Blur
Blut (1982) 45:23-32
© Springer-Verlag 1982
Enhanced Acceptance of Regenerating Bone Marrow of Random Bred Newborn Mice by Random Bred Adult Mice* Z. Ben-Ishay1, G. Prindull z, S. Yankelev 1, and A. Shushan 1 1 Laboratory of Experimental Hematology, Department of Anatomy and Embryology, Hebrew University-Hadassah Medical School, Jerusalem, Israel 2 Department of Pediatrics, University of GSttingen, D-3400 GSttingen, Federal Republic of Germany
Summary. Regenerating bone marrow of newborn random bred Sabra mice (9-13 days old) was obtained by the administration of two consecutive i. p. injections of hydroxyurea (HU) (2 x 1000 mg/kg body wt), three days prior to collection of the marrow cells. The bone marrow of HU-treated newborn mice was assayed for CFU-S, CFU-C and plasma-clot-diffusion-chamber (PCDC) progenitor cells. A fourfold content of CFU-S was found in the regenerating bone marrow compared with that of the control marrow, while the level of CFU-C and PCDC progenitor cells was the same in treated and untreated newborn mice. In lethally irradiated adult, random bred Sabra recipient mice, transfused with regenerating bone marrow from newborn mice, the initial survival rate was greater than in irradiated animals receiving normal newborn marrow (75% as against 50%); marrow repopulation, 10-14 days after transfusion, was also greater in the former than in the latter group of animals (1.5-2 x 106 nucleated cells per femur as compared with 0 . 8 - 2 x 105). The bone marrow of these groups of mice was assayed for CFU-S, CFU-C and PCDC progenitor cells; with a cell inoculum of 5 X 104 i.v., 105 in vitro and 5X 104 per DC, respectively, pluripotent and committed stem cells were detected in the experimental group and were lacking in control recipients. Regenerating bone marrow of newborn mice was also transfused into lethally irradiated splenectomized recipients. In this experimental group there was high mortality, low marrow repopulation and lack of CFU-S ( 5 - 1 0 x 104 cell inoculum). The results of this study indicate that, despite genetic differences among random bred Sabra mice, regenerating bone marrow of newborn mice "takes better"
* Supported by a grant from Das Ministerium fi~rWissenschaft uud Kunst, Land Niedersachsen, Federal Republic of Germany Offprint requests to: Prof. Z. Ben-Ishay, MD (address see above)
0006-5242/82/0045/0023/$2.00
24
Z. Ben-Ishay et al. than normal marrow in lethally irradiated recipients. Improved marrow acceptance is possibly due to the increased content o f activated CFU-S a n d / o r preCFU-S in the regenerating bone marrow.
Key words: Regenerating bone marrow - Marrow transplantation - Newborn mice - CFU-S - Pre-CFU-S - P C D C - CFU-C Transfusion o f normal murine bone marrow of adult animals into irradiated recipients provides a restricted number of cycling pluripotent stem cells (CFU-S). Under the steady state of hematopoiesis, approximately 90% o f CFU-S o f mature mice are resting cells while only 10%, or less, are in cycle [11, 17]. The fate of noncycling CFU-S, transfused into the recipients, is uncertain. Normal donors, therefore, provide a relative small n u m b e r of cycling pluripotent stem cells which seems to obviate a desired amplification o f the transfused stem cell pool. Brecher et al. [6] suggested that part of the difficulty in the taking of normal bone marrow transplant could be due do absence of stimuli required to trigger resting CFU-S into cycle. These investigators showed that a better take is achieved in partially shielded recipient mice when the marrow transfusion precedes the irradiation. According to Brecher et al. the enhanced marrow take seems to be due to irradiation-induced activation o f transfused resting CFU-S [6]. The object of the present study is to suggest an experimental model wherein lethally irradiated mice receive a transfusion of bone marrow enriched with pluripotent stem cells. Such bone marrow is obtained by pretreating the donors with hydroxyurea (HU), a potent D N A inhibitor. Following HU-induced initial damage, the ensuing regenerating marrow becomes highly enriched in activated CFU-S. The results of this investigation indicate that regenerating bone marrow "takes" better than normal marrow in a steady state of hematopoiesis.
