Original Manuscript International Journal of Cell Cloning 7314-321 (1989) Rapid Decline of Clonogenic Hemopoietic Progenitors in Semisolid Cultures of Bone Marrow Samples Derived from Patients with Chronic Myeloid Leukemia U. Wnd14 H.A. Messnerb 'Universitatsklinikum Essen, Innere Klinik und Poliklinik. Essen. Federal Republic of Germany; bontario Cancer Institute, Toronto, Ontario, Canada Key Words. Clonogenic hemopoietic progenitors Cell culture CML * Recloning Abstract. Bone mam samples, from newly diagnosed patients with chronic myeloid leukemia (CML) and normal individuals, were grown in methylcellulose and serially recultured under identical conditions. Specimens derived from normal individuals gave rise to multilineage and megakaryocyte colonies for one to two sequential cultures. Erythroid bursts and granulocyte-macrophage colonies were observed for three to five sequential cultures. Cultures initiated from samples of patients with CML showed a rapid decline of all types of colonies. Colonies were rarely seen for more than two sequential cultures. When pooled colonies and background cells were recloned separately, secondary colonies were mainly seen in cultures of background cells. This observation is consistent with the view that secondary colonies are more likely to arise from dormant clonogenic progenitors, rather than through cells that have formed primary colonies through a self-renewal process. Introduction Chronic myeloid leukemia (CML) is a clonal hemopathy [l] originating in transformed pluripotent stem cells. The stem cell nature of this disease was documented by observations that myeloid and lymphoid cells are part of the malignant clone [2-4]. The malignant clone gains dominance during the evolution of the disease and suppresses the production of hemopoietic cells by residual normal progenitors. The total number of early hemopoietic progenitors [5-71 and their more differentiated progeny increase significantly. In spite of this observation, it is difficult to sustain proliferation of CML cells in long-term liquid suspension Correspondence: H.A. Messner, Ontario Cancer Institute, 5 Sherbourne Street, Toronto, Ontario M4X lk9, Canada. Received April 27, 1989; provisionally accepted May 26, 1989; accepted for publication June 15, 1989. 737-1454189/$2.1 oalphamed Press
Rapid Decline of Hemopoietic Precursors in CML 315 culture [8]. On the contrary, it appears to be feasible to ablate the malignant clone in long-term culture and to reestablish polyclonal hemopoiesis from residual normal hemopoietic progenitors [9]. Using short-term colony assays, we examined whether or not the proliferation of progenitors in samples from patients with CML could be sustained in replating experiments. In agreement with the liquid suspension culture system, we observed a rapid reduction of clonogenic progenitors in semisolid cultures when compared to normal bone marrow samples. Materials and Methods Patients Bone marrow samples were procured from consenting patients with newly diagnosed CML in chronic phase and normal bone marrow transplant donors. The bone marrow samples were aspirated into heparinized syringes. Primary Cell Cultures Mononuclear cells of density less than 1.77 glml were obtained by centrifugation in Pexcol. The cells were cultured in Iscove's modified Dulbecco's medium (IMDM; GIBCO, Grand Island, NY) supplemented with 3% human plasma, 5% medium conditioned by phytohemagglutinin-stimulated leukocytes (PHA-LCM; PHA, HAS; Wellcome Diagnostics, Starkford, England), 5 X M 2-mercaptoethanol (2ME; Sigma Chemical Co., St. Louis, MO) and.9% methylcellulose (Dow Chemicals, Midland, MI) [lo, 111. Samples from normal individuals were plated in 35 mm Petri dishes (Lux, Miles Scientific, Naperville, IL) at a final cell concentration of 2 X lo5 mononuclear cells per ml. Specimens from patients with CML were grown at 5 X 14/ml. The plasma was obtained from a patient with severe aplastic anemia and contained the equivalent of 2-3 U/ml of erythropoietin (Epo). Culture plates were incubated at 37 C in humidified air supplemented with 5% cop The plates were examined with an inverted microscope of high optical resolution to determine the frequency of multilineage colonies (CFU-gemm), megakaryoqte colonies (CFU-meg), erythroid bursts (BFU-e) and granulocyte-macrophage colonies (CFU-gm) using described criteria [El. Serial Replating Experiments Primary cultures were usually initiated in 6-8 35 mm Petri dishes. "ha of the dishes were maintained for 14 days and scored as described above. The remaining dishes were replated on day 1 of culture. The content of each dish was harvested by adding 2 ml of IMDM to the culture mixture. The plates were gently agitated and the contents removed by pipette. The material of all available culture plates within each experimental group was pooled. Cells were pelleted by centrifugation at 2, rpm for 1 min, resuspended in MDM, counted and replated under identical culture conditions. The final volume of the culture mixture of each group WBS equivalent to the total volume of all pooled dishes used in each experiment. Aliquots of 1 ml were plated as described above. The replating experiments were repeated every 1 days until no further cells could be recovered. In some experiments, the cell number rather than the plated volume was kept constant for each subsequent generation. Both experimental designs yielded similar data.
