Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia

Abstract

The interaction of stem cells with their bone marrow microenvironment is a critical process in maintaining normal hematopoiesis. We applied an approach to resolve the spatial organization that underlies these interactions by evaluating the distribution of hematopoietic cell subsets along an in vivo Hoechst 33342 (Ho) dye perfusion gradient. Cells isolated from different bone marrow regions according to Ho fluorescence intensity contained the highest concentration of hematopoietic stem cell (HSC) activity in the lowest end of the Ho gradient (i.e., in the regions reflecting diminished perfusion). Consistent with the ability of Ho perfusion to simulate the level of oxygenation, bone marrow fractions separately enriched for HSCs were found to be the most positive for the binding of the hypoxic marker pimonidazole. Moreover, the in vivo administration of the hypoxic cytotoxic agent tirapazamine exhibited selective toxicity to the primitive stem cell subset. These data collectively indicate that HSCs and the supporting cells of the stem cell niche are predominantly located at the lowest end of an oxygen gradient in the bone marrow with the implication that regionally defined hypoxia plays a fundamental role in regulating stem cell function.

Fig. 1.

Separation of different bone marrow cell (BMC) fractions according to a Ho dye diffusion gradient. (A) Blue versus red fluorescence intensity of BMCs after i.v. infusion of Ho dye (0.8 mg per mouse at 5 and 10 min before killing) with R2-R7 gates representing sorted cell fractions of diminishing fluorescence. (B) CAFC frequencies versus time in culture for the different cell fractions. (C) Early- and late-forming CAFC frequencies as a function of Ho red mean fluorescence intensity (MFI) determined from analysis of postsorted cells. Downward arrows indicate that the CAFC frequencies were below the limit of detection.

Fig. 2.

Long-term repopulation for donor cells (CD45.1) sorted from high (R3) and low (R7) Ho-perfused bone marrow. Sorted bone marrow cells from donor (CD45.1) mice were transplanted into 10 Gy irradiated recipients (CD45.2) along with 200,000 recipient-type, non-SP cells to provide for short-term repopulation. Donor cell chimerism in the recipient mice was analyzed at 18 weeks and 12–14 months posttransplant. (A) Myeloid engraftment in the blood after 18 weeks posttransplant is shown. The number of mice for each cell dose group is indicated. The frequencies of repopulating cells (LTRUs) capable of producing >50% myeloid engraftment with 95% confidence limits (L-Calc program; Stem Cell Technologies) were 67 (27–166), 4.5 (–10), and 1.1 (0.34–3.35) per 10⁶ sorted cells from R7, whole Ho gradient (WBM), and R3 regions, respectively. (B) High (R3) versus low (R7) Ho dye perfused bone marrow populations for frequency of in vivo 12–14 month posttransplant LTRUs. Frequencies of donor cell chimerism in peripheral blood (PB) T cells, PB myeloid cells, bone marrow (BM) c-kit⁺ cells, and BM LTC-CFC are shown. Error bars represent 95% confidence limits.

Fig. 3.

PIM adducts in vivo in bone marrow SP cells. Mice were injected with saline or 120 mg/kg PIM (n = 5), and after 3 h, bone marrow samples were collected and subjected to Ho staining in vitro to isolate SP cells. Representative PIM staining of SP and non-SP cells in the bone marrow is shown. Three different regions of bone marrow cells based on Ho dye efflux, non-SP (R1), top SP (R2), and bottom SP (R3) were sorted. The sorted cells were analyzed for PIM binding by using flow cytometry (A) as well as immunofluorescence microscopy (B). (A) For flow cytometric analysis of PIM binding, the sorted cells were fixed and intracellularly stained by using anti-PIM primary antibody and a goat anti-mouse IgG F(ab′)₂ Alexa Fluor 488 secondary antibody. Similar positive staining of SP cells were obtained in two other separate experiments, including cells stained by using the FITC-conjugated anti-PIM antibody. (B) For visualization of PIM binding by microscopy, cytospin slides of non-SP, top SP, and bottom “tip” SP were immunostained for the adduct. Representative images stained for PIM (green) and nucleus (DAPI, blue) are shown. (Scale bar: 10 μM.)

Fig. 4.

The hypoxic cytotoxin TPZ is highly toxic to thymus and selectively depletes late-forming CAFCs in the bone marrow. TPZ was administered i.p. to C57BL/6J recipients in four daily doses of 30 mg/kg. On the day after the last dose, thymus and bone marrow were harvested and compared with cells obtained from saline-injected control mice. (A) Thymus weight (Left) and cell yield (Right) (trypan blue excluding) were estimated from three separate experiments in mice treated with 4 × 30 mg/kg over 4 days showing marked toxicity of TPZ. (B) Bone marrow cells were pooled from four mice and plated for estimate of CAFC content per hind limb (HL). Error bars represent 95% confidence intervals.