1 phase of the cell cycle, whereas only 1´6 6 0´4% were in the S-phase and 2´4 6 2´3% in the G2/M phase, thus con®rming that most of CB-derived progenitor cells were in the G0/G1 phase (Table III). To investigate further the cell cycle distribution of freshly harvested CD34 cells, these were stained with the PE-conjugated antistatin monoclonal antibody S-44 and the proportion of ¯uorescent cells was evaluated by means of a FACStar ¯ow cytometer. Expression of statin, a 57-kDa Table III. Cell cycle distribution of freshly harvested CD34 cells derived from cord blood.

% of CD34 cells

G0

G1

S

G2/M

68´4 6 7

27´6 6 2

1´6 6 0´5

2´4 6 2

Results are expressed as a means 6 SD of seven different experiments. G0 cells were calculated on the basis of the percentage of CD34 cells expressing the nuclear protein statin. Table IV. Cell cycle distribution of CB-derived cultured CD34 cells after incubation with IL-3, G-CSF and SCF.*

Freshly harvested After 6 h After 12 h After 24 h

% G0/G1

%S

% G2/M

95 6 6 77´4 6 8 82´4 6 6 81 6 5

1´8 6 0´8 11 6 3 9´1 6 2 8´5 6 3

3´2 6 1 11´6 6 2 8´5 6 3 11´5 6 4

* CD34 cells were incubated in a serum-free medium in the presence of IL-3, G-CSF (20 ng/ml each factor) and SCF (100 ng/ml) and the DNA content assessed by cyto¯uorimetric assay after 6, 12 and 24 h of incubation. Results are expressed as a means 6 SD of three different experiments.

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Fig 2. Cytocentrifuge preparation of CB-derived CD34 cells stained for statin by the immunoalkaline phosphatase reaction. Two cells show intense granular staining of the nucleus (1200´).

nuclear protein, has been recognized as a unique marker of quiescent cells (Wang, 1985): the protein is found in resting (G0) cells and is rapidly down-regulated when cells progress to the G1 phase. Thus, expression of statin can be used to identify cells that are in the G0 phase of the cell cycle; this can be assessed both by immunocytochemistry (Fig 2) and by ¯ow cytometry. Using the latter, we found that 68´4 6 7% of freshly harvested CD34 cells expressed statin (n 7), indicating that about two-thirds of CD34 cells in the G0/ G1 region of the ¯ow cytometric DNA histograms were in the G0 phase and the remaining cells were in the G1 phase of the cell cycle (Table III). We also assessed the proportion of CD34 cells that were in the different phases of the cell cycle at different time points (after 6, 12 and 24 h) during incubation in a serum-free medium in the presence of IL-3, SCF and G-CSF. As shown in Table IV, a discrete proportion of CD34 cultured cells had rapidly progressed into the S-phase after only 6 h of incubation (11%) and a similar proportion was also detectable after 12 and 24 h of incubation (9´1% and 7´5% respectively). Importantly, the proportion of CD34 cells did not change signi®cantly during the 24-h culture (97´3 6 2% and 95´5 6 3% at the beginning and after 24 h of incubation with growth factors respectively; P > 0´05), con®rming that the changes observed in the cell cycle distribution were effectively dependent on the progression through the cell cycle of CD34 cells. We also observed an increase in the absolute number of CD34 cells after the 24h incubation, but this did not reach statistical signi®cance. This last observation suggests that, although recruited in the S-phase within 24 h of culture, CD34 cells require a longer time to complete cell division. DISCUSSION In this work, we have investigated the cycling status of CBderived progenitor cells and their kinetics of recruitment into the S-phase of the cell cycle. We have studied both the committed progenitor cells (CFU-GEMM, CFU-GM and BFU-E, all together de®ned as CFCs) and the early progenitor LTC-ICs,


