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BRIEF REPORT
From the Terry Fox Laboratory, British Columbia Cancer
Agency, and the Department of Medical Genetics, University of British
Columbia Vancouver, BC, Canada.
Transplantable human hematopoietic stem cells (competitive
repopulating units [CRU]) can be quantitated based on their ability to produce large populations of lymphoid and myeloid progeny within 6 weeks in the marrow of intravenously injected, sublethally irradiated NOD/SCID mice. It is shown that the proportions of total
injected human fetal liver and cord blood CRU in the marrow of mice 24 hours after transplantation are 5% and 7%, respectively, as
determined by limiting-dilution assays in other primary and secondary
NOD/SCID mice. The similarity in these 2 seeding efficiency values
suggests that mechanisms regulating the ability of human hematopoietic stem cells to enter the marrow from the blood, at least in this xenotransplant model, do not change between fetal life and
birth. In addition, it appears that previously reported human stem cell frequencies and their in vivo self-renewal activity measured in NOD/SCID mice have been markedly underestimated.
(Blood. 2000;96:3979-3981) During the lifetime of a person, both lymphoid and
myeloid cells are continuously generated from a small population of
multipotent hematopoietic stem cells.1-3 However, their
low frequency and phenotypic heterogeneity4,5 has made
them a difficult human cell population to study directly. On the other
hand, they can engraft the marrow of sublethally irradiated
immunodeficient mice for prolonged periods, even after the intravenous
injection of limiting numbers of cells. This, in turn, has allowed
their quantification using the in vivo competitive repopulating unit
(CRU) assay,4,6 first developed for murine stem cell
enumeration.7 All repopulation assays likely underestimate
hematopoietic stem cell frequencies for 2 reasons. First, only a
fraction of the injected stem cells would be expected to seed into the
extravascular space of the bone marrow (even when syngeneic or
autologous myelo-ablated hosts are used), and, second, not all might be
stimulated to produce measurable levels of lymphoid and myeloid
progeny. Although the marrow-seeding efficiency of murine repopulating
stem cells in vivo has not yet been measured, the high purities of such
cells that can be obtained (greater than 20%)8,9 indicate
that their in vivo seeding fraction must be at least as high. In
support of this is the demonstration that approximately 20% of
intravenously injected murine week 5 cobblestone area-forming cells
(CAFC-week 5, a closely related cell type detectable in vitro), are
found 24 hours later in the marrow of syngeneic
recipients.10
The purpose of the current experiments was to obtain a direct measure
of the marrow-seeding efficiency of human repopulating stem cells in
sublethally irradiated NOD/SCID mice. This involved measuring the total
number of human stem cells initially injected into a group of primary
test mice (by limiting-dilution analysis of another set of primary mice
injected with much smaller numbers of the same human cells) and
comparing this value with the number of human stem cells in the marrow
of the test mice killed 1 day later (by limiting-dilution analysis of
another set of secondary mice).
Preparation of human cells
Competitive repopulating unit assays
Table 1 shows the CRU
frequencies determined for the low-density human CB and FL cells used
in the current studies. These values, which are similar to previously
published values,4,6,11 were used to calculate the number
of CRU injected at the same time into other recipients of much larger
numbers of the same cells. The latter mice were killed 1 day later, and
the number of human CRU detectable in their marrow was assessed by a
second series of limiting-dilution assays in NOD/SCID mice. These
numbers and the derived marrow seeding efficiencies for human CB and FL CRU in irradiated NOD/SCID mice are summarized in Table
2. The values obtained for human CRU from
both sources were similar (7% for CB and 5% for FL) and also
remarkably close to the value of 4% to 5% reported recently by Van
Hennik et al13 for human CB CAFC-week 6 in NOD/SCID mice
given 9 cGy. The fraction of human CFC that could be recovered from the
marrow of the CB-injected mice (1 experiment) and FL-injected mice (3 experiments) in the current studies (assessed using conditions that are
nonpermissive for murine CFC14) was lower (approximately
2%, data not shown). The corresponding value reported by Van Hennik et
al13 was approximately 4%.
These findings bear out the prediction that human CRU and CAFC/LTC-IC-week 6 would have similar seeding efficiencies in the NOD/SCID xenotransplant model based on a growing body of evidence that the human CRU and CAFC/LTC-IC-week 6 assays detect highly overlapping populations.4,15-17 However, CRU frequencies would also be expected to be at least 10 to 20 times lower because of the in vivo parameters that specifically reduce their efficiency of detection. Thus, in the absence of rigorous quantitative studies, LTC-IC/CAFC-week 6 may be obvious in populations in which CRU appear undetectable. The finding that human CB and FL CRU seed to the marrow of NOD/SCID mice with approximately equal efficiency suggests that the molecular mechanisms regulating this property are similar for these 2 sources of human CRU. This does not preclude the possibility that such mechanisms are subject to variation or that the seeding efficiency and developmental potential of stem cells may vary independently of each other. Interesting examples of populations that show large differences in repopulating activity that cannot be readily explained by changes in cell numbers include stem cells progressing from G0 to G1 and from G1 to S/G2/M.17-20 In addition, brief exposure of murine cells to IL-3 or to IL-3 in combination with SF and IL-12 was found to decrease the in vivo seeding efficiency of CAFC-week 6.10 Conversely, others21-24 have reported enhanced repopulating activity of marrow cells pretreated with these or other cytokines. A 5% to 7% seeding efficiency of CRU in NOD/SCID mice means that their previously reported frequencies represent underestimates by a factor of 14 to 20. This also impacts on previous measurements of human stem cell amplification in NOD/SCID mice (up to 5-fold over input levels at 4 to 6 weeks after transplantation11,25). These would now be seen to represent net increases up to 100-fold and to indicate an ability of human stem cell self-renewal to be potently stimulated in vivo by broadly species cross-reactive mechanisms.