Material and Methods I. Experiments with Normal, Nonsplenectomized Recipients (outlined in Fig. 1) A total of eight experiments were carried out. Animals. All animals used, both donors and recipients, were random bred Sabra mice. Newborn mice (9-13 days old) of both sexes were used as donors of bone marrow. They were divided into two groups: (1) 40 hydroxyurea-treated and (2) 24 untreated animals. Five hydroxyurea-treated and three untreated donors were used in each experiment. Adult males (2-3 months old) were used as first and second recipients in the spleen colony assay. One to two months old mice of both sexes were used as hosts for the plasma-clot-diffusion-chamber cultures (PCDC). Regenerating Donor Bone Marrow. Bone marrow was obtained from newborn mice following hydroxyurea1 (HU) administration. Each animal received two consecutive i.p. injections of HU (1000 mg/kg body wt), freshly dissolved in 0.1 ml sterile water, 6 h apart. Three days later, the animals were killed and bone marrow was obtained for transfusion (see below). Control newborn mice received 2 consecutive i.p. injections of 0.1 ml sterile water with the same time interval between injections and were killed three days later. Irradiation and Transfusion Protocol of First and Second Recipients in the Spleen Colony Assay. A total of 72 first recipients (nine in each experiment) were transfused i.v. with marrow from 1 Kindly supplied as a gift by the Squibb Comp., Princeton, NJ, USA
Transplantation of Regenerating Bone Marrow of Newborn Mice
25
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HU-treated donors; an equal number of second recipients received an i.v. transfusion of bone marrow of first recipients. Similarly, 72 first recipients received a transfusion of marrow from control donors and 72 second recipients were transfused with bone marrow of first recipients. Twenty-four hours prior to transfusion, first and second recipient mice received total body irradiation of 850 rad in a single dose (120 rad/min at a SSD of 50 cm, Philips RT 250 machine, 200 KeV, 1.2 mm Cu HVL, 0.5 mm Cu Filter). For the purpose of studying the development of endogenous colonies, a tenth animal, irradiated but not transfused, was added to each group of first or second recipients (experimental and control).
Bone Marrow Suspension: Preparation and Cell Counts. Bone marrow of (1) donor newborn mice (HU-treated and controls) and of (2) first recipients (experimental and control groups) was flushed from femurs with M 199 culture medium. Single cell suspensions were obtained by repeated passage through a syringe (4~ 25 needle gauge). Total counts of nucleated cells per femur were carried out in a hemocytometer. Bone marrow cells were cytocentrifuged (Shandon), stained with the Wright stain and differentially counted. The cells were classified as (1) erythroid (replicative and non-replicative), (2) granuloid (replicative and non-replicative) and (3) lymphoid.
CFU-S Assay (Spleen Colony Technique). Twenty-four hours after the irradiation the mice were lightly anaestesized by ether and the first recipients received an i.v. transfusion (in the lateral tail vein) of 2.5 X 104 HU-treated marrow cells or 5 x 104 normal marrow cells in 0.3 ml M 199 culture medium. (Preliminary experiments with an inoculum of 5 x 104 HU-treated marrow cells gave rise to countless and confluent spleen colonies.) Each of the second recipients, both experimental and controls, received 5 x 104 cells. All recipients (and also the irradiated but nontransfused animals) were caged individually and were given pellets containing antibiotics and drinking water containing 6% HC1. The first recipients were killed 10-14 days after the marrow transfusion. This time interval between transfusion and killing was selected in view of previously reported increased number of spleen colonies and increased spleen weight in recipient mice, 8-14 days after a bone marrow transfusion of 5-Fluorouracil (another DNA inhibitor) treated donors [10]. Second recipients were sacrificed 8 days after the marrow transfusion for conventional spleen colony technique. Surviving first and second recipients were killed by
26
Z. Ben-Ishay et al.
neck dislocation and the spleens were removed and fixed in a mixture of 85% ethanol, 10% formatin and 5% acetic acid. Spleen colonies on the surface were counted 24 h after fixation at a magnification of x 30.
