Colony forming units and haematopoietic stem cells in osteoclaspoeosis

Metab.

Bone

Metabolic Bone Disease 8 Related Research 0 by S.N.P.M.D. (Paris

Dis. & Rel. Res. 3, 131-133 (1981)

1981)

c Stem

nel II SiS

J.F.

LOUTIT

‘, J.

PETERS 1 and

M.J.

MARSHALL2

1 MRC Radiobiology Unit, Harwell, Didcot, Oxon, OX11 ORD, Great Britain. 2 Charles Salt Research Centre, Robert Jones and Agnes Hunt Orthopaedic Britain.

Abstract HSC and CFUs are not identical. HSC are no longer considered to be a homogeneous population but an age-structured spectrum of ceils (Schofield, 1978). CFUs, which have been identified onlly in rodents and with certainty only in mice, may be a sub-set of HSC required by mice, perhaps controlled by the W locus and virtually eliminated in doubie W mutants. A dichotomy of CFUs and HSC has also been demonstrated by WiktorJedrzejczak et al. (1977) ; + + bone marrow treated with anti-Thy 1.2 serum lost its curative properties for W WV anaemic mice with CFUs being unaffected. Key Words : Osteopetrosis Osteoclast.

- Radiation

chimaeras

-

The term colony forming unit ,(CFU) is often used synonymou’sly with haemato,poi,etic stem cells (HSC). The former are cells present in myeioid tissue of mice (bone marrow, spleen and fetal liver) and are sparse in blood. When given in modest doses to lethally irradiated mice, ‘CFUs produce colonies of differentiat,ed haematopoietic cells visi,ble in the spleen after 8-10 ‘days. HSC are ceils in the same tissues which, when given in larger doses to ,lethally irraldiated mice, permit recovery through adopted haematopoiesis. We show here that lethally irradiated osteopetrotic mi mi mice injected intravenously with ,bone marrow from We WV mice, an animal s.everely ,deficient in CFUs, recover from the radiation syndrome and are cured of osteopetrosis. Thus, the bone marrow of We WV mice, despite paucity of CFUs, contains adequat,e numbers of haematopoietic and osteoclastopoietic stem cells. Microphthalmic mice (mi mi) are osteo.petrotic because of defective resorption of Ibone associated with abnormal osteoclasts (Marks and Walker, 1978). Th,e osteopetrosis is cured ,by engraftment of bone marrow or spleen or fetal ‘liver ceils from syngeneic normal siblings (Loutit and Sansom, 1976) in appropriately large doses (Nisbet et al., 1979) or, after lethal irradiation, by intravenous edministration of

Hospital,

Oswestry,

Salop,

SYlO

7AG,

Great

H-2 compatible allogeneic myeloid cells (Nisbet et al., 1979 ; Wal,ker, 1975). When myeloid &is of beige mice are given to lethally irradiated mi mi mice, giant lysosomes, the marker of beige cells appear i,n osteoclasts of the recovered mice probably through the chain HSC -_) monocyt,es + phagocytes with fusion -_, osteoclasts (Loutit and Nisbet, 1979). One ‘would Ilke to known whether marrow from mice doubly mutant at the W locus (chromosome 5) would, like normal marrow, cure osteopetrosis. These mice are congenitally anaemic (macrocytic) (Russell, 1979) and deficient in CFUs (McCulloch et al., 1964) though with normal complements of granulocytemonocyte precursors ,(Eennett et al., 1968). Harrison (1972) had shown that despite the paucity of CFUs, the marrows from W mutant animals were only marginally less effective than normal in curing the radiation syndrome of hemi-syngeneic irradiated normal counterparts, and that the recovered reci,pients manifested the macrocytosis of the donor 6 months ‘later. In the present study radiation chimaeras were established with mi’mi host mice and donor bone marrow derived from either We W or + + animals (actually +Ph, Ph being closely linked to W in a triplet) (Searle and Truslove, 1970). Donors and recipients were allogeneic but both H-2k. Table I gives the 28 day survival of the two groups after doses of bone marrow from lo5 to 5 X lo6 cells, the former value usually the slowest dose to effect survival after a lethal irradiation. Uneven survival was attri,buted to the general unthriftiness of the host stock, but th,ere was little difference between the two grouips. All survivors were cured of osteopetrosis and have remained cured up ‘until 6 months after treatment with the exception of one mouse, treated with We WV, that showed radiological relapse. Nevertheless, the two groups differed as to origin of red corpuscles. Two months after treatment, neither group was clinically anaemic. Formal blood counts were done only on the groups receiving We W marrow giving figures ; RBC ml--’ - 9.32 f 0.30 (S.E.M.) X 109, PCV’O/o - 45.6 + 1.0, MCV pm3 - 49.18 * 0.83. Two individuals had red blood corpuscles around 7 X lo9 ml--’ with mean corpuscular volumes of 51 and 56 pm3. However, the two groups differed

