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Reply to letter by Weissman et al.
Immunology Today, Vol. 10, No. 6, 1989
Reply to letter by Weissman et aL Sir, For whatever reasons, the article by Spangrude et al. 1 received wide press coverage. Indeed, we took the title of our commentary 2 from a Science editorial in which Weissman is quoted a; saying: "This is the end of the road that was the search for the stem cell." We naturally asked if this (and the more sensational claims, not of course attributed directly to the scientists concerned) was justified. The commentary, however, was based solely on our understanding of the data presented in the paper under consideration and was not influenced by articles in the popular press. We apologize if our commentary caused distress to the authors; it certainly was not meant to. In fact, we clearly stated that many of the claims made in the article were fully justified and that the paper contained valuable information regarding the simultaneous myeloerythroid and lymphoid developmental potential of the appropriate cell population. Having said this, some of our conclusions m~y perhaps have been influenced by insufficient detail in the original manuscript. For example, Fig. 4 (Ref. 1) shows that th~ tractionated ceiis can give rise to T cells, B cells and neutrophils. This was for only one time point and it was not stated if this applied to all mice injected with the cells. Subsequently, Spangrude et al. showed that when several mice were injected with 100 Thv-110 Lin- Sca-1+ cells and examined 8-12 weeks later 80% of
B
the animals contained donor-derived T cells. No data was given for myeloid cells but parity was implied by the subsequent statement that survivors of (potentially) lethal radiation were almost completely reconstituted in the T, B, granulocyte, macrophage lineages. As we stressed at the time, longer intervals are probably necessary to establish the fidelity and origin of reconstitution. Furthermore, we certainly did not suggest that all CFU-S12 are equivalent to haematopoietic stem cells. It may be pointed out however, that Spangrude et al., in fact, presented no evidence that the sorted cells could give rise to CFC-S12. By definition, the cells are CFC-S12 because they generate colonies at !2 days. An earlier cell was implied by Mulder and Vissers using ,,,uu..m.'~ -'--" ....... 123 labelling and a more recent paper by Ploemacher and Brons6 clearly identifies an earlier popuiation of cells which does generate CFC-S~2. Unfortunately, tota/recovery of d 12 CFU-S after the fractionation procedures used was not g!ven in the original article by Spangrude eta/. With this information, it would be possible to estimate the percentage of d12 CFU-S which possess the characteristics under examination. Final!y, a word about 'enrichm,ent'. ;n the mouse ~train used~. there was a relatively low concentration of d12 CFU-S (about 15/10 s bone marrow cells). This must be compared with other laboratories (see Ref. 3 for example) where d12 CFU-S are present in the order of 50/10 s bone marrow cells. Clearly, enrichment of d12 CFU-S (or for any other cell population) cannot exceed
~" B
purity. Thus, the enrichments obtained in the two studies mentioned 3.4 give comparably 'pure' populations (at least in terms of d12 CFU-S) as the 1000-fold enrichment in Fig. 2 of the study of Spangrude et a/. (see Table 1). In other words, since cla;ms based upon enrichment values reflect the concentration of cells in the starting population, it is unfair to claim that the Thy-1 ~° LinSca-1 + cells are 5-fold enriched compared with other studies. We hope that these comments may in some way help to avert a diplomatic incident; Irving Weissman can perhaps be reassured by the fact that we have not been invited a second time by immun ;logy Today to comment on their more recent paper.
T.M.Dexter B.l.Lord PatersonInstitute for CancerResearch, ChrsstieHospitaland Holt RadiumInstitute, Wilmslow Road, ManchesterM20 9BK, UK.
References 1 Spangrude,G.J., Heimfeld, S. and Weissman, I.L.(1988) Science 241, 58-62 2 Lord, B.I. and Dexter,T.M. (1988) Immunol. Today9, 376-377 Visser, JWM, Bo.umaP,J.G.. Mulder A.H., Eliasor.,J.F.and de Leeuw, A.M. (1984)2. Exp. Med. 59, 1576-1590 4 Lord, B.I. and Spooncer, E. (1986) Lymphokine Res. 5, 59-72 5 Mulder. A.H. and Visser,J.W.M. (1987) Exp. Hematol. 15, 99-104 6 Ploemacher,R.E.and Brons, N.H.C. (1988)J. Cell. Physiol. 136, 531-536 7 Lord, B.I., Hendry,J.H., Keene,J.P.et al. (1984) Cell Tissue Kinet. 17, 323-334
Tabie 1. Relativeenrichmentsof day-12CFC-S Source
CFU-S/IOs
f No.a
CFC-S/105
Relativeenrichment Enrichment Relativeinitial factor CFC-Sconcentration
Spangrude etal. (1988) (Fig.2)
U F
15 9200
0.1
150 9.2 x 104
613
1.0
613(1.4x)
Lordand Spooncer (1986) (Table1)
U F
30.5 5890
0.06
484 9.4 x 104
193
3.23
623 (1.4x)
Visser U 49 0.1 490 125 3.27 etal. (1984) F 0600 6.6 x 104 (Table1) aThelowerseedingefficiency(f No.) usedfor the Lordand Spooncerdata is the resultof low dose-rateirradiation;. U: unfractionatedmarrow; F: fractionatedmarrow. (~ 1989, Elsevier Science PublishersLtO,UK. 0167-4919/89/50";.00
