Original Papers. Characteristics of Proliferation and Differentiation of Spleen Colony-Forming Cells from Bone Marrow.

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1 Original Papers International Journal of Cell Cloning 2: (1984) Characteristics of Proliferation and Differentiation of Spleen Colony-Forming Cells from Bone Marrow Wu Chu-tse, Liu Min-pei Department of Experimental Hematology, Institute of Radiation Medicine, Beijing, China Key Words. CFC-S. CFU-S. Lymphoid stem cell. Myeloid stem cell Lymphoid-myeloid stem cell. Hemopoietic stem cell Abstract. By using c-band staining or sex chromosome identification techniques, we have demonstrated that some of the spleen colony-forming cells from normal bone marrow have the potential to form both myeloid and lymphoid elements. Introduction The experimental study of hemopoietic stem cell research has been accelerated by Till and McCulloch [I], who developed the technique of spleen colony formation from hemopoietic stem cells. By using radiationinduced aberrant chromosomes as a cell marker, Becker et al. [2] demonstrated that each spleen colony is derived from the proliferation and differentiation of a single cell, which is defined as spleen colony-forming cell (CFC-S) or a spleen colony-forming unit (CFU-S). By using the chromosome marker technique Barnes et al. [3], Wu et al. [4], and Nowell et al. [S] consistently showed the existence of a cell population in hemopoietic tissue which is multipotential in that it may differentiate either to the lymphoid system or to the myeloid blood cell lines. In 1977, Abramson et al. [6] further suggested the existence of three types of stem cells in hemopoietic cell populations; i.e., lymphoid-myeloid stem cell, which is similar to the stem cell defined by Wu et al. [4]; restricted myeloid stem cell; and restricted lymphoid stem cell (particularly the T-lymphocyte stem cell) AlphaMed Press, Inc /83 /$2.00/0

2 Chu-tse/Min-pei 70 In examining the model as described above, the following problems require serious consideration: (a) Chromosome aberration induced by irradiation may or may not interfere with normal activity of the cell, especially with respect to proliferation and differentiation; (b) some authors believe that spleen colony formation in vivo and mixed colonies formed in vitro are derived from myeloid stem cells and therefore regard myeloid stem cells as identical with CFC-S [7-lo]; and (c) whether or not murine lymphoid-myeloid stem cells are capable of producing spleen colonies in lethally irradiated recipient mice [8]. All these problems are not only very important but remain unsolved. In the present communication, we use the sex chromosome c-band staining technique to further explore the characteristics of proliferation and differentiation of spleen colony-forming cells from murine bone marrow. Materials and Methods Animals All animals used were inbred LACA mice (MRC Laboratory Centre, England). At the beginning of the experiments, the mice were 70 days old. Irradiation Condition Gamma rays from a T o source were used with the dose rate of 0.65 to 0.70 Gy per min. Preparation of Cell Suspension Bone marrow from femurs, tibias, and sternum were flushed into 6 ml of RPMI-1640 culture medium. A single cell suspension of bone marrow was prepared by passing the cells through a syringe with a 4-gauge needle. Colchicine (0.125 pg, Fluka AG, Chemische Fabrik, Switzerland) was.added to 4.0 ml of bone marrow cell suspension. After 4 h of incubation at 37"C, the cells were collected for oband staining and chromosome karyotype analysis. Lymphoid cells from the spleen or lymph nodes of mesenteric, axillaries and cervical regions were each pressed through a single nylon net to separately prepare cell suspensions in 5 ml of RPMI-1640 culture medium. After centrifugation, the sedimented cells were resuspended in 2.5 ml of culture medium. Transformation of T-Lymphocytes Each culture consisting of 3.0 ml culture medium containing 10% newborn calf serum, 0.4 ml concanavalin A (Con A; 40 Fg in 1 ml saline, Sigma Chemical Company, St. Louis, MO) and 0.6 ml of spleen or lymph node cell suspension were added. In a parallel control culture system, 3.4 ml of culture medium with 10% newborn calf serum was added to 0.6 ml of spleen or lymph node cell sus-