We thank Yvonne Yang for expert secretarial assistance in preparing the manuscript.
Submitted March 20, 2000; accepted July 24, 2000.
Supported by a grant from the National Cancer Institute of Canada (NCIC) with funds from the Terry Fox Run (Toronto, ON) and by grant P01-HL55435 from the National Institutes of Health (Bethesda, MD). C.J.E. was Terry Fox Research Scientist of the NCIC.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Connie J. Eaves, Terry Fox Laboratory, 601 W 10th Ave, Vancouver, BC V5Z 1L3, Canada; e-mail: ceaves{at}bccancer.bc.ca.
1. Prchal JT, Throckmorton DW, Caroll AJ, Fuson EW, Gams RA, Prchal JF. A common progenitor for human myeloid and lymphoid cells. Nature. 1978;274:590-591[Medline] [Order article via Infotrieve]. 2. Raskind WH, Fialkow PJ. The use of cell markers in the study of human hematopoietic neoplasia. Adv Cancer Res. 1987;49:127-167[Medline] [Order article via Infotrieve]. 3. Turhan AG, Humphries RK, Phillips GL, Eaves AC, Eaves CJ. Clonal hematopoiesis demonstrated by X-linked DNA polymorphisms after allogeneic bone marrow transplantation. N Engl J Med. 1989;320:1655-1661[Abstract].
4.
Conneally E, Cashman J, Petzer A, Eaves C.
Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice.
Proc Natl Acad Sci U S A.
1997;94:9836-9841 5. Bhatia M, Bonnet D, Murdoch B, Gan OI, Dick J. A newly discovered class of human hematopoietic cells with SCID-repopulating activity. Nat Med. 1998;4:1038-1045[Medline] [Order article via Infotrieve].
6.
Wang JCY, Doedens M, Dick JE.
Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay.
Blood.
1997;89:3919-3924
7.
Szilvassy SJ, Humphries RK, Lansdorp PM, Eaves AC, Eaves CJ.
Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy.
Proc Natl Acad Sci U S A.
1990;87:8736-8740 8. Wolf NS, Kone A, Priestley GV, Bartelmez SH. In vivo and in vitro characterization of long-term repopulating primitive hematopoietic cells isolated by sequential Hoechst 33342-rhodamine123 FACS selection. Exp Hematol. 1993;21:614-622[Medline] [Order article via Infotrieve].
9.
Spangrude GJ, Brooks DM, Tumas DB.
Long-term repopulation of irradiated mice with limiting numbers of purified hematopoietic stem cells: in vivo expansion of stem cell phenotype but not function.
Blood.
1995;85:1006-1016
10.
Van der Loo JCM, Ploemacher RE.
Marrow- and spleen-seeding efficiencies of all murine hematopoietic stem cell subsets are decreased by preincubation with hematopoietic growth factors.
Blood.
1995;85:2598-2606 11. Holyoake TL, Nicolini FE, Eaves CJ. Functional differences between transplantable human hematopoietic stem cells from fetal liver, cord blood, and adult marrow. Exp Hematol. 1999;27:1418-1427[Medline] [Order article via Infotrieve]. 12. Boggs DR. The total marrow mass of the mouse: a simplified method of measurement. Am J Hematol. 1984;16:277-286[Medline] [Order article via Infotrieve].
13.
van Hennik PB, de Koning AE, Ploemacher RE.
Seeding efficiency of primitive human hematopoietic cells in nonobese diabetic/severe combined immune deficiency mice: implications for stem cell frequency assessment.
Blood.
1999;94:3055-3061
14.
Cashman JD, Lapidot T, Wang JCY, et al.
Kinetic evidence of the regeneration of multilineage hematopoiesis from primitive cells in normal human bone marrow transplanted into immunodeficient mice.
Blood.
1997;89:4307-4316
15.
Conneally E, Eaves CJ, Humphries RK.
Efficient retroviral-mediated gene transfer to human cord blood stem cells with in vivo repopulating potential.
Blood.
1998;91:3487-3493
16.
van Hennik PB, Verstegen MMA, Bierhuizen MFA, et al.
Highly efficient transduction of the green fluorescent protein gene in human umbilical cord blood stem cells capable of cobblestone formation in long-term cultures and multilineage engraftment of immunodeficient mice.
Blood.
1998;92:4013-4022
17.