441 (1.0x)
185
Reply to letter by Weissman et aL Sir, For whatever reasons, the article by Spangrude et al. 1 received wide press coverage. Indeed, we took the title of our commentary 2 from a Science editorial in which Weissman is quoted a; saying: "This is the end of the road that was the search for the stem cell." We naturally asked if this (and the more sensational claims, not of course attributed directly to the scientists concerned) was justified. The commentary, however, was based solely on our understanding of the data presented in the paper under consideration and was not influenced by articles in the popular press. We apologize if our commentary caused distress to the authors; it certainly was not meant to. In fact, we clearly stated that many of the claims made in the article were fully justified and that the paper contained valuable information regarding the simultaneous myeloerythroid and lymphoid developmental potential of the appropriate cell population. Having said this, some of our conclusions m~y perhaps have been influenced by insufficient detail in the original manuscript. For example, Fig. 4 (Ref. 1) shows that th~ tractionated ceiis can give rise to T cells, B cells and neutrophils. This was for only one time point and it was not stated if this applied to all mice injected with the cells. Subsequently, Spangrude et al. showed that when several mice were injected with 100 Thv-110 Lin- Sca-1+ cells and examined 8-12 weeks later 80% of
B
the animals contained donor-derived T cells. No data was given for myeloid cells but parity was implied by the subsequent statement that survivors of (potentially) lethal radiation were almost completely reconstituted in the T, B, granulocyte, macrophage lineages. As we stressed at the time, longer intervals are probably necessary to establish the fidelity and origin of reconstitution. Furthermore, we certainly did not suggest that all CFU-S12 are equivalent to haematopoietic stem cells. It may be pointed out however, that Spangrude et al., in fact, presented no evidence that the sorted cells could give rise to CFC-S12. By definition, the cells are CFC-S12 because they generate colonies at !2 days. An earlier cell was implied by Mulder and Vissers using ,,,uu..m.'~ -'--" ....... 123 labelling and a more recent paper by Ploemacher and Brons6 clearly identifies an earlier popuiation of cells which does generate CFC-S~2. Unfortunately, tota/recovery of d 12 CFU-S after the fractionation procedures used was not g!ven in the original article by Spangrude eta/. With this information, it would be possible to estimate the percentage of d12 CFU-S which possess the characteristics under examination. Final!y, a word about 'enrichm,ent'. ;n the mouse ~train used~. there was a relatively low concentration of d12 CFU-S (about 15/10 s bone marrow cells). This must be compared with other laboratories (see Ref. 3 for example) where d12 CFU-S are present in the order of 50/10 s bone marrow cells. Clearly, enrichment of d12 CFU-S (or for any other cell population) cannot exceed
~" B
purity. Thus, the enrichments obtained in the two studies mentioned 3.4 give comparably 'pure' populations (at least in terms of d12 CFU-S) as the 1000-fold enrichment in Fig. 2 of the study of Spangrude et a/. (see Table 1). In other words, since cla;ms based upon enrichment values reflect the concentration of cells in the starting population, it is unfair to claim that the Thy-1 ~° LinSca-1 + cells are 5-fold enriched compared with other studies. We hope that these comments may in some way help to avert a diplomatic incident; Irving Weissman can perhaps be reassured by the fact that we have not been invited a second time by immun ;logy Today to comment on their more recent paper.
T.M.Dexter B.l.Lord PatersonInstitute for CancerResearch, ChrsstieHospitaland Holt RadiumInstitute, Wilmslow Road, ManchesterM20 9BK, UK.
References 1 Spangrude,G.J., Heimfeld, S. and Weissman, I.L.(1988) Science 241, 58-62 2 Lord, B.I. and Dexter,T.M. (1988) Immunol. Today9, 376-377 Visser, JWM, Bo.umaP,J.G.. Mulder A.H., Eliasor.,J.F.and de Leeuw, A.M. (1984)2. Exp. Med. 59, 1576-1590 4 Lord, B.I. and Spooncer, E. (1986) Lymphokine Res. 5, 59-72 5 Mulder. A.H. and Visser,J.W.M. (1987) Exp. Hematol. 15, 99-104 6 Ploemacher,R.E.and Brons, N.H.C. (1988)J. Cell. Physiol. 136, 531-536 7 Lord, B.I., Hendry,J.H., Keene,J.P.et al. (1984) Cell Tissue Kinet. 17, 323-334
Tabie 1. Relativeenrichmentsof day-12CFC-S Source
CFU-S/IOs
f No.a
CFC-S/105
Relativeenrichment Enrichment Relativeinitial factor CFC-Sconcentration
Spangrude etal. (1988) (Fig.2)
U F
15 9200
0.1
150 9.2 x 104
613
1.0
613(1.4x)
Lordand Spooncer (1986) (Table1)
U F
30.5 5890
0.06
484 9.4 x 104
193
3.23
623 (1.4x)
Visser U 49 0.1 490 125 3.27 etal. (1984) F 0600 6.6 x 104 (Table1) aThelowerseedingefficiency(f No.) usedfor the Lordand Spooncerdata is the resultof low dose-rateirradiation;. U: unfractionatedmarrow; F: fractionatedmarrow. (~ 1989, Elsevier Science PublishersLtO,UK. 0167-4919/89/50";.00
441 (1.0x)
185