Plasma-clot-diffusion-chamber (PCDC) Cultures of Marrow Cells. These were prepared with marrow from two groups: (a) newborn donor mice (HU-treated and controls) and (b) first recipients, 10-14 days after a marrow transfusion (HU-treated or normal). PCDC were made and processed as described by Steinberg et al. [16]. A total of 104 bone marrow cells of newborn mice or 5 × 104 cells of first recipients were enclosed in each DC. A single chamber was inserted into the peritoneal cavity of each host. Host mice received an i.p. injection of cyclophosphamide (150 mg/kg), 24 h prior to chamber insertion. The PCDC were harvested 5 days after implantation into the peritoneal cavity. Granulocytic and/or macrophage aggregates of up to 20 cells were scored as clusters and those larger than 20 cells, as colonies. Clusters of benzidinepositive cells grouped together were classified as erythroid bursts derived from BFU-d-e [8].
Assay of CFU-C was carried out on bone marrow of (a) the normal and hydroxyurea-treated newborn mice and (b) the first recipients (of normal and of HU-treated marrow). The culture medium consisted of Eagle's MEMc~ modification (Flow) with 20% Foetal Calf Serum (Gibco), 0.3 % bacto-agar and 20 U Gentamycine per ml. Suspensions of bone marrow cells were first incubated for 30 rain at 37°C to eliminate adherent cells and then seeded in 35 mm plastic Petri dishes at a concentration of 10~ cells/m1. An amount of 0.1 ml colony stimulating factor (CSF) was added to each dish, which thus contained a total of 1.1 ml final medium. The CSF consisted of post-endotoxin serum of mice injected Lipopolysaccharide B (Salmonella Abortus Equi, Difco), as described by Byrne et al. [7]. The Petri dishes were placed in humidified "cake" boxes (Stewart Plastic Ltd., Purley Way Croydon, London). The boxes were gased for 15 min. with a composition of 85% N2 + 10% CO2 + 5% Q , sealed and placed in an incubator at 37°C for a period of 8 days. The cultures were examined by means of an inverted microscope (Olympus): aggregates of more than 50 cells were scored as colonies and those smaller than 50 cells, as clusters.
II. Experiments with SplenectomizedFirst Recipients A group of 60, one and a half to two mouths old male mice were splenectomized. Two weeks after the operation they were divided into two groups: (1) 30 recipients of HU-treated bone marrow of newborn mice and (2) 30 recipients of control marrow. Twenty-four hours prior to transfusion, the splenectomized mice received total body lethal irradiation (850 rad). Nine to eleven days after transfusion, the splenectomized recipients were sacrificed. Marrow cells of splenectomized recipients were (a) counted (total counts per femur) and (b) assayed for CFU-S by the day-8 spleen colony technique (i. e., transfusion of 5-10 x 104 marrow cells of splenectomized first recipients into previously lethally irradiated uon-splenectomized second recipients).
Results
Regenerating Bone Marrow of Newborn Mice Three days after two consecutive injections of H U the total nucleated cell count was reduced from 4 - 5 x 106 per femur in untreated animals to 2 - 3 x 106. Differential cell counts indicated that the marrow population became p r e d o m i n a n t l y early myelopoietic with a shift of the erythroid/granuloid ratio from 1.6/1 (control values) to 1/5.3.
