While investigators in a number of laboratories have documented that the hematopoietic stem cells (HSCs) of fetal and adult mice are CD38+, no information is available about CD38 expression by HSCs of newborn and juvenile mice. We used a murine transplantation model to examine HSC CD38 expression. First, we observed that all HSCs from newborn bone marrow are CD38−. Next, it was determined that the majority of HSCs in the bone marrow of 5-week-old mice are CD38−, with a minority being CD38+. These observations indicated that the CD38+ subpopulation of HSC appears before the age of 5 weeks and expands during adolescence. However, the majority of HSCs of 5-week-old mice became CD38+ following injection of 5-fluorouracil, indicating that activation of juvenile stem cells enhances CD38 expression. These observations may have implications for CD38 expression by HSCs from human umbilical cord blood and bone marrow of young children in steady state and under pathological conditions.
Characterization of the surface phenotypes of hematopoietic stem cells (HSCs) and development of techniques for their purification are important subjects in stem cell transplantation and gene therapy. Transplantation of purified human HSCs that are free of T lymphocytes could minimize graft-versus-host disease in allogeneic transplantation. In autologous situations, transplantation of HSCs that are free of tumor cells may reduce the risk of recurrence of malignancies.
For many years, CD34 and CD38 have been regarded as important markers for HSC. It was generally accepted that human HSCs and primitive progenitors are CD34+CD38−/low,1 however, recent studies of murine stem cells caused controversies about surface phenotypes of HSCs. It was documented that the majority of adult murine HSCs are CD34−.2,3,4 In our laboratory, we observed that the developmental change of CD34 expression from positive to negative state takes place between 7 and 10 weeks of age for most of murine HSCs.5 We also discovered that CD34 expression by adult stem cells is influenced by the activation state of the HSCs.2 Regarding stem cell CD38 expression, investigators in four different laboratories including ours reported that HSCs in yolk sac, fetal liver and adult mouse bone marrow express CD38.6,7,8,9 We demonstrated that activation of adult murine HSCs causes reversible changes in CD38 expression.6
Unknown has been the state of CD38 expression by HSCs of newborn or juvenile mice. This question has significant clinical relevance because cord blood cells recently have emerged as an important source for stem cell transplantation.10 In this paper, we used a mouse transplantation model to examine CD38 expression by HSCs in the bone marrow of newborn and 5-week-old mice.
Materials and methods
Fluorescein isothiocyanate (FITC)-conjugated NIM-R5 (anti-CD38; rat IgG2a) was purchased from Southern Biotechnology Associates (Birmingham, AL, USA), biotin-conjugated RAM34 (anti-CD34; rat IgG2a), FITC-conjugated A20 (anti-Ly-5.1; rat IgG2a), phycoerythrin (PE)-conjugated RB6-8C5 (anti-Gr-1; rat IgG2b), PE-conjugated 53-2.1 (anti-Thy-1.2; rat IgG2a), PE-conjugated RA3-6B2 (anti-CD45R/B220; rat IgG2a) from BD Pharmingen (San Diego, CA, USA), and PE-conjugated M1/70 (anti-Mac-1; rat IgG2b), streptavidin-conjugated PE from Caltag Laboratories (San Francisco, CA, USA).
C57BL/6-Ly-5.1 mice originally obtained from Jackson Laboratories (Bar Harbor, ME, USA) and bred in the Animal Research Facility of The Veterans Affairs Medical Center were used as newborn and juvenile donors. Ten to 14-week-old C57BL/6-Ly-5.2 female mice (Charles River Laboratories, Raleigh, NC, USA) were used as irradiated recipients and as the source for radioprotective cells. In some experiments, the juvenile donor mice were treated with an intravenous injection of 5-fluorouracil (5-FU) at 100 mg/kg body weight 48 h before death. Bone marrow cells from Ly-5.1 mice were flushed from femurs and tibiae, pooled, and washed twice with phosphate-buffered saline containing 0.1% bovine serum albumin. The samples were then made into single cell suspension by repeated pipetting and filtering through 40 μm nylon mesh. The cells with densities ranging from 1.063 to 1.077 g/ml were collected by gradient separation using Nycodenz (Accurate Chemical and Scientific Corp, Westbury, NY, USA). The resulting mononuclear cells (MNCs) were stained with FITC-conjugated anti-CD38 and biotin-conjugated anti-CD34 followed by streptavidin PE. After addition of propidium iodide at a concentration of 1 μg/ml, the cells were washed twice, resuspended in phosphate-buffered saline containing 0.1% bovine serum albumin and kept on ice until cell sorting. Fluorescence-activated cell sorting (FACS) was performed on a FACS Vantage (Becton Dickinson, San Jose, CA, USA), with appropriate isotype-matched controls.