3 Characteristics of CFC-S from Bone Marrow 71 pension. After gassing the culture bottles with air containing 5% COz for about 10 seconds, the bottles were closed tightly and incubated at 37 C for 62 h; then pg of colchicine was added to each bottle, and the bottles were incubated an additional 10 h at 37OC. Finally, the cells were collected and smeared on slides for c-band staining and chromosome analysis. Transformation of B-Lymphocytes In a small glass bottle, 2.4 ml of culture medium (17.8 ml RPMI-1640 medium, 2.0 ml newborn calf serum, 0.2 m12-mercaptoethanol (2-ME) in saline solution at a concentration of 5 x lo-sm), 1.0 ml E. coli endotoxin solution (200 pg refined endotoxin in 1.0 ml culture medium, Beijing Institute of Biological Preparations, Beijing, China) and 0.6 ml of spleen or lymph node cell suspensions were added. In the control culture system, only 3.4 ml of culture medium containing 10% newborn calf serum and 0.6 ml of the spleen or lymph node cell suspension were mixed. The culture condition and preparation of c-band staining were the same as described previously. Chromosome Karyotype Analysis of Single Spleen Colonies Thirteen days after intraveneous injection of murine bone marrow cells into a group of 8.0 Gy irradiated mice, colchicine was given intraperitoneally in a dose of pg per gram of body weight, and the mice were sacrificed 3 to 4 h later. Single spleen colonies were excised and suspended in 2 ml of saline solution to produce a single cell preparation for c-band staining and chromosome karyotype analysis. Chromosome Analysis by C-Band Staining Technique The method of c-band staining was similar to that described previously [ 111. After stopping the mitotic cells in metaphase by colchicine, the collected lymphocytes, bone marrow cells, or cells from a single spleen colony were spun down and dispersed into 2 ml of M of KCl solution at 37 C for 60 min. After hypotonic treatment, the cells were spun down again and resuspended in 5 ml of a fixing solution of methanol and acetic acid in a 3:l ratio. Cells were smeared on clean slides and immersed into 0.2 N of HCI solution for min and transferred into 5% Ba(0H)z solution for another min. After washing and drying, the slides were treated in 2 X SSC solution at 60 C for min and finally stained by Giemsa solution. Thirty well dispersed mitotic cells in metaphase were chosen arbitrarily and subjected to karyotype analysis. Results Clonogenic Nature Of Spleen Colony Formation After chromosome c-band staining, all of the 40 chromosomes from female mice showed clear-cut centromeres. However, in the Y chromosome of male mice the centromere was not marked (Fig. 1).

4 Chu-tse/Min-pei 72 Fig. 1. Chromosome karyotype after c-band staining. (A) 40 chromosomes from female mice with clear cut centromeres. (B) Y chromosome from male mice with unmarked centromere. Arrow indicates Y chromosome. A mixed bone marrow cell suspension with equal numbers of nucleated bone marrow cells (0.75 X 104) from either sex of mice was injected into a group of 8.0 Gy-irradiated recipient mice. Thirteen days later, their chromosome karyotypes were analyzed from the randomly excised spleen colonies. Table I shows that in 33 colonies examined, 20 were composed of cells with XX chromosomes, and 13 were XY type, Not one of them was