CFU-S and Diffusion-chamber-progenitor-cells Developing from Bone Marrow of HU-treated and Untreated Newborn Mice, Assayed by Day 10-14 Spleen Colonies in First Recipients and by PCDC Cultures (Fig. 2) CFU-S. Transfusion of 2.5 × l04 HU-pretreated donor marrow cells gave rise to a m e d i a n n u m b e r of 6.7 colonies per spleen. A characteristic individual colony had a
Transplantation of Regenerating Bone Marrow of Newborn Mice
27
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diameter of 2-3 m m and the mean spleen weight of unfixed specimens was 70 mg. By contrast, transfusion of twice as many bone marrow cells (5 x 104) from untreated donors gave rise to a median number of 3.4 colonies per spleen; most of the colonies were 0.5-2 m m in diameter and the mean spleen weight was 35 mg. Furthermore, the survival rate of recipients of control marrow was lower than that of the experimental group (50% as against 75%). For this reason several animals who received control marrow were sacrificed for the spleen colony scoring already on the 10-1 lth day after marrow transfusion. No endogenous colonies were observed in irradiated but not transfused animals.
PCD C The results obtained with PCDC cultures of bone marrow from hydroxyurea treated and untreated newborn mice indicate a mean value of 10 diffusion-chamber-progenitor cells of granuloid/macrophage (G/M) colonies per 104 cells inoculated. In addition, 4-5 erythroid colonies derived from Burst-forming-units in PCDC (BFU-d-e) (day 5 of culture) were detected per 104 cells seeded in each chamber (Fig. 2). It is noteworthy that erythroid bursts developed in PCDC of bone marrow of normal and also of HU-treated newborn mice in the absence of specific erythropoietic stimuli applied to host or donor animals.
CFU-C of Normal and Hydroxyurea-treated Newborn Mice ,~n average of 38 CFU-C/105 marrow cells were detected in both hydroxyurea-treated (3 days after 2 injections of HU), and untreated newborn mice. However, in the case of marrow from HU-treated animals, approximately 10% of CFU-C gave rise to "macroscopic" G / M colonies (Fig. 3).
Cell Counts of Femoral Bone Marrow of First Recipients Twelve to fourteen days after the transfusion, a total of 1.5-2 x 10a nucleated ceils per femur was found in recipients of marrow from HU-treated newborns as compared with 0.8-2 x 105 nucleated cells per femur in recipients of marrow from untre-
28
Z. Ben-Ishay et al. 50-
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Fig. 4. Data on plasma-clot-diffusion-chamber cultures (PCDC) and CFU-S (the spleen colony technique) of first recipients bone marrow, 10-14 days after a transfusion of hydroxyurea-treated (HU) or control (C) marrow of newborn mice
ated animals (days 10-12 after transfusion). (The figure for normal adult male Sabra mice is 20-25 x 106 [2]). Differential counts showed a high content of early myelopoietic cells (56%) in recipients of marrow from HU-treated newborns and over 50% lymphocytes in recipients of normal marrow.
CFU-S and Diffusion-chamber-progenitor Ceil Content of Femoral Bone Marrow of First Recipients, Assayed by Day-8 Spleen Colonies in Second Recipients and by PCDC Cultures (Fig. 4). CFU-S. Transfusion of 5 X 104 bone marrow cells from recipients of HU-pretreated marrow into lethally irradiated second recipients gave rise to a median n u m b e r of 6 colonies per spleen. In contrast, no spleen colonies were formed in those second recipients (after transfusion of 5 x 104 cells) whose donors, i. e., first recipients, had received bone marrow of control newborn mice. Previously published studies in which
29
Transplantation of Regenerating Bone Marrow of Newborn Mice 100
-
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adult untreated donor Sabra mice were used, had indicated an average of four median number o f spleen colonies (day 8 spleen colonies) in lethally irradiated recipients following a transfusion of 5 × 104 marrow cells [2].