Ly-5.2 mice were administered a single 950-cGy dose of total body irradiation using a 4 × 106 V linear accelerator. Sorted Ly-5.1 cells were injected into the tail vein of the irradiated Ly-5.2 mice along with 2 × 105 bone marrow cells from adult Ly-5.2 mice as radioprotective cells. In all experiments, the number of cells transplanted reflected the ratios of cells in the sorting windows. For analysis of engraftment, peripheral blood was obtained from the retro-orbital plexus using heparin-coated micropipets (Drummond Scientific, Broomall, PA, USA). The samples were stained with FITC-conjugated anti-Ly-5.1 and red blood cells were lysed using FACS Lysing Solution (Becton Dickinson). Analysis for donor-derived cells was carried out on a FACS Calibur (Becton Dickinson). Donor (Ly-5.1) cells in granulocyte and monocyte/macrophage, T-cell, and B-cell lineages were analyzed by staining with PE-conjugated anti-Gr-1 and PE- conjugated anti-Mac-1, PE-conjugated anti-Thy-1.2 and PE-conjugated anti-B220, respectively.
Data presentation and statistical analysis
The number of repopulating units (RU) in each test samples was calculated from the percentage donor cells engrafted, D, according to the formula: RU = competitor RU × D/(100−D), described by Harrison et al.11 After donor RU were calculated relative to RU of 2 × 105 bone marrow cells in each recipients, mean ± s.d. percentages of RU in the CD38− and CD38+ cell populations were computed. Levels of significance were determined using the Student's t-test.
CD38 expression by HSCs of newborn mice
Earlier, we and other investigators documented that all HSCs of 5-week-old and younger mice are CD34+.5,12 We therefore tested the engraftment capabilities of CD38− and CD38+ subpopulations of CD34+ bone marrow cells of newborn mice using the FACS regions shown in Figure 1. Because the ratio of the CD34+CD38− (R1) cells to CD34+CD38+ (R2) cells was 20:1, 9.0 × 103 CD34+CD38− or 4.5 × 102 CD34+CD38+ cells were transplanted into each recipient mouse. The levels of engraftment at 6 months after transplantation, determined by measuring the percentages of donor (Ly-5.1)-derived peripheral blood nucleated cells are shown in Figure 2. Only CD34+CD38− cells engrafted. The evidence for multilineage engraftment in individual mice is presented in Table 1.
CD38 expression by HSCs of 5-week-old mice
Next, we analyzed the CD38 expression by long-term engrafting cells of 5-week-old mice. Because we and other investigators documented that all bone marrow HSCs of 5-week-old and younger mice are CD34+,5,12 we set electronic sorting gates as shown in Figure 3. Since the ratio of the CD34+CD38− (R3) cells to the CD34+CD38+ (R4) cells was 5:1, we transplanted 8.0 × 104 CD34+CD38− cells or 1.6 × 104 CD34+CD38+ cells per mouse. The levels of engraftment are presented in Figure 4. Mice that had been transplanted with CD34+CD38− cells showed higher engraftment levels (18.9 ± 12.7%) than mice transplanted with CD34+CD38+ cells (2.8 ± 2.0%) (P < 0.005). Multilineage engraftment was seen in all mice. Calculation of RU indicated that the majority (90%) of the long-term engrafting cells are CD38− in the bone marrow of 5-week-old mice.