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which are considered to be the most immature progenitor cells that can be investigated in vitro (Sutherland et al, 1989). Whereas the CFCs are responsible for short-term engraftment, the LTC-ICs are thought to represent a more reliable approximation of the haematopoietic stem cells responsible for long-term engraftment of a transplant. In order to assess the proportion of these progenitor cells in the S-phase of the cell cycle, we used the Ara-C suicide technique, which has been shown to give reliable and reproducible results when compared with [3H]-thymidine incorporation suicide (Preisler & Epstein, 1981; Dresch et al, 1983). Our results show that after 24 h of liquid culture in the presence of FBS there was no statistical difference between the number of CFCs and LTC-ICs grown in the presence or absence of Ara-C, clearly indicating that very few committed and early CB-derived progenitor cells are in the S-phase of the cell cycle. The same results were observed when similar experiments were performed in serum-free conditions, ruling out the possibility that the presence of FBS could have affected the cycling status of the progenitor cells (Cashman et al, 1990; Sitnicka et al, 1995). Our results on the proportion of CFCs and LTCICs in the S-phase con®rm those of a recent study by Movassagh et al (1997), who used the [3H]-thymidine incorporation suicide technique and demonstrated that the Ara-C approach is similar to the thymidine suicide approach for the assessment of the proportion of haematopoietic progenitor cells in the S-phase of the cell cycle. These data were further strengthened by our ®nding that 96% of the CD34 cells derived from CB were found in the G0/G1 phase of the cell cycle and only 1´6% in the S-phase, according to the ¯ow cytometric assessment of the DNA content. Using this technique, however, it is not possible to distinguish, among the cell fraction with a DNA content typical of the G0/G1 phase, cells that are really quiescent (G0) from those that are cycling (G1). Thus, we took advantage of an antistatin monoclonal antibody in order to discriminate between resting (statin positive) and cycling (statin negative) cells. We found that about 60% of freshly harvested CB-derived CD34 cells were resting, being out of the cell cycle, whereas most of the remaining progenitor cells were actually cycling and were in the G1 phase of the cell cycle. Our observation that incubation of CB progenitor cells in the presence of FBS for 36 h was associated with the progression into the S-phase of a signi®cant proportion of both CFCs and LTC-ICs can be interpreted as that the cells entering the S-phase derive from those in the G1 phase of the cell cycle. Our data are substantially in agreement with previous reports which showed, using the evaluation of DNA content by ¯ow cytometry, that more than 90% of CD34 cells derived from CB are in the G0/G1 phase of the cell cycle (Traycoff et al, 1994; Hao et al, 1995; Roberts & Metcalf, 1995). However, because of the technical approach that was used in these studies, the whole CD34 cell population rather than the single types of progenitor cells were evaluated. Most importantly, we have been able for the ®rst time to discriminate between the proportion of these cells in the G0 and G1 phase before treatment with cytokines.

Another novel ®nding of our work emerges from the assessment of the kinetics of recruitment of CB-derived progenitor cells into the S-phase of the cell cycle. To study the kinetics of recruitment into the S-phase, we incubated the mononuclear cell fraction derived from CB with a combination of cytokines and then assessed the proportion of CFCs and LTC-ICs entering the S-phase of the cell cycle after 12, 24 and 36 h exposure to these cytokines. We decided to use the combination of G-CSF, IL-3 and SCF because it has been previously shown (Ponchio et al, 1995) that these cytokines are able to recruit a relevant proportion of PB-derived CFCs and LTC-ICs into the cell cycle. Under these experimental conditions, we observed that 70% and 81% of CFC and LTCIC, respectively, entered the S-phase of the cell cycle after 24 h. Furthermore, more than 90% of both progenitor cells were killed by Ara-C upon continuous exposure to G-CSF, IL3 and SCF (up to 36 h), suggesting that a proportion of both CFCs and LTC-ICs can be rapidly recruited into the S-phase of the cell cycle in vitro in the presence of these cytokines. Cyto¯uorimetric assessment of the DNA content con®rmed that CB-derivedCD34 cells in the presence of this combination of cytokines start rapidly to progress into the S-phase. Interestingly, our data showing that incubation of CB progenitor cells in serum-free culture and haemopoietic growth factors is associated with a more rapid induction of the cell cycle compared with incubation in the presence of FBS is in keeping with results reported by Jordan et al (1996), who found that serum-free conditions are preferable for activating quiescent cells. Taken together, our results are in apparent contrast to parallel experiments performed with bone marrow (BM)- and PB-derived progenitor cells, which showed that a longer exposure to these cytokines (up to 48±72 h) was needed to trigger such cells into the cell cycle (Ponchio et al, 1995). The difference between the kinetics of recruitment into the cell cycle of CB and adult progenitor cells can be explained assuming that the rate of exit from the G0/G1 phase in response to cytokines is faster for at least a proportion of CBderived progenitor cells than for PB- or BM-derived progenitor cells (Traycoff et al, 1994). We do not have a de®nitive explanation for this different behaviour, but we can formulate two not mutually exclusive interpretations. (i) CB-derived progenitor cells may respond differently to haematopoietic growth factors; it has been shown that the proliferative response of CB-derived CFCs and LTC-ICs to SCF is higher than their PB- or BM-derived counterparts (Roberts & Metcalf, 1995; Tanaka et al, 1995; Weeks et al, 1998). (ii) At least a proportion of the progenitor cells rapidly recruited into the S-phase is derived from those in the G1 phase of the cell cycle, which represent a substantial amount of CBderived CD34 cells, as shown by our experiments performed with statin. We also found that the absolute number of CFCs and LTCICs after 24 and 36 h of exposure to IL-3, SCF and G-CSF (in the absence of Ara-C) was not statistically different from baseline levels (at the beginning of incubation). Moreover, we found that differentiation of cultured CD34 cells, if any, was minimal. In fact, both their proportion and absolute number did not signi®cantly change after 24 h of incubation


