Circulating pluripotent hematopoietic progenitor cells in neonates

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Bancalari et al.

REFERENCES 1. Frantz ID III, Wethammer J, Stark AR. High-frequency ventilation in premature infants with lung disease: adequate gas exchange at low tracheal pressure. Pediatrics 1983; 71:483. 2. Carlo WA, Chatburn RL, Martin R J, et al. Decrease in airway pressure during high-frequency jet ventilation in infants with respiratory distress syndrome. J PEDIATR 1984;104:101. 3. Trindade O, Goldberg RN, Bancalari E, Dickstein P, Ellison J, Gerhardt T. Conventional vs high frequency jet ventilation in a piglet model of meconium aspiration: comparison of pulmonary and hemodynamic effects. J PEOIATR 1985; 107:115. 4. Weisberger SA, Carlo WA, Fouke JM, Chatburn RL, Tillander T, Martin RJ. Measurement of tidal volume during high-frequency jet ventilation. Pediatr Res 1986;20:45. 5. Gillespie DJ. High frequency ventilation: a new concept in mechanical ventilation. Mayo Clin Proc 1983;58:187. 6. Rouby J J, Simonneau G, Benhamou D, et al. Factors influencing pulmonary volumes and CO2 elimination during highfrequency jet ventilation. Anesthesiology 1985;63:473. 7. Beamer WC, Prough DS, Royster RL, Johnston WE, Johnson JC. High-frequency jet ventilation produces auto-PEEP. Crit Care Med 1984;12:734. : 8. Frantz ID III, Close RH. Elevated lung volume and alveolar pressure during jet ventilation of rabbits. Am Rev Respir Dis 1985;131:134. 9. Banner M J, Gallagher T J, Banner TC. Frequency and percent inspiratory time for high-frequency jet ventilation. Crit Care Med 1985;13:395.

The Journal of Pediatrics April 1987

10: Saari AF, Rossing TH, Solway J, Drazen JM. Lung inflation during high-frequency ventilation. Am Rev Respir Dis 1984;129:333. 11. Crosfill ML, Widdicombe JG. Physical characteristics of the chest and lungs and the work of breathing in different mammalian species. J Physiol (Lond) 1961;158:1. 12. Geubelle F, Senterre J. Methods of investigation of the mechanics of breathing in the artificially ventilated newborn. Biol Neonate 1970;16:35. 13. Butler W J, Bohn DJ, Bryan AC, Froese AB. Ventilation by high-frequency oscillation in humans. Anesth Analg 1980; 59:577. 14. Bohn D J, Miyasaka A, Marchak BE, Thompson WK, Froese AB, Bryan AC. Ventilation by high-frequency oscillation. J Appl Physiol 1980;48:710. 15. Benhamou D, Ecoffey C, Rouby J J, Fuseiardi J, Viars P. Impact of changes in operating pressure during high-frequency jet ventilation. Anesth Analg 1984;63:19. 16. Tran N, Lowe C, Sivieri EM, Shaffer TH. Sequential effects of acute meconium obstruction on pulmonary function. Pediatr Res 1980;14:34. 17. Tyler DC, Murphy J, Cheney FW. Mechanical and chemical damage to lung tissue caused by meconium aspiration. Pediatrics 1978;62:454. 18. Yeh T-F, Lilien LD, Barathi A, Pildes RS. Lung volume, dynamic lung compliance, and blood gases during the first 3 days of postnatal life in infants with meconium aspiration syndrome. Crit Care Med 1982;10:588.

Clinical and laboratory observations Circulating pluripotent hematopoietic progenitor cells in neonates Robert D. Christensen, M,D. From the Division of Human Development and Aging, Departments of Pediatrics and Internal Medicine, University of Utah School of Medicine, Salt Lake City

Supported by Grant RO-1-HD-220830-01 from the National Institutes of Health. Submitted for publication Aug. 11, 1986; accepted Oct. 27, 1986. Reprint requests: Robert D. Christensen, M.D., Division of Human Development and Aging, University of Utah School of Medicine, 50 N. Medical Drive, Salt Lake City, UT 84132.

CFU-GEMM CFU-GM 3HTdr MEM

Colony- forming-unit-granulocyte, erythrocyte, monocyte, megakaryocyte Colony-forming-unit-granuloeyte, monocyte Tritiated thymidine Minimum essential medium

Volume 110 Number 4

Red blood cells, platelets, neutrophils, monocytes, and macrophages are all derived from a common progenitor cell. Johnson and Metcalf ~and Hara and Ogawa 2 independently devised tissue culture assays for these progenitor cells in mice, and later Fauser and Messner 3 described a similar assay for human cells. The hematopoietic progenitors were termed CFU-GEMM, indicating that the colonies of cells that they formed contained granulocytes, erythrocytes, monocytes, and rnegakaryocytes. It was subsequently demonstrated that CFU-GEMM were present in the peripheral blood and the bone marrow of human adults4 and that, whether in the blood or marrow, they were in a quiescent, nonproliferating state. 5 Neonates are not infrequently afflicted with hematologic disorders or infectious diseases that might affect the kinetics of their CFU-GEMM. However, neither the blood concentration nor the proliferative state of C F U - G E M M has been reported in human fetuses or neonates. Therefore, this study was designed to compare CFU-GEMM number and proliferative rate in normal adults and in term and preterm neonates.