WandlIMessner 3 16 lsble I. Number of clonogenic progenitors in serial replating studies of bone marrow samples derived from normal individuals and patients with CML Number of No. of colonies' Source of generations cells in culture CFU-gemm CFU-meg BFU-e CFU-gm Total Normal PE1 Controls PE2 (n = 1) PE3 PE4 PE5 CML PEl (n = U) PE2 PE3 PE4 PE5 3 f 2 I f 1 6 f 13 4f3 4f31 33f16 8f52 If1 21f22 3f18 53f37 2f2 9f8 11f9 2f6 3f3 3 f 3 l f 2 l f 2 9 f 17 181 f 218 199 f 254 395 f 529 2 f 4 5 f 85 52 f 86 'Per 5 X lo4 MNC (mean f SE) Replating of Colonies and Background Cells In some experiments, all colonies were removed individually by micropipette, pooled and replated. The residual culture medium included individual cells and small cell clusters. It was collected as described above and plated in separate dishes. Statistical Analysis The frequency of multi- and single-lineage hemopoietic colonies for each sequential culture was recorded and expressed as plating efficiency (PE). The frequency of colonies observed in primary cultures (PE1) was considered to be 1%. The Kruscal-Wallis test was employed to compare the PE observed in each of the sequential cultures [13]. Results Colony Frequency Afer Replating Bone marrow samples of 1 normal individuals and 15 patients with CML were used to initiate short-term cultures in methylcellulose. Primary cultures of all patients were subjected to serial replating studies. The frequency of multi- and single-lineage colonies was recorded for each culture. The pooled data of all patients entered into the study are summarized in nble I. Cultures initiated from normal samples in general show a lower number of colonies per 5 X lo4 colonies compared to CML. A decline of all colony types was observed during the serial replating process. CFU-gem and CFU-meg were usually only seen for 1-2 sequential cultures. BFU-e and CFU-gm were observed for as many as 3-5 consecutive cultures. In contrast, cultures of samples obtained from patients with CML usually yielded a higher than normal frequency of BFU-e and CFU-gm in primary cul-
Rapid Decline of Hemopoietic Precursors in CML 317 Fig. 1. Serial recloning studies demonstrate differences in the rate of decline of clonogenic hemopoietic progenitors in bone marrow samples of normal individuals and patients with CML. tures. This was followed by a rapid decline. Colony growth was rarely demonstrated after the second culture. The difference in the rate of decline is displayed in Figure 1 for the total number of all colonies scored in the respective culture dishes. The proportion of surviving colony-forming cells in the second and third generation is significantly lower for patients with CML (p <.1 and < O.OOO1, Kruscal-Wallis). We questioned whether or not the decrease of colony growth in the recloning experiments was a technical problem due to decreasing cell numbers. This was examined by linearity studies performed for the second culture of bone marrow samples from normal individuals. A total of 2 X lo5 mononuclear cells was plated either in 1,2,4, 8 or 16 dishes. The number of colonies was counted for each dish as well as for the total number of dishes plated for each cell concentration. The data of a representative experiment are shown in Figure 2. A linear relationship was identified between the number of plated cells and frequency of colonies observed. In addition, the total number of colonies grown from 2 X los cells was similar and independent of the cell concentration per dish. Replating of Colonies and Culture Medium Containing Single Cells and Clusters Experiments were performed to examine whether or not clonogenic cells observed in serial cultures were derived from primary clonogenic cells or whether they may have been present in a dormant status during the primary culture. All
WandUMessner 318 Fig. 2. Linearity study of normal clonogenic progenitors during the second generation of culture. The numbers listed in brackets indicate the total number of colonies observed for 2 X los cells. The total number of colonies was a function of the total number of cells plated and independent of the cell density per plate. readily identifiable colonies were removed by micropipette, pooled and plated. The remaining culture medium that contained single cells, as well as small clusters, was harvested and replated under identical culture conditions. All cells collected by the 2 different procedures were plated. The results of experiments performed on samples of 3 normal individuals and 2 CML patients are presented in 'hble 11. Cells derived from the background yielded a significantly larger number of colonies than those obtained from primary colonies. Delayed Addition of PHA-LCM to Cultures In a subset of experiments, PHA-LCM was added with a delay of 2-1 days after initiation of the cultures. These studies were performed on normal bone marrow cells to determine whether or not clonogenic cells would be able to survive for prolonged periods of time without proliferation in semisolid cultures. The data of these experiments are shown in 'hble III. PHA-LCM can be withheld from these cultures for 8-1 days without substantially altering the frequency of CFUmeg, BFU-e and CFU-gm. The frequency of CFU-gemm colonies, however, was more susceptible to the delayed addition of PHA-LCM.
