British Journal oftlaematology, 1981, 48, 147-153.

Characterization of a Developmentally Early Macrophage Progenitor

Found in Normal Mouse Marrow

PAUL BAINES, SIMON BOL* AND MARTIN ROSENDAAL

Department of Anatomy and Embryology, University College London, Gower Street, London, and *Radiobiological Institute TNO, 151 Lunge Kleiweg, Rijswijk, ZH, Holland

(Received 8 September 1980; accepted for publication 27 October 1980)

SUMMARY. In tissue culture high proliferation potential colony forming cells (HPP-CFC, Bradley & Hodgson, 1979) formed large colonies in which macro- phages predominated. Granulocyte/macrophage colony forming cells (GM-CFC, Bradley & Metcalf, 1966) formed smaller colonies of granulocytes and macrophages. We compared HPP-CFC from normal adult mouse bone marrow with GM-CFC from the same source. We found that HPP-CFC differed from GM-CFC in the following ways: (1) HPP-CFC formed larger colonies containing more than 5 x lo4 cells. (2) They required spleen conditioned medium (Metcalf & Johnson, 1978) as wellas pregnant mouse uterine extract (PMUE, Bradley et al, 1971) as stimulants. (3) Only 9% of HPP-CFC were killed by the phase specific drug hydroxyurea. 36-55% ofGM-CFC were killed by the same treatment. (4) The modal density of HPP-CFC was 1.070 g/cm3. That of GM-CFC was 1.080 g/cm3.

Recently Bradley & Hodgson (1979) reported that a mouse macrophage progenitor, cultured in soft agar, formed large colonies which contained more than 5 x lo4 cells. 5 x lo4 cells is equivalent to more than 16 doublings of the cell from which the colony formed. Consequently this progenitor has been called the high proliferation potential colony forming cell

Bradley & Hodgson (1979) found a high proportion of HPP-CFC in the marrow of mice pretreated with the cytotoxic drug 5-fluorouracil. HPP-CFC could also be found in normal bone marrow though their incidence was low there.

Two properties of HPP-CFC suggest that it probably represents a developmentally early cell: (i) it has a high capacity for proliferation; (ii) it is relatively resistant to treatment with 5-fluorouracil in viva, i.e. it is not killed by this cytotoxic drug (Rosendaal et al, 1979). However, since most of our information about HPP-CFC has been obtained with cells from donors treated with 5-fluorouracil, we wanted to test this hypothesis on undisturbed HPP-CFC from normal untreated bone marrow. We assessed their capacity to replicate and form other haemopoietic precursors in vitro; we measured their cycling status and their modal

Correspondence: Dr Martin Rosendaal, Department o f h a t o m y and Embryology, University College London, Gower Street, London WClE 6BT.

(HPP-CFC).

0007-1048/81/05ocrO147$02.00' 0 1981 Blackwell Scientific Publications

147

148 P. Baines, S . Bol and M . Roseridaal

density. We compared these properties with those of the granulocyte/macrophage colony forming cell (GM-CFC, Bradley & Metcalf, 1966), a committed precursor which forms smaller colonies in culture.

Ths paper reports our findings which lend further support to the proposal that HPP-CFC represent developmentally early haemopoietic cells.

MATERIALS AND METHODS

Animals. We reared C57Bl/lOScSn x Balb/c or vice versa (BlocFI) mice for this work in our animal unit. When these mice were 10 weeks or older we collected their femoral marrow cells for these studies.

Cell culture. We cultured GM-CFC in alpha medium (Flow) to which 25% heat-inactivated pooled human serum was added. The stimulant for these cells was pregnant mouse uterine extract alone (PMUE, Bradley et al, 1971) (6% ofthe whole culture). HPP-CFC were cultured with the same medium. 6% PMUE and 6% rat spleen conditioned medium were used as stimulants. PMUE was prepared as described by Bradley et al(l971) and rat spleen conditioned medium as described by Metcalf& Johnson (1978) with 1/300 pokeweed mitogen (Difco). We prepared post-endotoxin serum and assayed GM-CFC, in the manner described by Bol et al (1979). The important and distinguishing difference between colonies derived from HPP-CFC and GM-CFC is their size. Our colonies were grown from single cell suspensions so that clumps of cells would not be mistaken for large colonies.