Clinical and laboratory observations

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METHODS Ten to 20 mL venous blood was obtained from each of 10 healthy adult volunteers and from a placental vessel after delivery of 10 neonates. None of the subjects had bacterial infection, anemia, or thrombocytopenia. All specimens were anticoagulated with 10 to 20 U preservativefree heparin/mL blood, and processed immediately. A nucleated cell count (Coulter Electronics, Hialeah, Fla.) and 200 cell differential were performed on each sample. CFU-GEMM were determined by plating 0.5 to 3 • 105 light density cells (<1.077 g/cm 3) in methylcellulosecontaining cultures, according to the method of Leary et a12 Plates were set up in quadruplicate, and colonies were scored after 14 days. Their mixed nature was confirmed by the method of Fauser and Messner,3,5 in which colonies are subjected to cytologic analysis after removal from the dish. The proliferative rate of CFU-GEMM was determined by the method of Iscove et al., 7 modified by Lu et al., 8 in which methyl-3H-thymidine, 0.1 mCi (specific activity, 79.9 Ci/mmol, New England Nuclear, North Billerica, Mass.) is added to one aliquot of light-density blood cells while an equal amount of nonradioactive thymidine is added to a second aliquot. After a 20-minute incubation, with agitation every 5 minutes, thymidine (4000 #g) in ice-cold alpha MEM (Flow General, Inc., McLean, Va.) is added. The cells are then washed twice in the thymidinealpha MEM solution and plated as above. The proliferative rate was calculated in the same manner reported for unipotent granulocytic precursors (CFU-GM). 9 In brief,

623

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I

40 38 35 33 30 WEEKS OF GESTATION

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Fig.

I. Blood concentration ofCFU-GEMM (cells/mL blood) in 10 adults compared with that of 10 newborn infants. Gestational age of neonates ranged from 26 to 43 weeks.

the number of colonies/106 plated cells subjected to 3HTdr were subtracted from the number of colonies/106 plated cells subjected to "cold" thymidine. The remainder was divided by the number of colonies/106 plated cells subjected to "cold" thymidine. The paired t test was used to evaluate differences between colony numbers formed after exposure to "cold" thymidine compared with 3HTdr. Statistical comparisons between the various age groups were performed using the Student t test. The study was approved by the Institutional Review Board of the University of Utah. RESULTS Circulating CFU-GEMM were detected in all subjects (Fig. I). In the adults, blood concentrations ranged from 57/mL to 300/mL. Significantly greater concentrations were detected in neonates (1500/mL to l l,500/mL, P <0.0001 vs adults). The blood concentration of CFUGEMM in the premature infants (8160 + 2950/mL, mean _+ SD) was not significantly greater than in the term babies (5490 + 3470 mL). However, the highest value

624

Clinical and laboratory observations

The Journal of Pediatrics April 1987

60=

adults had a CFU-GEMM 3HTdr suicide rate of 0%; the other had a rate of 13%. Therefore, in the adults CFUGEMM were generally in a quiescent, nonproliferating state. In contrast, in all but one of the neonates, CFUGEMM were actively proliferating (33 + 15% 3HTdr suicide rate for the entire group of neonates, mean _+ SD, P <0.0001 compared with adults). The preterm infants had a proliferative rate (37% _+ 15%) not significantly greater than that of the term babies (29% + 19%). However, the highest proliferative rate observed (53%) was in the infant with lowest gestational age (26 weeks). The neonate with the lowest proliferative rate (40 weeks gestation, Fig. 2) had the highest CFU-GEMM blood concentration of any term infant (10,200/mL, Fig. 1).

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Fig. 2. Rate of proliferation of CFU-GEMM, as assessed by tritiated thymidine suicide rate, in 10 adults compared with that in 10 newborn infants. CFU-GEMM were seen to be quiescent, or proliferating at a slow rate, in adults, but were actively cycling in most neonates.

observed ( l l , 5 0 0 / m L ) was from the most premature infant (26 weeks gestation), and the lowest (1500/mL) from the infant with longest gestation (43 weeks). The 3HTdr suicide rate reflects the relative proliferative rate of the cells tested. A high rate of cell killing by 3HTdr correlates with rapid cell proliferation; conversely, a low rate signifies a slowly proliferating, quiescent population of cells. In the adult cells, no diminution in colony formation was observed after exposure to 3HTdr. Light-density adults blood cells that were exposed to "cold" thymidine formed 46 _+ 11 colonies/106 plated cells (mean _+ SEM), compared with 51 + 14 colonies/106 cells exposed to 3HTdr. In contrast, in term neonates, significant killing of the colonies was observed after exposure to 3HTdr (803 _+ 130/106 cells exposed to 3HTdr, compared with 1200 _+ 160/106 cells exposed to "cold" thymidine, P <0.05). Similarly, in preterm infants, significant diminution in colony formation was observed after exposure to 3HTdr (940 _+ 220/106 cells exposed to 3HTdr compared with 1640 + 430/106 cells exposed to "cold" thymidine, P <0.05). The CFU-GEMM proliferative rates, calculated for each individual tested, are shown in Fig. 2. Nine of the 10