PCDC. In the cultures of bone marrow of first recipients who had received a transfusion of marrow f r o m HU-treated newborns, a mean value of 15 G / M colonies per 5 × 104 seeded cells was found; in addition, 3 - 4 erythroid bursts per chamber were also observed. Taken together, G / M and erythroid colonies in PCDC amounted to a total of ~ 18 per 5×104 cells (i.e. ~ 360 per 10a cells) compared with ~ 1250 PCDC colonies per 106 bone marrow cells of normal adult Sabra mice (unpublished observations). In contrast, in PCDC cultures of first recipients, which were initially transfused with control marrow, no G / M or erythroid colonies were observed.
CFU-C of First Recipients An average of 92 CFU-C/10 ~ bone marrow cells were found in the recipients transfused with hydroxyurea-treated marrow of newborn mice i. e., 920 CFU-C/106 cells compared with 1500 CFU-C/106 marrow cells from normal adult Sabra mice (unpublished observations). No G / M colonies were detected in cultures o f bone marrow of first recipients who had received a transfusion of marrow from untreated newborn mice (Fig. 5).
Experiments with Splenectomized First Recipients. Cell Counts of Femoral Bone Marrow and CFU-S Content of Splenectomized Recipients Sixty per cent of previously splenectomized and lethally irradiated first recipients who received a transfusion of either HU-treated or normal bone marrow of newborn mice survived 9-11 days after the transfusion. Surviving mice had approximately 1-2 × l0 s nucleated cells per femur, irrespective of whether they had received HU-treated or normal bone marrow. Zero to one, day-8 colonies per spleen were produced in second recipients by transfusion of the bone marrow ( 5 - 1 0 x 104) of splenectomized first recipients. The results were similar in splenectomized mice which had been transfused with HU-treated or with normal marrow of newborn mice.
30
Z. Ben-Ishay et al.
Discussion
During the early stages following administration of single or multiple injections of DNA inhibitors, the bone marrow contains an admixture of different subpopulations of hematopoietic stem cells. These include primitive pluripotent stem cells with a high self-replicating capacity (?pre-CFU-S) [10], primitive diffusion-chamber-progenitor-cells (?pre-DCPC) [3] and a class of erythropoietic stem cells, possibly burst-forming-units (BFU-E), which require large amounts of erythropoietin for in vitro colony formation [13]. Under the steady state of hematopoiesis, the bone marrow of normal adult mice contains approximately 90% of CFU-S in a resting phase [17] while under conditions of marrow regeneration, resting CFU-S decrease to an average of 70% with a concomitent increase in cycling CFU-S [1]. The large majority of CFU-S in the bone marrow of newborn mice are in cell-cycle [12, 14, 15] thus highly sensitive to the effects of HU. Following HU administration to infant mice, increased proliferative capacities of CFU-S are elicited which become apparent during the regenerating phase. In fact, regenerating bone marrow from newborn mice has been shown in the present study to give rise in first recipients to bone marrow with a higher cellularity, consisting predominantly of early myelopoietic cells, than in recipients of bone marrow from normal newborn mice. Furthermore, the spleen colonies in first recipients of marrow of HU-treated newborn donors were four times as numerous, and also larger in size, than in first recipients of normal newborn marrow. The occurrence of large spleen colonies is indicative of the marked capacity of CFU-S for self-replication, a characteristic feature of primitive stem cells (or pre-CFU-S). Mobilization of primitive CFU-S by 5-Fluorouracil treatment was also reported by Hodgson and Bradley [10]. We suggest that pre-CFU-S, which potentially feed into the CFU-S pool, rarely do so physiologically in newborn mice. Primitive CFU-S are however mobilized during severe depletion of CFU-S and progenitor cells. The PCDC cultures of regenerating bone marrow of newborn mice indicated a similar content of diffusion-chamber-progenitor cells (DCPC) as in control marrow. In a previous study on newborn mice given two consecutive injections of HU (2 x 1000 mg/kg body wt) 6 h apart, the bone marrow was collected, in contrast to the 3-day period in the present study, 2 h after the second injection, i. e., during the phase