CD38 expression by HSCs of 5-FU-treated 5-week-old mice
We have shown in previous studies that activated adult HSCs were CD38−.6 To determine whether there are similar effects on stem cells in 5-week-old mice, we tested CD38 expression by HSCs of 5-week-old mice following 5-FU injection. Because the effects of 5-FU injection on the expression of CD34 by HSCs in 5-week-old mice are not known, we separated bone marrow cells on the basis of CD38 expression alone. The sorting regions are presented in Figure 5. Since the ratio of the CD38− (R5) to CD38+ (R6) cell populations in the bone marrow from 5-FU-treated mice was 1:1, we transplanted 3 × 105 cells of both populations per mouse. The average engraftment levels of the recipients transplanted with CD38− cells and CD38+ cells were 10.4 ± 9.0% and 43.8 ± 14.7% at 6 months post-transplantation, respectively (Figure 6). These values were different at P < 0.002. Again, multilineage engraftment was seen in all recipient mice.
In this report, we studied CD38 expression by HSCs in the bone marrow of newborn and 5-week-old mice. The results clearly demonstrated that all HSCs of newborn mice and the majority of HSCs in the bone marrow of 5-week-old mice are CD38−. We also observed that CD38+ marrow cells from 5-FU-treated 5-week-old mice show higher engraftment levels than CD38− cells. The latter observation indicated that activation of juvenile HSCs enhances CD38 expression. This direction of changes is opposite to that of the changes seen in adult HSCs.6
Earlier, Randall et al7 demonstrated that only CD38high population, but not CD38− population, in Sca1+, c-kit+, Linlow/− liver cells from 14.5 days post coitum (dpc) mice and adult mice are capable of long-term hematopoietic reconstitution. Dagher et al8 also demonstrated that only CD38+ population in c-kit+, Lin− yolk sac cells derived from 9 dpc mice, as well as adult mice are capable of long-term reconstitution. Based on these observations, it was concluded that CD38 is expressed by HSCs throughout murine ontogeny.8 However, our results clearly indicated that all HSCs of newborn mice and the majority of HSCs in the bone marrow of 5-week-old mice are CD38−. The first ontogenetic conversion of HSC CD38 expression from CD38+ to CD38− state must take place between 14.5 dpc and the perinatal period. Earlier, we documented that a minority of adult HSCs are CD38−.6 It appears that the second ontogenetic conversion from CD38− to CD38+ stem cells begins before the age of 5 weeks.
In earlier reports from our laboratory,2,5 we described that CD34, another important surface molecule of HSCs, is also under developmental control and affected by the activation state of HSCs. HSCs in fetal and juvenile mice are CD34+.5 The change of HSC CD34 expression from the positive to negative state takes place between 7 and 10 weeks of age for the majority of murine HSCs.5 The physiological significance of the developmental change in stem cell CD34 expression is unclear because genetically engineered CD34-null mice show normal hematopoiesis in vivo.13
The physiological significance of the bidirectional ontogenic changes in CD38 expression of HSCs we described here is also unknown. CD38 is an ectoenzyme that utilizes NAD+ (nicontinamide adenine dinucleotide) and is expressed by many hematopoietic cells.14 However, CD38-null mice exhibited a normal compartment of all hematopoietic lineages,15 indicating that CD38 is dispensable for lymphohematopoiesis. It is possible that the absence of stem cell abnormalities seen in the CD38- and CD34-null mice may be due to the presence of other molecules with functional redundancy. Both CD38 and CD34 are considered to be adhesion molecules.13,14 The fact that they are ontologically regulated may indicate an important function that has yet to be elucidated.
Murine CD38 cDNA shows 70% sequence homology with that of human CD38.16 Thus, our observations in a mouse model may possess implications for CD38 expression by HSCs from human umbilical cord blood and the bone marrow of young children under steady-state and pathological conditions.
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This work was supported by NIH grants RO1-DK54197 and PO1-CA78582 and by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs. The authors thank Anne G Livingston, Karen A Rivers and Dr Tsukasa Higuchi for assistance in preparation of this manuscript, and the staff of Radiation Oncology Department of the Medical University of South Carolina for assistance in irradiation of the mice.
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Higuchi, Y., Zeng, H. & Ogawa, M. CD38 expression by hematopoietic stem cells of newborn and juvenile mice. Leukemia 17, 171–174 (2003) doi:10.1038/sj.leu.2402785
- hematopoietic stem cells
- developmental changes
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