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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.
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© 2000 by The American Society of Hematology.
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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.