~ ~~~ Rapid Decline of Hemopoietic Precursors in CML 3 19 nble 11. Replating of pooled colonies and pooled background cells' Source of Source of No. of colonies bone marrow cells for cells replating CFU-gemm CFU-rneg BFU-e CFU-gm Normal Exp. #1 Pooled colonies 28 controls background 4 28 16 98 Exp. #2 Pooled colonies 47 background 2 5 85 14 Exp. #3 Pooled colonies 3 background 1 I5 8 CML Exp. #l Pooled colonies background 75 Exp. #2 Pooled colonies 19 background 17 8 'All cells harvested by both methods from individual plates were replated separately under otherwise identical culture conditions. nble III. Colony formation after delayed addition of PHA-LCM PHA-LCM added No. of coloniesa on day of culture CFU-gemm CFU-meg BFU-e CFU-gm Experiment 1 2 2 23 27 15 4 9 4 38 62 6 2 41 11 143 8 3 76 21 Experiment 2 4 2 5 126 49 6 6 11 129 85 1 3 11 89 24 'Per 2 x 15 MNC Discussion Short-term colony assays and long-term liquid suspension cultures have contributed to the understanding of differentiation events in normal and abnormal hemopoiesis. Both types of culture appear to favor differentiation. It remains difficult to achieve sustained self-renewal of hemopoietic progenitors. Our replating studies were designed to answer the question of whether or not recognizable clonogenic progenitors in patients with CML are able to increase their frequency by a self-renewal process. Our data indicate that the frequency of pluripotent as well as committed progenitors decreases rapidly during the first
WandllMessner 32 generation in culture and that essentially no colony-forming cells survive a second transfer. The total length of time that CML-derived clonogenic progenitors can be maintained in short-term semisolid culture systems is significantly shorter when compared to normal samples. The data are consistent with similar observations made for CFU-gm [14]. The reason for the difference between normal and CMLderived precursors in both culture systems is not readily obvious. Clonogenic cells derived from patients with CML may require different culture conditions for prolonged survival compared to normal cells. Differences in the requirements for various hemopoietic growth factors, such as Epo, are well recognized [l5, 161. Alternatively, the number of early progenitors representing a bone marrow reserve may be reduced or altered in their physiological state. The latter view is supported by differences in the cell cycle status of clonogenic progenitors in CML. Precursors in patients with CML appear to be in cell cycle [16], while in normal individuals a proportion of clonogenic precursors assumes a resting phase. Recruitment of these resting cells into cell division may require time, and thus may occur in subsequent culture generations. Future studies on bone marrow samples derived from patients with polycythemia rubra Vera (PRV) may provide further insight into the generality of this proposed mechanism since clonogenic cells in patients with PRV also appear to be in cell cycle [17]. The emergence of new colonies late after initiating a cell culture has been well established for normal blast colonies with self-renewal capability [18]. Our experiments in which pooled colonies and cells obtained by harvesting the remaining culture medium were recultured separately. The majority of colonies observed beyond the first generation may indeed be derived from previously dormant clonogenic progenitors and not from cells generated by a self-renewal process in primary colonies. These studies suggest strongly that clonogenic progenitors tested in this system do not appear to be autonomous and do not seem to have extensive self-renewal capability. They are likely downstream from the more primitive progenitors that are capable of sustaining the CML clone. Acknowledgments This work was supported by grants from the Medical Research Council of Canada and the National Cancer Institute. The authors would like to thank Anita Abele for her carehi preparation of this manuscript. References 1 Fialkow PJ, Jacobson RJ, F'apayannopoulou T. Chronic myelocytic leukemia: clonal origin in a stem cell common to the granulocyte, erythrocyte, platelet and monocytelrnacrophage. Am J Med 1977;63:125-13. 2 Fialkaw PJ, Denman AM, Jacobson RJ, Lmenthal MN. Chronic myelocytic leukemia. Origin of some lymphocytes from leukemic stern cells. J Clin Invest 1978;62:815-823.
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