Gothot A, Van der Loo JCM, Clapp W, Srour EF.
Cell cycle-related changes in repopulating capacity of human mobilized peripheral blood CD34+ cells in non-obese diabetic/severe combined immune-deficient mice.
Blood.
1998;92:2641-2649
18.
Habibian HK, Peters SO, Hsieh CC, et al.
The fluctuating phenotype of the lymphohematopoietic stem cell with cell cycle transit.
J Exp Med.
1998;188:393-398
19.
Oostendorp RAJ, Audet J, Eaves CJ.
High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo.
Blood.
2000;95:855-862 20. Glimm H, Oh I-H, Eaves C. Human hematopoietic stem cells stimulated to proliferate in vitro lose engraftment potential during their S/G2/M transit and do not re-enter G0. Blood. In press. 21. Fabian I, Bleiberg I, Riklis I, Kletter Y. Enhanced reconstitution of hematopoietic organs in irradiated mice, following their transplantation with bone marrow cells pretreated with recombinant interleukin 3. Exp Hematol. 1987;15:1140-1144[Medline] [Order article via Infotrieve].
22.
Tavassoli M, Konno M, Shiota Y, Omoto E, Minguell JJ, Zanjani ED.
Enhancement of the grafting efficiency of transplanted marrow cells by preincubation with interleukin-3 and granulocyte-macrophage colony-stimulating factor.
Blood.
1991;77:1599-1606
23.
Zanjani ED, Ascensao JL, Harrison MR, Tavassoli M.
Ex vivo incubation with growth factors enhances the engraftment of fetal hematopoietic cells transplanted in sheep fetuses.
Blood.
1992;79:3045-3049
24.
Peled A, Petit I, Kollet O, et al.
Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4.
Science.
1999;283:845-848
25.
Cashman JD, Eaves CJ.
Human growth factor-enhanced regeneration of transplantable human hematopoietic stem cells in nonobese diabetic/severe combined immunodeficient mice.
Blood.
1999;93:481-487
© 2000 by The American Society of Hematology.
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  M. Inaba, Y. Adachi, H. Hisha, N. Hosaka, M. Maki, Y. Ueda, Y. Koike, T. Miyake, J. Fukui, Y. Cui, et al. Extensive Studies on Perfusion Method Plus Intra-Bone Marrow-Bone Marrow Transplantation Using Cynomolgus Monkeys Stem Cells, August 1, 2007; 25(8): 2098 - 2103. [Abstract] [Full Text] [PDF]
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|
Q. Li, H. Hisha, R. Yasumizu, T.-X. Fan, G.-X. Yang, Q. Li, Y.-Z. Cui, X.-L. Wang, C.-Y. Song, S. Okazaki, et al. Analyses of Very Early Hemopoietic Regeneration After Bone Marrow Transplantation: Comparison of Intravenous and Intrabone Marrow Routes Stem Cells, May 1, 2007; 25(5): 1186 - 1194. [Abstract] [Full Text] [PDF]
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 |
|
 Y. Liang, G. Van Zant, and S. J. Szilvassy Effects of aging on the homing and engraftment of murine hematopoietic stem and progenitor cells Blood, August 15, 2005; 106(4): 1479 - 1487. [Abstract] [Full Text] [PDF]
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T. Yahata, K. Ando, T. Sato, H. Miyatake, Y. Nakamura, Y. Muguruma, S. Kato, and T. Hotta A highly sensitive strategy for SCID-repopulating cell assay by direct injection of primitive human hematopoietic cells into NOD/SCID mice bone marrow Blood, April 15, 2003; 101(8): 2905 - 2913. [Abstract] [Full Text] [PDF]
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 J. Cashman, B. Dykstra, I. Clark-Lewis, A. Eaves, and C. Eaves Changes in the Proliferative Activity of Human Hematopoietic Stem Cells in NOD/SCID Mice and Enhancement of Their Transplantability after In Vivo Treatment with Cell Cycle Inhibitors J. Exp. Med., November 4, 2002; 196(9): 1141 - 1150. [Abstract] [Full Text] [PDF]
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A. Jetmore, P. A. Plett, X. Tong, F. M. Wolber, R. Breese, R. Abonour, C. M. Orschell-Traycoff, and E. F. Srour Homing efficiency, cell cycle kinetics, and survival of quiescent and cycling human CD34+ cells transplanted into conditioned NOD/SCID recipients Blood, March 1, 2002; 99(5): 1585 - 1593. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
CD19/20+) and myeloid
(CD45/71+CD15/66b+) cells (5 or more of each)
per 2 × 104 propidium iodide [PI] events) in
suspensions prepared from the marrow of femurs and tibias of each
mouse. Human CRU frequencies were then calculated from the proportions
of negative mice (mice not considered positive for both human lymphoid
and myeloid cells) with L-calc software (StemCell), which uses
Poisson statistics and the method of maximum likelihood. Human CRU
numbers per total primary mouse bone marrow were calculated by
multiplying the determined frequency by the total number of cells
recovered from 2 femurs and 2 tibias by 4, on the assumption that 2 femurs and 2 tibias comprise 25% of the total
marrow.