Cell Cycle of Cord Blood-derived Haematopoietic Progenitors in the presence of growth factors. This suggests that the combination of cytokines used in our experiments not only is able to trigger the cell cycle of CB-derived progenitor cells but also does not induce a decline in their number (possibly because of their differentiation or death), at least for 24 h. This last observation could have a practical implication, e.g. when CB-derived haematopoietic progenitor cells should be used as a target for retroviral-mediated gene transfer protocols. Finally, our results can also be interpreted taking into account the results previously reported by Gothot et al (1997), who found that in steady-state bone marrow the turnover of primitive haematopoietic cells was lower than their committed counterpart and that a direct relationship existed between the rate of cycling and the degree of lineage commitment. In freshly harvested CB, we found that very few LTC-ICs are in the S-phase of the cell cycle; however, in contrast to what was observed in the bone marrow, also very few CFCs were found in the S-phase of the cell cycle. A possible explanation is that a different regulation exists for bone marrow haematopoiesis and for CB haematopoiesis, but the factors that can determine this difference are actually unknown. Also, Gothot et al (1997) found that cultured CD34 cells re-entering G0 have a shorter prereplicating phase than freshly isolated G0 CD34 cells. This observation could explain the high proportion (up to 90%; Fig 1) of LTC-ICs and CFCs entering the S-phase when incubated in the presence of cytokines for 36±48 h. In summary, our data show that very few CB-derived CFCs and LTC-ICs are in the S-phase of the cell cycle. Furthermore, results on cell cycle distribution and statin expression of CD34 cells, while con®rming the low numbers of progenitor cells in the S-phase of the cell cycle, clearly indicate that a signi®cant number of CD34 cells are in the G1 phase of the cell cycle. Our results also provide new information on the kinetics of recruitment into the S-phase of the cell cycle of CB-derived CFCs and LTC-ICs, and on culture conditions that can modify their cycling status without reducing their numbers. These ®ndings could play an important role in the engineering of CB-derived progenitor cells for gene therapy and in the designing of clinically relevant protocols for their ex vivo expansion.

ACKNOWLEDGMENTS This work was supported by Fondazione Italiana per la Ricerca sul Cancro (A.I.R.C.) and from IRCCS Policlinico San Matteo (grant 261RFM 95/01). The authors thank Dr G. Comolli, Flow Cytometry Unit at Service of Virology and Research Laboratory of Biotechnology, IRCCS Policlinico San Matteo, Pavia, Italy, for FACS analysis; Dr E. Wang, The Bloom®eld Centre for Ageing, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, for her generous gift of the antistatin monoclonal antibody; and Dr C. Dellavecchia, Department of Internal Medicine and Medical Therapy, IRCCS Policlinico San Matteo, Pavia, Italy, for helpful discussion.

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