I t is more convenient to assay HPP-CFC whose incidence is low in normal marrow in large culture dishes. In this way the cellular concentration can be kept down, which is necessary for good growth, and sufficient colonies can be cultured to obtain reliable results. We used 60 mm diameter dishes (Sterilin) and a double-layer technique. We added the stimulants to make up 3 ml nutrient solid agar (Bacto agar, Difco, 0.5%) in the bottom layer. We put 2 x lo4 cellsinto 2 ml semi-solid agar (0.3%) in the upper layer. For retransplantation experiments we grew cells in a single layer of 0.8% methocel (A4M, premium grade, Dow).

After 10 d of culture in a fully humidified atmosphere of 5% 02, 10% COz and 85% N2 we counted our colonies under x 10 magnification. We used an eyepiece graticule to assess colony size. We scored colonies greater than 2 mm diameter as HPP-CFC.

Drug treatments. Hydroxyurea (Sigma) was injected intraperitoneally, 1 mg/g body weight; and 5-fluorouracil (Sigma) intravenously, 150 mg/kg body weight.

Eqtrilibriuni derisify cetitrifiirgation. We separated cells on the basis of differences in their buoyant density (Shortman, 1968; Bol et al, 1977).

Spleen co lony fbr r i i i rg units. We assayed these by the method ofTill& McCulloch 9 d after the graft. We irradiated mice with 8.5 grays X-irradiation: Marconi source, 230 kVp, 15 mA, 45 TSD, HVL 1 mm Al, 1 mm Cu, a t about 1.2 grays/min.

RESULTS

We found the incidence of HPP-CFC in the marrow of normal untreated BIOCFl mice cultured with rat spleen conditioned medium and human serum to be about one cell in 1500. Our HPP colonies consisted of a mixture of blast cells, macrophages and granulocytes for the

Early Macrophage Progenitors 149

first week of culture but macrophages predominated after 1&12 d. They grew most quickly for the first 9 d of culture and we calculate that over this period the doubling time of the precursor in t d v o was 13.5 h (Table I ) .

Since we grew these large colonies from normal untreated mouse marrow we wished to establish that the cell from which the colonies arose was notjust a normal granulocyte/macro- phage colony forming cell (GM-CFC) forming large colonies in response to especially favourable culture conditions. Accordingly we treated normal mouse marrow in uivo with the S phase specific drug hydroxyurea and compared the proportion ofGM-CFC lost by exposure to the drug with the proportion of HPP-CFC lost (Table 11). Hydroxyurea killed 36% of GM CFC which formed colonies 1-2 mm in diameter and 55% of GM-CFC whlch formed small colonies less than 1 mm in diameter, but only 9% of HPP-CFC. Clearly, HPP-CFC and GM-CFC were different cells. The low figure of 9% kill by hydroxyurea compares with the proportion of spleen colony forming units (CFUs, Till & McCulloch, 1961) in normal marrow killed in vivo by hydroxyurea (lo%, Rosendaal & Dixon, in preparation).

We centrifuged normal mouse marrow on linear BSA density gradients (Bol et al, 1977) in order to find out if it were possible to separate GM-CFC and HPP-CFC. The modal buoyant density ofHPP-CFC was 1.070 g/cm3 and that of GM-CFC 1.080 (Fig 1). We also observed in these experiments that it was not possible to separate HPP-CFC 'from either spleen colony forming units (CFUs) or from developmentally early GM-CFC, on the basis of their buoyant density. The density of all these haemopoietic cells was about 1.070 g/cm3.

