J Clin Pathol 1985;38:68-72

Sideroblastic colonies in erythroid cultures grownfrom normal human marrow

S KAABA, A JACOBS, K BARNES

From the Department ofHaematology, University of Wales College ofMedicine, Cardiff

SUMMARY Normal human erythroid progenitor cells from bone marrow were grown in cultureusing a methyl cellulose clonal assay technique. Sideroblastic erythroid cells were found in themajority of colonies examined at 14-17 days, and a few sideroblasts were found in some of thecolonies examined after shorter periods of culture. Electron microscopy confirmed the presence

of both intramitochondrial iron deposits and cytoplasmic ferritin aggregates. These morphologi-cal appearances probably represent an abnormality induced by the in vitro culture conditions andcannot be used as evidence for an intrinsic defect in haem synthesis.

The appearance of sideroblastic erythroid cells incolonies grown in vitro from the blood' or bonemarrow2 of patients with idiopathic acquiredsideroblastic anaemia has been held to confirm thatthe erythroid progenitors in this condition areabnormal and give rise to progeny expressing thespecific metabolic defect of haem synthesis thoughtto be characteristic of this condition. The presentstudy shows that progenitors from normal marrowmay give rise to sideroblastic colonies in culturewhen they are incubated under standard conditionsin methylcellulose.

Material and methods

BONE MARROWNormal bone marrow samples were obtained duringthe course of surgery from the femur of patientsundergoing total hip replacement or from the ster-num of patients undergoing cardiac surgery. In somecases aspirates from the iliac crest were obtainedunder anaesthesia from patients undergoing minorsurgery. All subjects were haematologically normal.Fully informed consent was obtained in all casesbefore the procedure.PREPARATION OF CELL SUSPENSIONBone marrow samples were collected in 10 mlEagle's minimum essential medium (FlowLaboratories) containing 2% heat inactivated, filtersterilised fetal calf serum and 15 U/ml preservativefree heparin at 4°C. The cells were immediately cen-trifuged at 2000 g for 7 min and the supematantdiscarded. The cell pellet was resuspended in 5 ml

Accepted for publication 24 September 1984

fetal calf serum and bone marrow granules brokendown by aspiration through a 25 G needle. Nucle-ated cells were separated by centrifugation on aFicoll-Hypaque (Pharmacia) discontinuous densitygradient (1-077 g/ml) at 400 g for 30 min at roomtemperature. The interface cells were collected,washed twice with Eagle's minimum essentialmedium at pH 7 4, and used for culture.

CULTURE OF CFU-E AND BFU-EColony forming unit-erythroid (CFU-E) and burstforming unit-erythroid (BFU-E) were cultured by amodification of the methylcellulose system of Iscoveet al.2a In brief, 2 x 105 nucleated marrow cells permillilitre were cultured in alpha-medium (Flow)with nucleosides containing 0-8% methylcellulose(Dow, A4M, Premium), 2 1 g/l bicarbonate (Well-come), 10' M beta-mercaptoethanol (BDH),10-7 M sodium selenite (BDH), 10 mg/lvitamin E (Eastman), 10-8 M human transferrin(Behringwerke), antibiotic antimycotic solution(Gibco), and 1-0 U/ml anaemic sheep erythropoietin(Step III, Connaught). Cultures were performed inthe presence of pooled human AB serum (33%).Replicate cultures were incubated at 37°C with 5%CO2 in air in a fully humidified incubator.

MORPHOLOGY OF CELLS IN COLONIES ANDBURSTSErythroid colonies and bursts derived from CFU-Eand BFU-E respectively were routinely removedfrom the culture plates and the morphology of thecells examined. A micromanipulator adapted to thestage of a Leitz (Diavert) inverted microscope was

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Sideroblastic colonies in erythroid cultures grown from normal human marrow

used to guide a fine pasteur pipette to individualcolonies and bursts, which were removed from theculture using a micrometer screw gauge actionHamilton syringe. Colonies and bursts were placeddirectly into a Shandon (Southern) cytocentrifugeplastic holder and after the addition of a few dropsof fetal calf serum or 2% bovine serum albumin inphosphate buffered saline the material was cytocen-trifuged at 650 rpm for 10 min. Slides were air driedand fixed in methanol for 10 min. Duplicate prep-arations were stained with Jenner-Giemsa and byPerls' reaction with a neutral red counterstain. Ery-throid colonies and bursts were examined under oilimmersion using a Zeiss Universal microscope.Erythroid cells containing six or more iron staininggranules were defined as sideroblasts.

TRANSMISSION ELECTRON MICROSCOPYColonies and bursts picked out of the culture plateswere placed in cone shaped plastic ampoules andfixed with 3% glutaraldehyde in 0-1 M cacodylatebuffer and 2% osmium tetroxide. Araldite sectionswere examined at magnifications up to x 130 000after staining with uranyl acetate and lead citrate.