of extensive damage. Such marrow was studied by DC cultures and it was observed that it contained twice as many DCPC as the control marrow [3]. It was concluded that in newborn mice, DCPC are most likely, non-cycling cells which are not damaged by HU but are triggered into cycle and self-replication by the sudden HUinduced depletion of cycling pluripotent stem cells and committed progenitors. However, by the time three days have passed, the DCPC in the marrow of treated newborns returns to control levels. It is of interest that, apart from G / M colonies in PCDC cultures of both regenerative and control marrow from newborn mice, day-5 BFU-d-e were also noted. The occurence of erythroid bursts in PCDC cultures developing from bone marrow of HU-treated or untreated newborn mice may thus be regarded as a normal feature distinguishes it from adult marrow [4]. More important is that PCDC of bone marrow of first recipients-which were transfused with regenerating marrow of newborn mice-indicated the presence of an average of 18 DCPC per 5 x 104 cells. In contrast, 5 x t04 marrow cells of controls did not give rise to
Transplantation of Regenerating Bone Marrow of Newborn Mice
31
colonies in PCDC. These findings parallel those obtained with CFU-S and they too indicate enhanced acceptance of regenerating versus normal donor bone marrow. Studies of CFU-C of regenerating bone marrow of newborn mice (3 days after HU administration), indicate concentrations of such stem cells similar to those in control marrow. In an investigation on adult mice given two consecutive doses of HU (2 x 1000 mg/kg body wt), Hodgson et al. [9] showed a 91.5% CFU-C killing, 2 h after the last HU injection, CFU-C rose exponentially thereafter until day 3 when they reached control levels. Based upon results obtained in adult mice by Hodgson et al., it is reasonable to assume that the response of CFU-C in newborn mice to HU administration may be similar to that in the adult animal, i.e., initial extensive CFU-C killing followed by recovery by day 3. As indicated above, regenerating marrow of newborn mice contained CFU-C of which 10% produced "macroscopic" G / M colonies in agar cultures, a finding also reported by Bradley et al. [5]. Occurrence of macroscopic G / M colonies is indicative of the presence of CFU-C with a high proliferative capacity in regenerating marrow. CFU-C assay of bone marrow of first recipients transfused with HU-treated donor cells indicated an average of 92 G / M colonies per 105 seeded cells, while in control recipients, G / M colonies were not observed with a similar inoculum. This finding indicates that CFU-S in regenerating marrow had repopulated the hematopoietic system of lethally irradiated recipients and had differentiated along the CFU-C pathway, in contrast to lack or poor take of control marrow. The experiments with splenectomized first recipients gave negative results. In these animals HU-treated bone marrow of newborn mice, although highly enriched with CFU-S, was able neither to repopulate the marrow of first recipients nor to produce spleen colonies in second recipients. These results show that pluripotent stern cells require the spleen "homing" [18] and/or a "proliferation stimulus" controlled by the spleen-bone marrow interdependence [19] in order to express their proliferative and differentiating capacities. The results of the present study indicate that bone marrow transplantation in mice can be improved if the donor marrow is in a state of regeneration. Newborn mice were used as donors because HU kills a large proportion of CFU-S, the majority of which are cycling cells in the infant animal [3]. The extensive initial damage constitutes a strong stimulus for regeneratio n and results in a marrow much enriched with CFU-S and/or more primitive CFU-S. Despite genetic differences among the random bred Sabra mice, HU-treated donor marrow displayed a higher initial repopulating capacity than the control marrow expressed by total cell number per femur and by the high proportion of myeloblasts in first recipients. At present, the reason for the enhanced take of regenerating bone marrow remains unclear. It is possible that newly activated CI~V-S in regenerating marrow partially or temporarily lose their antigenicity and this permits them to self-replicate and differentiate in non-syngeneic recipients.
