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Modification of Rhodamine Staining Allows Identification of Hematopoietic Stem Cells with Preferential Short-Term Or Long-Term Bone Marrow-Repopulating Ability J

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Modification of Rhodamine Staining Allows Identification of Hematopoietic Stem Cells with Preferential Short-Term Or Long-Term Bone Marrow-Repopulating Ability J Proc. Natl. Acad. Sci. USA Vol. 92, pp. 8901-8905, September 1995 Medical Sciences Modification of rhodamine staining allows identification of hematopoietic stem cells with preferential short-term or long-term bone marrow-repopulating ability J. MARK J. M. ZIJLMANS*t, JAN W. M. VISSER*t, KARIN KLEIVERDA§, PHILIP M. KLUIN§, ROEL WILLEMZE*, AND WILLEM E. FIBBE* *Laboratory of Experimental Hematology, Department of Hematology, and §Department of Pathology, University Medical Center, 2300 RC Leiden, The Netherlands; and tLindsley F. Kimball Research Institute, Laboratory of Stem Cell Biology, New York Blood Center, New York, NY 10021 Communicated by J. J. van Rood, University Hospital, Leiden, The Netherlands, June 12, 1995 ABSTRACT We have developed a modified rhodamine In addition to methods exploiting surface marker expres- (]Rho) staining procedure to study uptake and efflux in sion, other techniques are based on functional differences. murine hematopoietic stem cells. Distinct populations of One of these involves the use of rhodamine 123 (Rho), a Rho++ (bright), Rho+ (dull), and Rho- (negative) cells could supravital fluorescent dye that accumulates intracellularly and be discriminated. Sorted Rho- cells were subjected to a that may bind to mitochondria (5, 6). Cells with smaller or less second Rho staining procedure with the P-glycoprotein block- active mitochondria exhibit reduced uptake of Rho (6). Rho ing agent verapamil (VP). Most cells became Rho positive efflux from the cell is mediated by the transmembrane carrier [Rho-/Rho(VP)+ cells] and some remained Rho negative protein P glycoprotein, encoded by the multidrug-resistance [Rho-/Rho(VP)- cells]. These cell fractions were character- gene (7-9). Hydrophobic drugs used in anticancer therapy such ized by their marrow-repopulating ability in a syngeneic, as vinca alkaloids, anthracyclines, and taxanes are extruded sex-mismatch transplantation model. Short-term repopulat- from the cell by P glycoprotein. P glycoprotein-mediated ing ability was determined by recipient survival for at least 6 transport can be blocked by verapamil (VP). Rho uptake weeks after lethal irradiation and transplantation-i.e., ra- studies have been used to purify murine HSCs (10-13). The dioprotection. Long-term repopulating ability at 6 months Rho' (dull) cells representing primitive stem cells exhibited after transplantation was measured by fluorescence in situ MRA, while Rho++ (bright) cells representing more mature hybridization with a Y-chromosome-specific probe, by graft progenitor cells had the ability to form spleen colonies or in function and recipient survival. Marrow-repopulating cells vitro colonies. were mainly present in the small Rho- cell fraction. Trans- Recent developments in HSC characterization have dem- plantation of 30 Rho- cells resulted in 50%o radioprotection onstrated heterogeneity within the cell population having and >80% donor repopulation in marrow, spleen, and thymus MRA. By using counterflow centrifugation, cells with short- 6 months after transplantation. Cotransplantation of cells term repopulating ability (STRA) can be distinguished from from both fractions in individual mice directly showed that cells with long-term repopulating ability (LTRA) (14). Recip- within this Rho- cell fraction, the Rho-/Rho(VP)+ cells ients transplanted with cells mediating LTRA only did not exhibited mainly short-term and the Rho-/Rho(VP)- cells survive lethal irradiation. These cells, however, could sustain exhibited mainly long-term repopulating ability. Our results long-term hematopoiesis ifcotransplanted with cells exhibiting indicate that hematopoietic stem cells have relatively high STRA. In the present study, we have developed a modified P-glycoprotein expression and that the cells responsible for Rho staining method selecting for Rho efflux capacity that long-term repopulating ability can be separated from cells allows identification of distinct populations of Rho++, Rho+, exhibiting short-term repopulating ability, probably by a and Rho- cells. The Rho- cells were further subdivided by a reduced mitochondrial Rho-binding capacity. second Rho staining with the use of VP in Rho-/Rho(VP)+ and Rho-/Rho(VP)- cells. Functional characterization of The lifelong production of mature lymphoid and myeloid these stem cell populations in a syngeneic mouse transplan- blood cells is derived from a small population of primitive tation model demonstrates highly purified cell fractions with hematopoietic stem cells (HSCs). The frequency of these cells preferential STRA or LTRA. in murine bone marrow is estimated to be 1/104-105 (1). HSCs are functionally characterized by their ability to repopulate the MATERIALS AND METHODS bone marrow including the HSC compartment of irradiated