Results

Cells from 15 normal marrow samples were cul-tured. In no case were any sideroblasts present inthe marrow before culture. Colonies were removedbetween the 4th and the 17th day. Table 1 showsthat sideroblastic colonies were present in 11 of 13cultures examined at 14-17 days, 1 of 2 culturesexamined at 11-12 days, and 3 of 7 cultures

examined at 4-8 days. Both ring sideroblasts andnon-ring forms were seen. The numbers of colonieswithin the cultures showing a sideroblastic appear-ance and the number of sideroblastic cells in thesecolonies increased from relatively low numbers forCFU-E to much higher numbers for BFU-E. In14-17 day cultures 55% of the total number of col-onies examined showed sideroblastic change andwithin these colonies 24% of the total cells weresideroblasts. Fig. 1 shows the percentage of the totalnumber of erythroid cells in each culture that weresideroblastic related to the day of examination.

Transferrin iron, transferrin saturation, and ferri-tin concentration in the medium of cultures from

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Days in cultureFig. 1 Percentage oferythroid cells containing six or moresiderotic granules after different incubation periods.

Table 1 Sideroblasts visualised in erythroid colonies examined after 7-16 days' culture ofnormal marrow mononuclearcells

Subject Day ofculture Colonies examined % sideroblastslcolony

Total Sideroblastic Mean Range

1 8 10 0 - -12 10 2 7 2-1215 10 8 33 11-53

2 4 10 0 - -7 10 4 6 3-11

11 10 0 - -3 7 3 0 -

14 6 6 54 15-1004 7 12 0 -

14 10 0 -5 7 10 1 25 -

14 10 2 27 4-506 14 7 6 33 10-587 16 3 3 32 18-438 8 10 2 8 -6-109 15 10 10 28 9-5010 17 10 5 1 1 5-20

1 1 14 10 0 -12 14 7 2 4 2-613 16 10 3 39 29-4514 14 12 7 24 8-6515 14 1 1 9 31 8-52

, ..

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70two normal marrows are shown in Table 2. Therewas no increase in iron or transferrin saturation dur-ing the 14 days, though there was a steady increasein ferritin, presumably due to synthesis and releaseby the culture cells. In subject 4, in whom the trans-ferrin saturation varied from 20% to 37%, nosideroblasts were seen in the 14 day colonies. Insubject 3, with transferrin saturation varying from69% to 91%, all six colonies examined at 14 dayscontained sideroblasts, 54% of all the cells in thesecolonies being sideroblastic. In one experiment,where the transferrin saturations of 29%, 32%, and62% in the original medium were compared, thepercentage sideroblasts in the resulting BFU-E col-onies examined after 14 days were 17%, 23%, and46% respectively.

Supplementation of the cultures at seven dayswith additional medium, beta-mercaptoethanol(10'4 M), or pyridoxal HCI (2 mg/l) did not appearto influence the appearance of sideroblasts in 14 daycolonies. Fig. 2 shows electron micrographs of typi-cal iron granules in erythroid cells from a normal 15day colony. Both mitochondrial (Fig. 2a) and ferri-tin type aggregates (Fig. 2b) were present and thesewere commonly found in the same cell. Sideroblastswere sometimes associated with morphological fea-tures of dyserythropoiesis, but this was not a consis-tent finding.

Discussion

Although the appearance of sideroblasts in ery-throid colonies grown in vitro has been accepted asevidence for an intrinsic metabolic defect in haemsynthesis in these cells,' 2 most patients with primaryacquired sideroblastic anaemia actually have greatlyreduced erythroid colony formation3 4 and manyhave no growth. The present study shows that earlyerythroid progenitor cells (BFU-E) from normalhuman bone marrow can mature into colonies inwhich a large number of morphologically recognis-able erythroblasts contain siderotic granules, oftenas perinqclear rings. The electron microscopic

Table 2 Iron and ferritin concentrations in the medium oferythroid cultures

Day of culture

0 4 7 11 14

Subject 4Transferrin iron (AmoVI) 18 18 18 17 21Transferrin saturation (%) 20 31 37 34 35Ferritin (,ug/1) 4 12 20 44 92

Subject 3Transferrin iron (Amol/1) 18 22 20 18 18Transferrin saturation (%) 75 91 77 75 69Ferritin (,ug/1) 40 44 44 54 72

Kaaba, Jacobs, Barnes

appearances are similar to those seen in the bonemarrow of patients with idiopathic acquired sidero-blastic anaemia5; both intramitochondrial irondeposits and cytoplasmic aggregates of ferritinmolecules are present, often in the same cell. Theoccurrence of this phenomenon in erythroid burstsarising from BFU-E after 14 days' culture, but onlyto a much lesser extent in seven day colonies arisingfrom CFU-E, suggests either that only the earlyprogenitors are sensitive to the conditions giving riseto sideroblastic change or that they need to beexposed to the culture conditions for a longer periodbefore a pathological effect is seen. The failure ofsupplementation of the cultures at seven days, eitherwith whole medium or pyridoxal HCI, to prevent thedevelopment of sideroblasts suggests that this doesnot depend on a simple nutritional deficiency. Bothsideroblastic and non-sideroblastic cells were foundin the same bursts and there was no apparent differ-ence in maturity between the two with respect tonuclear morphology or haemoglobinisation. Theappearances were not those to be expected from aseparate clone of sideroblastic cells.