5 Characteristics of CFC-S from Bone Marrow 73 Table I. Karyotype analysis of cells in a single spleen colony Experiment Bone marrow cells Number of Karyotype analysis of No. injected colonies colonies tested XY xx XX+XY x 103 (MI x 103(~) 7.7 x 103(~) 5, x 103(~) 7.4 x 103(~) x 103(~) ~ M = Male; F =Female composed of cells from both sexes. The above experimental results provided unequivocable evidence that each spleen colony originated from the proliferation and differentiation of a single stem cell. The Characteristics of CFC-S in Proliferation and Diferentiation The total number of CFC-S in the hemopoietic tissue is very low. It is therefore impossible to separate a pure population of CFC-S from the bulk of hemopoietic cells. As described above, each spleen colony is derived from a single CFC-S which possesses the properties of self-replication and differentiation. Thus, from the formation of a spleen colony it may provide an effective method to increase the number of this cell population, thereby facilitating their study. With this idea in mind, we designed an experiment to study the proliferation and differentiation of a single CFU-S (Fig. 2). Normal bone marrow cells (1.5 x 104) were injected intravenously into a group of 8.0 Gy-irradiated first recipient mice. Thirteen days later, a single spleen colony was randomly excised, dispersed into 0.8 ml culture medium, and injected into an 8.0 Gy-irradiated second recipient mouse. After an interval of 7-8 weeks, the second recipient was sacrificed and the chromosome karyotypes of the cells in the lymph nodes and spleen as well as the bone marrow cells were analyzed. For a step further in the exploration of the

6 Bone Marrow Cells x 1O'(F) 8.0 G y 8.0 Gy 8.0 Gy (M) First recipient - 0Weeks T B a- BM (M) CFU-S Second recipient Third Recipient Fig. 2. Experimental design for observation of the proliferation and differentiation of a single CFC-S (M =male).

7 Characteristics of CFC-S from Bone Marrow 75 potential of CFC-S in proliferation and differentiation, bone marrow cells (5 x 106) from the second recipient were injected subsequently into a third 8.0 Gy-irradiated recipient. Eight weeks later, the chromosome karyotypes were analyzed again as described above. Since we always used animals of the opposite sex as donor and recipient, it is possible to trace the behavior of the hemopoietic stem cells from generation to generation regarding their potential of self-replication and differentiation to both the myeloid and lymphoid series. Table I1 shows that CFC-S not only were capable of re-establishing the myeloid but also the lymphoid system in the lethally-irradiated second recipient. Radiation chimerism was observed in lymphoid cells stimulated by either Con A or 2-ME plus endotoxin from cells derived from mice of the opposite sex. The potential of re-establishment of the myeloid and lymphoid systems was also evident in the third recipient after the transplantation of 5 X 106 bone marrow cells from the second recipient. On day 13 after transfusion of 1.5 x 104 bone marrow cells in the first recipient, a piece of spleen tissue from a site other than the spleen colony was similarly treated and injected into an 8.0 Gy-irradiated mouse. Nine weeks later, no reconstitution of either myeloid or lymphoid systems was observed (Table 111). These results showed that the potential of self-replication and differentiation into myeloid and lymphoid systems is restricted to certain cells contained within the spleen colony, and indeed the spleen colony-forming cell itself is responsible for the re-establishment of hematological and immunological functions in lethally-irradiated animals. From another experiment, we have seen that cell suspensions from 15 single colonies derived from lethally-irradiated recipient mouse spleens on day 13 after injection of 1.5 X 104 normal bone marrow cells were incubated at 37 C for 62 h with Con A or 2-ME plus endotoxin; no mitotic cells were observed whatsoever. It is evident that after 13 days of in vivo growth, no cells sensitive to specific mitogens as T- and B-lymphocytes were produced from CFC-S (Table IV). The above experimental results also demonstrated that although spleen colony-forming cells possess the potential of re-establishment of myeloid and lymphoid functions in lethally-irradiated mice, the environment within the spleen colony is not suitable for the differentiation and maturation of lymphoctyes. This is consistent with the histological observation, i.e., no lymphocytes are found in spleen colonies. We have assayed five CFC-S from normal bone marrow of different individuals and found that most of them have the potential for re-estab-

8 Table II. The potential of a single CFC-S derived from the bone marrow of a female mouse in re-establishment of lymphoid and myeloid cells of a 8.0 Gy-irradiated male recipient Exp. Donor cells in second recipient Donor cells in third recipient No. % CFU-S donor % CFU-S donor karyotype/ total karyo type /total ST LT SB LB BM ST LT SB LB BM Spleen colony / / / / 12 ST = T-lymphocyte in spleen; LT = T-lymphocyte in lymph nodes; SB = B-lymphocyte in spleen; LB = B-lymphocyte in lymph nodes; BM = bone marrow; CFU-S =spleen colony B 4 t 0 i? F.?.