32
Z. Ben-Ishay et al.
References 1. Becker AJ, McCulloch EA, Siminovitch L, Till JE (1965) The effect of differing demands for blood cell production on DNA synthesis by haemopoietic colony-forming cells of mice. Blood 26:296 2. Ben-Ishay Z, Sharon S, Shorr A (1980) Stimulating factor (s) of hematopoiesis in the bone marrow of mice administered hydroxyurea. Effects on diffusion chamber progenitor cells (DCPC) and observations of pluripotent stem cells (CFU-S). In: Cronkite EP, Carsten AL (eds) Diffusion chamber culture. Springer, Berlin Heidelberg New York, p 124 3. Ben-Ishay Z, Prindull G, Ben-Israel D (1981) Effect of hydroxyurea on two different types of hematopoietic stem cells (CFU-S and DCPC) of newborn mice. Blut 42:165 4. Boyum A, Carsten AL, Laerum OD, Cronkite EP (1972) Kinetics of cell proliferation of routine bone marrow cells cultured in diffusion chambers: effect of hypoxia, bleeding, erythropoietin injections, polycythemia and irradiation of the host. Blood 40:174 5. Bradley TR, Hodgson GS, Bertoncello I (1980) Characteristics of primitive macrophage progenitor cells with high proliferative potential: relationship to cells with marrow repopulating ability in 5-Fluorouracil treated mouse bone marrow. In: Baum SJ, Ledney GD, Van Bekkun (eds) Experimental hematology today. S Karger, Basel Miinchen Paris London New York, p 285 6. Brecher G, Tjio JH, Haely JE, Narla J, Beal SL (1979) Transplantation of routine bone marrow without prior host irradiation. Blood Ceils 5:237 7. Byrne PV, Heit W, Kubanek B (1979) The proliferative states of density subpopulations of granulocyte-macrophage progenitor cells. Exp Hematol 7:105 8. Gerard E, Carsten AL, Cronkite EP (1978) The proliferative potential of plasma clot erythroid colony forming cells in diffusion chambers. Blood Cells 4:105 9. Hodgson GS, Bradley TR, Martin RF, Sumner M, Fry P (1975) Recovery of proliferating hemopoietic progenitor cells after killing by hydroxyurea. Cell Tissue Kinet 8:51 10. Hodgson GS, Bradley TR (1979) Properties of haematopoietic stem cells surviving 5-Fluorouracril treatment: evidence for a pre-CFU-S cell? Nature 281:381 11. Lajtha LG (1979) Stem cell concepts. Differentiation 14:23 12. Lucarelli G, Porcellini A, Cannevali C, Carmen A, Stohlman Jr F (1968) Fetal and neonatal erythropoiesis. Ann NYAcad Sci 149:544 13. Monette FC, Quellette PL, Thorson JA, Handsdorff W, Weiner EJ, Jarris Jr RF (1980)The in vitro erythropoietin sensitivity of late erythroid progenitors subjected to opposing physiologic demands. Exp Hematol 8:947 14. Porcellini A, Delfini C, Lucarelli G (1976) Kinetics of erythroid cells precursors in the newborn rat. Proc Soc Exp Biol Med 153:125 15. Rencricca NJ, Howard D, Kubanek B, Stohlman Jr F (1976) Erythroid differentiation of foetal, newborn and adult haemopoietic stem cells. Scand J Haematol 16:189 16. Steinberg HN, Handler ES, Handler EE (1976) Assessment of erythrocytic and granulocytic colony formation in an in vivo plasma clot diffusion chamber culture system. Blood 47: 1041 17. Till JE, McCulloch EA (1961) A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res 14:213 18. Trentin JJ (1970) Influence of hematopoietic organ stroma (Hematopoietic inductive microenvironment) on stem cell differentiation. In: Gordon AS (ed) Regulation of hematopoiesis, vol I. Appleton-Century-Crofts Medical, p 161 19. Wangenheim VKH, Siegers MP, Feinendegen LE (1980) Repopulation ability and proliferation stimulus in the hematopoietic system of mice following gamma-irradiation. Exp Hematol 8:694 Received August 9, 1981/Accepted March 20, 1982