In those cultures investigated, transferrin satura-tion in the medium appeared to remain fairly con-stant throughout the period of incubation whilethere was a steady rise in ferritin concentration, pre-sumably reflecting ferritin synthesis in macrophagesand, possibly, other cells. Cells incubated withhighly saturated transferrin appear to have a highincidence of sideroblasts at 14 days, while in cultureswith a low transferrin saturation this is less obvious.While a high transferrin saturation alone may resultin cellular iron loading and cytoplasmic ferritinaggregates in vivo it will not normally producemitochondrial iron deposits. It is possible, however,that the localised iron loading may stimulate secon-dary intracellular free radical generation with con-sequent membrane damage and this might be poten-tiated by the effects of oxygen toxicity known tooccur in marrow cultures.6The possible influence of culture conditions on the

morphological appearance of erythroid cells in cul-ture makes it difficult to interpret studies in patientswith sideroblastic anaemia. The paucity of colonygrowth in such patients and the occurrence ofsideroblasts in BFU-E from normal marrow mighteven suggest that when sideroblastic colonies areseen in these patientse cultures they are as likely tohave arisen from normal progenitors as from anabnormal clone. Ogawa and his colleagues' 8 notedthe presence of sideroblastic cells in erythroid col-onies from normal human marrow samples after7-10 days' growth in methylcellulose, but in a laterpaper' they considered the presence of such cells inperipheral blood BFU-E from a patient with

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72

sideroblastic anaemia to confirm the underlyingdefect of haem synthesis. Similarly, Mizoguchi et al2found ring sideroblasts only occasionally in CFU-Ebut more commonly in the BFU-E colonies of apatient with sideroblastic anaemia. This is the samepattern observed in our normal subjects and maywell have represented growth of a small remainingpopulation of normal erythroid progenitors. Thesignificance of sideroblasts in culture from patientswith abnormal erythropoiesis can be evaluated onlywhen parallel studies on normal subjects are avail-able to control the possible effect of culture condi-tions. Reid et a!9 have shown dyserythropoieticchanges in BFU-E derived colonies and related thisto co-culture with monocytes. The phenomenon weobserved was not consistently related to dysery-thropoiesis, but although the mechanism may be dif-ferent, it could well be relevant to the pathogenesisof mitochondrial iron deposits.

References

'Hutcheson JR, Ogawa M, Eguchi M, Spicer SS. Idiopathicsideroblastic anaemia: presence of sideroblastic changes in theerythropoietic precursors cultured from peripheral blood. ExpHaematol 1979;7:328-33.

2 Mizoguchi H, Kubota K, Suda T, Takaku F, Miura Y. Erythroidand granulocyte/macrophage progenitor cells in primaryacquired sideroblastic anaemia. International Journal of CellCloning 1983; 1: 15-23.

Kaaba, Jacobs, Barnesza Iscove NN, Sieber F, Winterhalter KH. Erythroid colony forma-

tion in cultures of mouse and human bone marrow. Analysis ofthe requirements for erythropoietin by gel filtration andaffinity chromatography on agarose concanavalin A. J CellPhysiol 1974;83:309-20.

3Ruutu T, Partanen S, Lintula R, Teerenhovi L, Knuutila S. Ery-throid and granulocyte-macrophage colony formation inmyelodysplastic syndromes. Scand J Haematol 1984;32:395-402.

4May SJ, Smith SA, Jacobs A, Williams A, Bailey-Wood R. Themyelodysplastic syndrome: analysis of laboratory characteris-tics in relation to the FAB classification. Br J Haematol (inpress).

May A, de Souza P, Barnes K, Kaaba S, Jacobs A. Erythroblastiron metabolism in sideroblastic marrows. Br J Haematol1982;52:611-21.

6Rich IN, Kubanek B. The effect of reduced oxygen tension oncolony formation of erythropoietic cells in vitro. Br J Haematol1982;52:579-88.

7 Ogawa M, Parmley RT, Bank HL, Spicer SS. Human marrowerythropoiesis in culture. I. Characterisation of methylcellu-lose colony assay. Blood 1976;48:407-17.

8 Parmley RT, Ogawa M, Spicer SS, Bank SL, Wright NJ. Humanmarrow erythropoiesis in culture: III. Ultrastructural andcytochemical studies of cellular interactions. Exp Haematol1978;6:78-90.

Reid CDL, Baptista LC, Deacon R, Chanarin I. Megaloblasticchange is a feature of colonies derived from an early erythroidprogenitor (BFU-E) stimulated by monocytes in culture. Br JHaematol 1981;49:551-61.

Requests for reprints to: Professor A Jacobs, Departmentof Haematology, University of Wales College of Medicine,Heath Park, Cardiff CF4 4XN, Wales.

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