9 Table III. The comparison of an individual spleen colony derived from normal bone marrow and a piece of similar spleen tissue other than colony in re-establishment of myeloid and lymphoid systems of 8.0 Gy-irradiated recipient $ Experiment Sex of Sex of Interval after Donor cells in second recipient (n No. donor recipient spleen colony s transfusion % CFU-Sdonor (weeks) karyotype/total 0 ST LT SB LB BM Ln? Spleen colony B 3-5 M F /5!? 1-2 M F /9 s 13-1 F M /10 z K 13-2 F M / F M /12 8 B 2. e. n a Control 9-1 M F o/ M F /6 ST = T-lymphocyte in spleen; LT = T-lymphocyte in lymph nodes; SB = B-lymphocyte in spleen; LB = B-lymphocyte in lymph nodes; BM = bone marrow; CFU-S = spleen colony

10 Chu-tse / Min-pei 78 Table IV. Comparison of mitotic indexes of normal spleen cells and cells from 13- day spleen colonies after 8.0 Gy irradiation and transfusion of 1.5 X lo4 normal bone marrow cells under the stimulation of specific mitogens Cells Mitogen used Mitotic index % Spleen cells - 0:2 Con- A ME + Endotoxin 2.6 Spleen colony cells Con-A 0 2-ME + Endotoxin 0.1 lishment of both myeloid and lymphoid functions. But in one case, the potential for reconstitution was obviously weak as compared with the other four cases (Table 111). Discussion Extensive studies of the properties and functions of hemopoietic stem cells have shown that cells in this pool are heterogeneous in nature [ 11-14]. This is important not only for the sake of studies of normal hemopoiesis, but also significant in the exploration of ideology of some hematological disorders. Spleen colony-forming cells are the earliest recognized hemopoietic stem cell populations. The fact that various myeloid components exist in spleen colonies can be identified directly by histological techniques. Whether or not the CFC-S could differentiate into lymphoid cells is a problem which remains to be clarified [ 15-18]. For the clarification of the underlying nature of CFC-S, some points of importance should be carefully observed: (a) It has become routine to use the lethally-irradiated mice to test the inherent potential of transplanted cells, but the possibility of disturbance caused by residual endog-

11 Characteristics of CFC-S from Bone Marrow 79 eneous hemopoiesis seems to be unavoidable. Some other compensatory measure such as the cytogenetic method must be undertaken to make it more reliable; (b) the marked cell induced by any means should not influence the innate nature of cellular proliferation and differentiation; (c) lymphoid cell lines at least involve T- and B-subpopulations. The reconstitution of immunological function in the irradiated recipient animals by transplanted CFC-S must include both T- and B-lymphocyte subpopulations. We treated this problem satisfactorily by using specific mitogens to stimulate appropriate target cells to produce respective mitotic indexes. In order to obtain a reliable and consistent conclusion regarding the nature of CFC-S, the above points must be taken into consideration. Our results clearly indicate that some of the CFC-S reconstitute hemopoietic and immunological deficiency; the latter include the recovery of both T- and B-lymphocytes. It is very interesting to note that among all the spleen colony-forming cells tested, not a single one has the ability to re-establish only the myeloid system. It is reasonable to conclude, therefore, that at least some of the colony-forming cells of bone marrow origin have the essential characteristics of pluripotent hemopoietic stem cells. Hence, it is misleading to look on the CFC-S as restricted to myeloid stem cells. Acknowledgment This work was supported in part by a grant from the Scientific Foundation Of Chinese Academy Of Sciences. Authors thank Prof. Chu Jen-pao for his encouragement and valuable comments in the preparation of this manuscript. The skillful technical assistance of Xue Hui-hua and Zhou Shuzhen is appreciated. References Till, J.E.; McCulloch, E.A.: A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res 14: (1961). Becker, A.J.; McCulloch, E.A.; Till, J.E.: Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature 197: 452 (1963). Barnes, D.W.H.; Ford, C.E.; Gray, S.M.; Loutit, J.F.: Spontaneous and induced changes in cell population in heavily irradiated mice. Prog Nuclear Energy 2: 1-10 (1959). Wu, A.M.; Till, J.E.; Siminovitch, L.; McCulloch, E.A.: Cytological evidence for a relationship between normal hematopoietic colony-forming cells and cells of the lymphoid system. J Exp Med 127: (1968).