TABLE I. Number ofcellularity ofHPP-CFC colonies from agar cultures of normal marrow at various times after initiation of

culture

No. ofcoloniesk SEM/Zx I@ cel ls Cells/colony, Day > 2 mm diameter mean+SEM ( x lop4)

~~~~~ ~

6 2.3k0.9 Not done 7 12.3 f 0.3 1.7 f0.7 9 24.7k2.0 7.4+ 1.1

12 23.5 f 1.5 5.6f2.2 15 25.7 k 1 .8 7.2 + 2.2

TABLE 11. Effect of in-uivo administration of hydroxyurea on femoral GM-CFC and HPP-CFC

ColonieslQ x 10' cells

GM-CFC+ SEM HPP-CFC+ SEM

Hydroxyurea < 1 mm Kill (%) 1-2 mtn Kill (%) > 2 mm Kill (%) ~~~~~

- 834 f 43 33+3 3 1 f 3 + 376f32 55 2 1 f 2 36 28f2 9

The results are the means of four separate experiments.

150 P. Baines, S . Bol and M . Rosendaal

Density ( g / ~ r n - ~ )

FIG 1. Density distribution of GM-CFC (0). HPP-CFC (A) and CFUs (0).

TABLE HI. HPP-CFC and GM-CFC content of HPP colonies from normal bone marrow and 5D 5-fluorourad marrow (NBM and 5D 5-FUBM)

C M - C F C / HPP colony HPP-CFC/ HPP colony ( f S E M ) ( f SEM)

Duration Cellularity of Derivation of culture HPP colony Colony diameter Colony diameter

Expt no. of HPP-CFC ( d ) ( x <0.2 mm > 0.2-< 2 mm > 2 mm

1 NBM

2 NBM

3 NBM

5D 5-FUBM

5D 5-FUBM

5D 5-FUBM 4 NBM

5D 5-FUBM

6 6 6 6 7 7

10 10

0.9 2.2 0.83 243 3 2.3 6.8 5.4

241 f 14 1321 f 71 145k 16

1243f56 167+64 351+110 390+ 104 408 62

2 5 k 5 431 f 13

1 1 f 3 158+6

2&2 66 f 28 18f 10 8 + 2

0 0.7 f 0.7 0.3 _+ 0.3 2.8k0.8

Not done Not done Not done Not done

Cells collected from high proliferation potential colonies grown in methocel were cultured again for 10 d in order to measure the number of HPP-CFC and GM-CFC in the colonies after different initial periods of culture. In experiments 1 and 2 more than 20 colonies of characteristic HPP appearance were collected individually. These colonies were pooled and the total number of cells counted and cultured. The result is the mean of the replicate dishes. In experiments 3 and 4 nine individual colonies each were collected separately, counted and cultured. In this experiment the mean result is that of each culture of each colony.

The mean number of cells in colonies less than 0.2 mm diameter is 138f 13 (seven doublings). In colonies 1-2 mm diameter we find a mean of 1 . 1 3 f 0 . 3 7 ~ lo4 cell/colony (14 doublings).

The numbers ofGM-CFC (24.2 mm and <0.2 mm diameter) formed by NBM and 5D 5-FUBM HPP-CFC in experiments 1 and 2 were significantly different at the 1% level ofprobability (Student’s ‘t’ test for small samples). The total number ofGM-CFC formed by NBM and 5D 5-FUBM HPP-CFC in experiment 3 was significantly different at the 5% level of probability.

Early Macrophage Progenitors 151

We wondered whether HPP-CFC could self-replicate or form GM-CFC in culture. At different durations of culture we collected all the cells of cultured HPP colonies, made single cell suspensions of them and cultured them under appropriate HPP or GM culture conditions. We established that HPP colonies grown from normal mouse marrow and from marrow from mice treated 5 d before collection with 5-fluorouracil(5D 5-FUBM) both contained cells with the capacity to form granulocyte/macrophage colonies when cultured in the presence of PMUE (Table 111). After 6 and 7 d of culture cells from 5D 5-FUBM-derived HPP colonies formed significantly more GM colonies than did cells from normal bone marrow HPP colonies. There was also a small number of HPP-CFC in HPP colonies which had only been grown for 6 d.

DISCUSSION

HPP-CFC formed colonies with a characteristic appearance in culture. They had a dense central core of cells and a more diffuse periphery. Colonies like these could be seen in our cultures from as early as 3 d until 18 d after the dishes were set up. GM-CFC colonies were smaller and uniformly dense.

Our results show that the large colonies could not have formed from GM-CFC in response to especially favourable culture conditions. We found that there were clear cellular distinctions between HPP-CFC and GM-CFC and established the following points of difference between them: (1) HPP-CFC formed large colonies containing more than 5 x lo4 cells. GM-CFC formed smaller colonies. (2) HPP-CFC required spleen conditioned medium as well as pregnant mouse uterine extract (PMUE) to form large colonies in culture. GM-CFC only required PMUE to form their colonies. (3) In normal marrow hydroxyurea only killed 9% of HPP-CFC whereas it killed 3655% of GM-CFC depending on the size of colony they could form. (4) The modal density of HPP-CFC was 1.070 g/cm3. That of GM-CFC was 1.080 g/cm3.