recipients (marrow-repopulating ability; MRA). For many Mice. BALB/c mice ranging from 8 to 12 weeks of age were years, purification of HSCs has been pursued, not only for purchased from Broekman (Someren, The Netherlands). Mice biological studies- e.g., susceptibility to hematopoietic growth were housed in laminar flow cabins for the duration of the factors-but also for clinical purposes-i.e., gene therapy, experiment and were fed commercially available rodent chow. transplantation without contaminating tumor cells, or trans- From 1 week before until 6 weeks after total body irradiation, plantation with in vitro amplified stem cells. Methods to purify the drinking fluid was supplemented with ciprofloxacin (85 HSCs rely on positive selection-i.e., binding of the lectin mg/liter) and polymyxin B (70 mg/liter). The experimental wheat germ agglutinin (WGA) (2,3) or expression of the Sca-1 protocol was approved by the institutional ethical committee antigen (4)-combined with depletion techniques based on on animal experiments. the absence of myeloid or lymphoid lineage markers. Using these techniques a 500- to 1000-fold enrichment of HSCs can Abbreviations: HSC, hematopoietic stem cell; Rho, rhodamine 123; be obtained (3, 4). VP, verapamil; MRA, marrow-repopulating ability; STRA, short-term repopulating ability; LTRA, long-term repopulating ability; WGA, wheat germ agglutinin; FISH, fluorescence in situ hybridization. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" in Hematology, University Medical Center Leiden, Building-1 C2R, accordance with 18 U.S.C. §1734 solely to indicate this fact. P.O. Box 9600, 2300 RC Leiden, The Netherlands. 8901 Downloaded by guest on September 27, 2021 8902 Medical Sciences: Zijlmans et al. Proc. Nati. Acad. Sci. USA 92 (1995) Stem Cell Purification. Mice were sacrificed by CO2 as- presence of donor cells in bone marrow (myeloid cells), thymus phyxia. Both femora were flushed using a syringe with a fine (T lymphocytes), and spleen (B lymphocytes). needle. Flushing, incubation, and washing procedures were In some experiments, a competitive repopulation assay was done in RPMI 1640 medium supplemented with 2% fetal done by transplanting female recipients with a combination of bovine serum, penicillin (500 ,ug/ml), and streptomycin (250 male and female cells from different stem cell fractions. In jig/ml) (GIBCO). Bone marrow cells were separated by density- these experiments, peripheral blood samples obtained by tail gradient centrifugation (Ficoll/Isopaque; 1.077 g/cm3). Low bleeding at intervals ranging from 3 to 16 weeks after trans- density cells were labeled with the monoclonal antibody 15-1.1 plantation were assayed for the presence of male donor cells. (rat IgG2b) binding to myelomonocytic cells (15). Cells were washed once and labeled with fluorescein isothiocyanate- RESULTS conjugated WGA (0.2 ,ug/ml; Vector Laboratories) and phy- coerythrin-conjugated goat anti-rat IgG (Caltag, South San Stem Cell Purification. Low density bone marrow cells Francisco, CA). WGA+/15-1.1- cells (±5%) were sorted (8-15%) were labeled with fluorescein isothiocyanate- using a FACStar flow cytometer (Becton Dickinson) equipped conjugated WGA, 15-1.1, and phycoerythrin-conjugated goat with a 5-W argon laser tuned at 488 nm (0.2 W). Sorting gates anti-rat IgG. The WGA+/15-1.1- cell population represented were set such that the WGAdim cells were also included. 4-6% of the cells. Rho staining (100 ng/ml, 20 min, 37°C, WGA+/15-1.1- sorted cells were stained with Rho (Molecular followed by washing twice at room temperature) of WGA+/ Probes) at a concentration of 0.1 ,gg/ml for 20 min at 37°C. 15-1.1- sorted cells allowed identification of Rho++ and Rho+ Cells were washed twice at room temperature, resuspended, cells (Fig. 1A). After incubation in Rho-free medium (37°C), and incubated in medium without Rho for 20 min at 37°C. To an additional Rho- population could be recognized. This improve the distinction between Rho- and Rho' cells, we Rho- cell population comprised 5-8% after 20 min (Fig. 1B) modified the procedure in that an excitation and emission and 30% after 60 min of incubation in Rho-free medium. For wavelength of 514 and 580 nm was used rather than the transplantation experiments, the Rho++, Rho+, and Rho- standard excitation and emission wavelengths of 488 and 530 cells were sorted after 20 min of incubation in Rho-free nm. An unstained control sample was used to set the sorting medium. In some experiments, the Rho- cell population was gate for Rho- cells (99% of unstained cells were within this sorted and restained with Rho. Now, the incubation in Rho- gate). Distinct populations of Rho-, Rho+, and Rho++ cells free medium (20 min, 37°C) was done in the presence of VP, were sorted. In some experiments, sorted Rho- cells (5-8%) with most cells (85-92%) becoming Rho(VP)+ and a minority were stained with Rho again (0.1 ,ug/ml; 20 min; 37°C), washed (8-15%) remaining Rho(VP)-. The Rho-/Rho(VP)+ cells twice at room temperature, and incubated in medium without represented 2-4/104 and the Rho-/Rho(VP)- cells repre- Rho, now in the presence of VP (10 ,uM) for 20 min at 37°C.
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