First published online August 30, 2007
Stem Cells
Vol. 25 No.
12
December 2007, pp.
3271
-3272
doi:10.1634/stemcells.2007-0477; www.StemCells.com
© 2007 AlphaMed Press
Severe Combined Immunodeficiency-Repopulating Cell Assay May Overestimate Long-Term Repopulation Ability
Peter A. Horn,
Rainer Blasczyk
Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany
Correspondence: Peter A. Horn, M.D., Institute for Transfusion Medicine, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Telephone: +49 511 532 8704; Fax: +49 511 532 2079; e-mail: Horn.Peter{at}mh-hannover.de
Received on June 20, 2007;
accepted for publication on August 17, 2007.
First published online in STEM CELLS EXPRESS August 30, 2007.
In their recent article [1], Kimura and colleagues describe the identification of long-term repopulating (LTR) potential of human cord blood-derived Lin–CD34–flt3– severe combined immunodeficiency (SCID)-repopulating cells by intra-bone marrow injection (IBMI). The authors demonstrate that the transplantation of Lin–CD34–flt3– cells in a SCID xenograft model by IBMI results in higher engraftment in secondary recipients than that of Lin–CD34+ flt3+ or Lin–CD34+flt3– cells, whereas the transplantation of Lin–CD34–flt3+ cells does not even result in engraftment by IBMI in nonobese diabetic (NOD)/SCID mice as primary recipients. In addition, it is shown that Lin–CD34– cells do not express detectable levels of c-kit. Based on these results, the authors conclude from the data presented "that the immunophenotype of very primitive human long-term repopulating hematopoietic stem cells (LTR-HSC) is Lin–CD34–c-kit–flt3–" and that "further studies will be required to identify positive markers [...] for these primitive human LTR-HSCs in the near future." This conclusion, however, would only be valid based on the prerequisite that no cell population except for true LTR-HSC is readout in the NOD/SCID xenotransplantation model.
However, this assumption is not completely certain, even though it is well accepted that the NOD/SCID xenotransplant model assays "a cell population different from most CFCs and LTC-ICs" [2] (reviewed in [3]) and is therefore regarded a valid test for the assessment of HSC by some. But there is ample evidence that committed progenitors also contribute to engraftment in this model [4–7]. One reason for that is likely the significantly lower proliferative demand placed upon the transplanted cells in a human versus a NOD/SCID mouse as a recipient of a hematopoietic stem cell transplant as well as the relatively short time to readout. After all, LTR-HSC in a human transplantation need to reconstitute hematopoiesis over the lifetime of the recipient, not just for a few months until readout, as is the case even in secondary SCID repopulating cells.
We here suggest a model of the repopulating capacity of cells at different stages in the stem cell hierarchy (Fig. 1) in which an overlap, but not identity, between long-term repopulating cells and SCID repopulating cells (including secondary transplantations) is seen, which is clearly separated from in vitro readouts such as colony-forming cells. This model is supported by the so far sole study in which a direct comparison between SCID repopulating capacity and engraftment after autologous transplantation was performed. When aliquots of the same virally transduced cell population were transplanted in NOD/SCID mice and nonhuman primates, it was clearly demonstrated that NOD/SCID repopulating clones were able to contribute to short-term repopulation in primates by tracking of vector integration sites using a linear amplification mediated-polymerase chain reaction [8]. This result supplied direct evidence that NOD/SCID mice are engrafted by cells also capable of engrafting after autologous transplantation in baboons and thus assay a cell population relevant to the transplantation of higher animal species such as nonhuman primates and most likely also humans. However, none of the NOD/SCID repopulating clones were found to contribute to hematopoiesis at 6 months or later after transplantation [4]. Together with the observation by Glimm et al. that none of more than 50 individual clones tracked by distinct vector insertion sites that were detected in the first month after transplant were active later [9], this suggests that, in nonhuman primates (and possibly also humans), short-term hematopoietic reconstituting cells are distinct from hematopoietic stem cells and that the former contribute significantly to NOD/SCID mice repopulation. These results should not be taken as evidence that long-term engrafting true HSC do not engraft in NOD/SCID mice but rather as evidence that (additional) cells that do not engraft long-term in an autologous transplantation in the baboon were readout by the NOD/SCID xenotransplant model, including secondary transplants [10].