132

J.F.

with post-irradiation tolerance and mutual H-2 compatilbility. Some animals showed both types of red cells and clonal variation with time. All animals but one exhibited permanent cure of osteopetrosis.

as to distribution of phosphoglucomutase-1 phenotypes (Pgm-1, chromosome 5). Mice treated with We WV marrow were predominantly of host type (PGlM-1B) whereas those treated with + f marrow were predominantly of donor type ‘(PGM-1A) (Table II).

The observations recorded are compatible with the hypothesis that osteoclasts arise from HSC through the chain monocytes + phagocytes + fusion of phagocytes. The observations are also compatible with osteoclasts arising from a stem cell independent of, but always associated with HSC. This would not necessarily involve the chain through monocytes. We conclufde that HSC and CFUs are not identical. HSC are no longer considered to Ibe a homogeneous population but an age-structured spectrum of cells (Schofield, 1978). CFUs, which have been identified only in rodents and with certainty only in mice, may be a sub-set of HSC required by mice, perhaps controlled by the W ,locus and virtually eliminated in double W mutants. A dichotomy of CFtJs and HSC has also been demonstrated by Wiktor-Jedrzejczak et al. ,(1977) ; + + bone marrow treated with antiThy 1.2 serum lost its curative properties for W WV anaemic mice with CFUs being unaffected.

The results indicate that We WV marrow containing from 10 to < 1 CFUs was about equally effective as + + l(+Ph) marrow with lo3 to 20 CFUs in curing the radiation syndrome, i.e., in [providing granulocytes, monocytes, ,lymphocytes and platelets in the first weeks after treatment, and in curing the osteopetrosis by providing competent osteoclasts. However, We WV marrow was much less effective in providing red corpuscles at a later time, 2 months. The explanation offered is that grafted HSC from We WV mice, though (perhaps marginally deficient in numbers, are well competent to produce leu’kocytes and platelets on demand, but are inferior to + + HSC in #producing erythrocytes to compete with the few host HS’C surviving the 600 rad (some 300 rad less than the lethal Idose for normal siblings). Residual cells and grafted donor stem cells must co-exist

Table

I. Survival

at 28 days of radiation

chimaeras

based

Loutit et al. : Stem Cells in Osteoclastopoiesis

on mi mi mice.

Genotype

of donated

ww Cell

dose

Deduced

++ content

Survival

Deduced content of CFU

of CFUs 5 1 5 1

x x x x

106 106

10 2 1 0.2

105

105

marrow.

316 414 6110 4110 Total

Survival

1000 200 100 20

9112 616 317 7110 25135

17130

Host mice were inbred stock derived originally from Griineberg or hybrids of this strain with CBA/H into which mi had been incorporated by repeated backcrossing (Loutit and Sansom, 1976). The LDo~ for X-rays (250 kV. 15 mA HVL 1.2 mm Cu) is 600 rad. Donor mice were produced by crossing (C3H/HeH x lOl/H)Fl mice bearing on the one hand + WV (a long established mutant), the + chromosome being marked by Ph, and on the other hand + We (a newly documented mutant) (Cattanach, 1978).

TaMe II. Phosphoglucomutase-I phenotypes blishment of chimaerism (600 rad). Red Host type (PGM-1B) Donor type (PGM-1A) Or mixture PGM-1A

corpuscles

in red

corpuscles

mi

of radiation

mi/WeWv 15

: PGM-18 Total

2 17

- chimaeras

at around

2 months

mimi/+

after

esta-

+

1 18 3 22 (3 not tested

?? )