Neutropenia, anemia, and thrombocytopenia are not uncommon problems in neonates. Although these hematologic findings may accompany a wide variety of neonatal diseases, the kinetics of the cytopenias have a common feature: a production rate insufficient to meet demands. In some instances this occurs because the production of neutrophils, erythrocytes, or platelets is actually decreased. In other cases, production, although increased, is not sufficient to compensate for a pathologic acceleration in removal of mature elements. For instance, a fetus with Rh hemolytic disease will develop anemia only after the erythrocyte production rate can no longer increase sufficiently to keep pace with the rate of hemolysis, a~ In this manner, a limitation in ability to increase production of any variety of blood cell will aggravate an otherwise mild case of accelerated destruction of that cell type. It has not been known whether human neonates possess an inherent limitation on increasing neutrophil, erythrocyte, or platelet production. From our studies, it appears that such a limitation may be present. In adults, if additional mature blood cells of any of the above varieties are required, such as during bacterial sepsis, chronic hemolysis, or isoimmune platelet destruction, the CFUGEMM proliferative rate: can be increased from the baseline rate of 0% up to at least 50% to 60%. 5 In contrast, in most neonates CFU-GEMM are already proliferating very rapidly in the baseline state, and therefore they have proportionately :less reserve with which to accommodate any additional production. In this study we observed a large concentration of CFU-GEMM in the blood of neonates. Similarly, in a previous study we observed a large number of circulating granulocyte-macrophage progenitor cells (CFU-GM) in neonates.9 Although it might be tempting to speculate that neonates have more hematopoietic progenitor cells per kilogram body weight than do adults, animal studies

Volume 1t 0 Number 4

suggest that this is not the case. In fact, neonatal rats have only about 10% as many C F U - G M per gram body weight as adults do." In summary, human neonates were observed to possess a sign!ficantly greater blood concentration of C F U - G E M M than that in adults. Whereas in adults C F U - G E M M were generally nonproliferating, they were seen to be proliferating actively in neonates. Perhaps this rapid baseline C F U - G E M M proliferative rate in neonates constitutes an inherent limitation in hematopoiesis, by impairing the ability to increase production of various blood cells significantly. I thank Suzanne Post and Buddy Nielsen for excellent technical assistance, and Neil K. Kochenour, M.D., Chief of the Division of Maternal-Fetal Medicine, under whose direction cord blood specimens were obtained. REFERENCES 1. Johnson GR, Metcalf D. pur e and mixed erythroid colony formation in vitro stimulated by spleen conditioned medium with no detectable erythropoietin, proc Natl Acad Sci USA i977;74:3879. 2. Hara H, Ogawa M. Murine hemopoietic colonies in culture containing normoblasts, macrophages and megakaryocytes. Am J Hematol 1978;4:23. 3. Fauser AA, Messner HA. Identification of megakaryocytes, macrophages and eosinophils in colonies of human bone

Clinical and laboratory observations

4.

5.

6.

7.

8.

9.

10.

11.

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marrow containing neutrophilic granulocytes and erythroblasts. Blood 1979;53:1023. Ganser A, Elstner E, Hoelzer D. Megakaryocytic cells in mixed haemopoietic colonies (CFU-GEMM) from the peripheral blood of normal individuals. Br J Haematol 1985;59:627. Fauser AA, Messner HA. Proliferative state of human pluripotent hemopoietic progenitors (CFU-GEMM) in normal individuals and under regenerative conditions after bone marrow transplantation. Blood 1979;5:1197. Leary AG, Strauss LC, Civin CI, Ogawa M. Disparate differentiation in hemopoietie colonies derived from human paired progenitors. Blood 1985;66:327. lscove NN, Till JE, McCulloch EA. The proliferative states of mouse granulopoietic progenitor cells. Proc Natl Acad Sci USA. 1970;134:33. Lu L, BroxmeYer HE, Meyers PA, Moore MAS, Thaler HT. Association of cell cycle expression of Ia-like antigenic determinants on normal human multipotental (CFUGEMM) and erythrgid (BFU-E) progenitor cells with regulation in vitro by acidic isoferritins. Blood 1983;61:250. Christensen RD, Harper TE, Rothstein G. Granulocytemacrophage progenitgr, cells in term and preterm neonates. J PEDIATR 1986;109:1047-51. Oski FA. The erythrocyte and its disorders. In Nathan DG, Oski F A, eds. Hematology of infancy and childhood. Philadelphia: WB Saunders, 1981;17-49. Christensen-RD, Hill HR, Rothstein G. Granulocytic stem cel! (CFUc) proliferation in experimental group B streptococcal sepsis. Pediatr Res 1983;17:278,