We consider that the most important difference between HPP-CFC and GM-CFC was that HPP-CFC formed larger colonies than GM-CFC when they grew in similar medium. We think this is a manifestation of a different doubling capacity of these precursors. According to the generation-age hypothesis (Rosendaal et al, 1979) the capacity of haemopoietic cells to self-replicate and form other haemopoietic cells indicates their place in the haemopoietic developmental lineage. Earlier cells have a greater doubling capacity than developmentally later cells. Consequently we consider that HPP-CFC are developmentally earlier than

I t is not yet clear how the HPP-CFC and the cells it forms on division use factors in spleen conditioned medium and in PMUE. We consider that the most probable explanation is that the HPP-CFC requires factor(s) in spleen conditioned medium for its initial divisions and that the cells which are formed in these divisions require factor(s) in PMUE. Iscove & Guilbert (1978) suggested an analogous mechanism for erythroid burst forming units. Spleen conditioned medium probably contains many different stimulatory factors which is why it is possible to culture erythroid, granulocytic and mixed colonies with it alone (Metcalf & Johnson, 1978). When spleen conditioned medium and PMUE are present in culture medium, macrophage development predominates.

GM-CFC.

152 P . Bainrs, S . Bid arid M . Roseridaal

The comparatively low kill (9”/0) by hydroxyurea of normal bone marrow HPP-CFC places them either in the group of haemopoietic progenitors which are not cycling actively or in a group of cells whose S phase is very brief. They are comparable with the normal marrow spleen colony forming units (CFUs) only 10% of which are killed by hydroxyurea. This low kill distinguishes them from more actively cycling precursors such as GM-CFC or erythropoietin responsive cells. O f these later precursors 45% and 76% respectively are killed by high specific activity tritiated thymidine (Lajtha et al, 1969) which kills cells forming DNA.

We found that HPP colonies from both normal marrow and 5-fluorouracil-treated marrow formed GM-CFC. Occasionally we found HPP-CFC in these HPP colonies too. The ability of HPP colonies to self-replicate and form GM-CFC is further evidence that HPP-CFC are developmentally earlier than GM-CFC.

Our experimental finding that GM-CFC are denser than HPP-CFC, GM-CFC, and CFUs is ‘consistent with other reports that other developmentally later cells are denser than developmentally earlier ones (Haskill et al, 1970; Bol et al , 1979). We were not able to separate HPP-CFC, GM-CFC, and CFUs, however, on the basis of their density. We are trying to do so by other means.

There was a significant difference between the number of GM-CFC formed by HPP-CFC derived from normal marrow and HPP-CFC derived from donors treated with 5-fluorouracil 5 d after collection (5D 5-FUBM). In our experiments, however, this difference was only clear after HPP-CFC had been in culture for 6 d. Later, differences were not apparent. We interpret this difference between HPP-CFC from 5-fluorouracil-treated marrow and normal marrow as follows. We propose that average HPP-CFC in the marrow of normal mice are develop- mentally later than average HPP-CFC in mice 5 d after 5-FU. Treatment of donor mice with 5-fluorouracil therefore probably lulls the developmentally later HPP-CFC which predomi- nate in normal marrow and ‘exposes’ developmentally earlier HPP-CFC which predominate after treatment with 5-fluorouracil.

ACKNOWLEDGMENTS

This work \vas supported by grants from the Medical Research Council and the Cancer Research Campaign. Dr Bol is supported by the Netherlands Foundation for Medical Research which is subsidized by the The Organization for the Advancement ofPure Research. Dr Baines would like to thank Radiobiological Institute T N O for their hospitality. We are grateful to Ms V. Slingerland and Mrs D. Frendo for their assistance.

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