12 Chu-tse/Min-pei 80 Nowell, P.C.; Hirsch, B.E.; Fox, D.H.; Wilson, D.B.: Evidence for the existence of multipotential lympho-hematopoietic stem cells in adult rat. J Cell Physiol 75: (1970). Abramson, S.; Miller, R.G.; Phillips, R.A.: The identification in adult bone marrow of pluripotent and restricted stem cells of the myeloid and lymphoid systems. J Exp Med 145: (1977). Kincade, P.W.; Jyonouchi, H.; Landreth, K.S.; Lee, G.: B-lymphocyte precursors in immunodeficient, autoimmune and anemic mice. Immuno Rev 64: (1982). Till, J.E.; McCulloch, E.A.: Hemopoietic cell differentiation. Biochem Biophys Acta 605: (1980). Quesenbeny, P.; Levitt, L.: Hematopoietic stem cells. N Engl J Med 301: (1979). Phillips, R.A.; Heterogeneity: Pluripotential and committed stem cells of the myeloid and lymphoid system; in Clarkson, Marks, Till, Differentiation of Normal and Neoplastic Hematopoietic Cells, pp (Cold Spring Harbor Laboratory, N.Y. 1978). Wu, Z.Z.; Shen, S.S.; Zhu, A.W.; Xue, H.H.; Yan C.S.; Zhu, R.B.: Studies on the property and transplantation of haemopoietic stem cells from peripheral blood. Sci Sin 24: (1981). Schofield, R.: The relationship between the spleen colony-forming cell and the haemopoietic stem cell: a hypothesis. Blood Cells 4: 7-25 (1978). Lajtha, L.G.: Stem cell concepts. Differentiation 14: (1979). Rosendaal, M.; Hodgson, G.S.; Bradley, T.R.: Organization of haemopoietic stem cells: the generation-age hypothesis. Cell Tissue Kinet 12: (1979). Lala, P.K.; Johnson, G,R.: Monoclonal origin of B-lymphocyte colony-formilrg cells in spleen colonies formed by multipotential hemopoietic stem cells. J Exp Med 148: (1978). Paige, C.J.; Kincade, P.W.; Moore, M.A.S.; Lee, G.: The fate of fetal and adult B-cell progenitors grafted into immunodeficient CBA/N mice. J Exp Med 150: (1979). Trentin, J.; Wolf, N.; Cheng, V.; Fahlberg, W.; Weiss, D.; Bonhag, R.: Antibody production by mice repopulated with limited numbers of clones of lymphoid cell precursors. J Immunol98: (1973). Yung, L.; Cheryl Why-Evans, T.; Diener, E.: Ontogeny of the murine immune system: development of antigen recognition and immune responsiveness. Eur J Immunol3: (1973). Received: April 8, 1983; accepted: August 25, 1983 Dr. Wu Chu-tse, Department of Experimental Hematology, Institute of Radiation Medicine, P.O. Box 130, Beijing (China)