Phenotypes of red cell phosphoglucomutase-1 were scored after electrophoretic separation followed by histochemical staining, by an unpublished method of Either. 10 pl of washed red cells were lysed in 40 PI of 0.003M EDTA (disodium salt) Ill) presoaked in Tris/glycine buffer pH 8.5 (0.025M and applied four times to a cellulose acetate gel (Helena Titan Trizma base, Sigma ; 0.19M glycine). This buffer was also used as the bridge buffer for electrophoresis which was carried out for 45 minutes at 200V. The staining mixture consisted of 0.4 ml 1M Tris/HCI pH 8.0 ; 0.1 ml 0.2M MgClz 6HzO ; 1 ml NADP (1 mg/ml) ; 0.08 ml glucose-l-phosphate (100 mg/ml) containing at least 1 % glucose 1,6 diphosphate ; 16U glucose; 0.04 ml MTT (10 mg/ml) ; 2 ml 1.5% agar. 6-phosphate dehydrogenase ; 0.04 ml PMS (2.5 mg/ml) The mi mi host mice carry the Pgm-76 allele. The WeWv and + + mice carry the Pgm-la allele. ?? 3 mice were examined at Oswestry quantitatively and serially for red cell phosphoglucose isomerase-1 (Marshall et al.. 1979), giving 1 100% donor type, 2 = 80:20 mixture of donor and host.

J.F. Loutit

et al.

: Stem Cells in Osteoclastopoiesis

References P., Loutit J.F. and Townsend K.M.S. : Osteoclasts derived from haematopoietic stem cells. Nature. 283 : 669-670, 1980. Bennett M.. Cudkowicz G., Foster Jr. R.S. and Metcalf D. : Hematopoietic progenitor cells of W anemic mice studied in viva and in vitro. J. Cell Physiol. 71 : 211-266, 1968. Cattanach B. : Mouse News Letter. 59 : 19, 1978. Harrison D.E. : Lifesparing ability (in lethally irradiated mice) of W/Wv mouse with no microscopic colonies. Radiat. Res. 52 : 553-562. 1972. Loutit J.F. and Nisbet N.W. : Hypothesis - Resorption of bone. Lancet I/ : 26-28, 1979. of Microphthalmic Loutit J.F. and Sansom J.M. : Osteopetrosis mice A defect of the hematopoietic stem cell ? Cakif. Tiss. Res. 210 : 251-259, 1976. Marks S.C. and Walker D.G. : Mammalian osteopetrosis A model for studying cellular and humoral factors in bone resorption. In : The Biochemistry and Physiology of Bone IV, G.H. Bourne. Ed., Academic Press, New York, pp. 227301, 1976. Marshall M.J., Menage P.J. and Nisbet N.W. : The dynamics of red cell chimerism in histoincompatable parabiosed mice. Exp. Hemat. 7 : 425-434, 1979.

Ash

133

McCulloch E.A., Siminovitch L. and Till J.E. : Spleen-colony formation in anemic mice of the genotype W/WV. Science. 144 : 844-846, 1964. Nisbet N.W., Menage J. and Loutit J.F. : Resolution and relapse of osteopetrosis in mice transplanted with myeloid tissue of variable histocompatibility. Transplantation. 28 : 285-290. 1979. Russell E.S. : Hereditary anemias of the mouse : A review for geneticists. Adv. Genet. 20 : 357-459. 1979. Schofield R. : The relationship between the spleen-colony-forming cell and the hematopoietic stem cells : A hypothesis. Blood Cells. 4 : 7, 1978. Searle A.G. and Truslove G.M. : A triple gene in the mouse. Genet. Res. 15 : 227-235, 1970. Walker D.G. : Bone resorption restored in osteopetrotic mice by transplants of normal marrow and spleen cells. Science. 190 : 784-785, 1975. Wiktor-Jedrzejczak W., Sharkis S., Ahmed A. and Sell K.W. : Theta-sensitive cell and erythropoiesis : Identification of a defect in W/WV anemic mice, Science. 196 : 313-315, 1977.

Accepted

:

June

13, 1980_

RESUME Les cellules souches h6matopoi6tiques (CSH) et les - Colony Forming Units (CFUs) = ne sont pas identiques. Ler CSH ne sent plus dCsormais consid&+es comme une population homog&ne, mair comma un specbs de cellules d%ges diff&ents (Schofield, 1978). Les CFUs, qui n’ont CU identiflies que chez la8 rongwrs et seulement avec certitude chew la souris, sont peutCtre une sous-classe de CSH dont a besoin la souris, peut-itre contrdl6es par le locus W, et virtuellement Blknin&ea chew les mutants a double W. Une dichotomie entre les CFUs et les CSH a aussi 6tC montrCe par Wiktor-Jedrzejczak et al. (1977) ; le moelle osseuse + + trait&e par le &rum anti-Thy 1.2 a perdu ses propridtbs curatives pour des souris W WV atimiques dont les CFUs sont affectbes.