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Figure 1. Model of repopulating potential of different stem cell/progenitor cell populations. According to this model, there is a large overlap, but not identity, between the experimental readouts of long-term repopulating cells in autologous transplantation and SCID repopulating cells. Both are clearly separated from in vitro readouts such as colony-forming cells, which mainly assess later progenitors. Abbreviation: SCID, severe combined immunodeficiency.
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However, it must be kept in mind that the readouts in the NOD/SCID mouse after transplantation with baboon stem and progenitor cells were different from those seen with human stem and progenitor cells. For example, in the NOD/SCID mice that received transplanted baboon cells, predominantly myeloid cells were detected, which is in contrast to NOD/SCID transplantation with human progenitor cells in which mainly B cells are typically detected. This may not be surprising since it seems possible that baboon and human cells have different sensitivities to mouse cytokines.
In addition to the highly limited number of animals frequently resulting in a low statistical power, autologous transplantation in a large animal model is associated with its own potential pitfalls as well. In this model, engraftment by transduced cells is the only possible readout, whereas there is no way to distinguish endogenous recovery, which is not unlikely even after supposedly "myeloablative" conditioning, from activity of infused nontransduced cells. It is therefore possible that a decrease in the percentage of transduced cells in the animal actually represents endogenous recovery competing with the infused graft [11].
After all, it must be kept in mind that the NOD/SCID xenotransplantations, but also autologous transplantations in a large animal, still are just surrogate assays, and results are not necessarily transferable to the human transplantation setting. Claims about HSCs measured by laboratory models should be made carefully and conservatively.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS
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P.A.H. is supported by the Else Kröner-Fresenius-Stiftung (Grant number: P 59/05//EKMS 05/25).
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REFERENCES
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- Kimura T, Asada R, Wang J et al. Identification of long-term repopulating potential of human cord blood-derived CD34-flt3- severe combined immunodeficiency-repopulating cells by intra-bone marrow injection. STEM CELLS 2007;25:1348–1355.[Abstract/Free Full Text]
- Larochelle A, Vormoor J, Hanenberg H et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: Implications for gene therapy. Nat Med 1996;2:1329–1337.[CrossRef][Medline]
- Coulombel L. Identification of hematopoietic stem/progenitor cells: Strength and drawbacks of functional assays. Oncogene 2004;23:7210–7222.[CrossRef][Medline]
- Horn PA, Thomasson BM, Wood BL et al. Distinct hematopoietic stem/progenitor cell populations are responsible for repopulating NOD/SCID mice compared with nonhuman primates. Blood 2003;102:4329–4335.[Abstract/Free Full Text]
- Ramirez M, Rottman GA, Shultz LD et al. Mature human hematopoietic cells in donor bone marrow complicate interpretation of stem/progenitor cell assays in xenogeneic hematopoietic chimeras. Exp Hematol 1998;26:332–344.[Medline]
- 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.[Abstract/Free Full Text]
- van Hennik PB, Verstegen MM, Bierhuizen MF 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.[Abstract/Free Full Text]
- Schmidt M, Zickler P, Hoffmann G et al. Polyclonal long-term repopulating stem cell clones in a primate model. Blood 2002;100:2737–2743.[Abstract/Free Full Text]
- Glimm H, Schmidt M, Fischer M et al. Efficient marking of human cells with rapid but transient repopulating activity in autografted recipients. Blood 2005;106:893–898.[Abstract/Free Full Text]
- Horn PA, Kiem HP. Expansion of SCID repopulating cells does not prove expansion of hematopoietic stem cells. Blood 2006;108:771–771.[Free Full Text]
- Tisdale JF. Modeling human hematopoiesis: Of mice and monkeys. Blood 2003;102:4254–4254.[Free Full Text]
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