Visualizing and analyzing Hematopoietic Stem Cell niches in situ

Hematopoietic stem cells reside in specific bone marrow niches, which are thought to be low in oxygen content. We employed advanced quantitative imaging techniques to comprehensively analyze the spatial distribution of stem and progenitor cells in the bone marrow, describe their interaction with distinct types of blood vessels, and determine that their characteristic hypoxic status is not dictated by localization in specific regions but is rather physiologically inherent to the hematopoietic stem cell pool.

HFSP Long-Term Fellow César Nombela-Arrieta and colleagues
authored on Mon, 13 May 2013

Hematopoietic Stem and Progenitor Cells have been proposed to strategically position in defined anatomical locations within the bone marrow stroma. These specialized niches provide hematopoietic stem cells with the necessary signals to maintain their properties. A clear understanding of the precise localization of stem cells within the intricate BM stroma is greatly hampered by the difficulties associated with imaging bone-enclosed tissues, the extremely low frequency of hematopoietic stem cell populations and the markers needed to discriminate them from the plethora of hematopoietic and non-hematopoietic cell subsets that populate the bone marrow parenchyma.


Figure: a. Image of a whole longitudinal bone marrow section stained with the nuclear die DAPI as it is scanned using Laser Scanning Cytometry. High-resolution images of the entire section are generated in different fluorescent channels by exciting with multiple lasers. The position of individual cells is recorded. b. Example of a three-dimensional reconstruction of confocal image stacks of bone marrow blood vessels (green) and c-kit+ hematopoietic progenitors. c. Examples of c-kit+Sca-1+ hematopoietic progenitors displaying a hypoxic status based on pimonidazole incorporation (top) and hypoxia inducible transcription factor (bottom), while lying adjacent to blood vessels (Laminin).

We took advantage of a powerful quantitative imaging technology, Laser Scanning Cytometry, to scan entire longitudinal sections of femoral bone marrow cavities, automatically identify hematopoietic stem and progenitor cells based on the selective expression of fluorescently labeled proteins, and therefore determine their spatial distribution within the bone marrow stroma. This comprehensive mapping revealed that stem cells are enriched in tissue areas close to inner bone surfaces (endosteum), decreasing in frequency towards bone distal regions. The majority of these cells were found to directly interact with different types of blood vessels, including arteries and sinusoids (specialized microvessels which constitute the venous equivalent of the bone marrow microvascular compartment). By using a novel experimental protocol we provide a three dimensional overview of the specialized and heterogeneous bone marrow vascular network.

Importantly, hematopoietic stem cells had been proposed to localize in areas where the oxygen content was minimal in the bone marrow (hypoxic), which was in apparent contradiction with the fact that they reside in close proximity to vascular structures where oxygen is exchanged from the blood into tissues. The hypoxic nature of hematopoietic stem cells was proposed based on their selective incorporation of pimonidazole, a probe that labels cells with low oxygen content, their expression of the hypoxia inducible transcription factor (HIF-1α), and their use of anaerobic glycolysis as the principal metabolic route to maintain cellular bioenergetics. Using imaging cytometry, we revealed that hematopoietic stem cells conserved this so-called hypoxic profile independently of their localization in the bone marrow and in other organs such as the spleen. Therefore, our results challenge the hypothesis of the super hypoxic niche, and open up exciting questions regarding the physiological relevance and molecular mechanisms that preserve the characteristic metabolic profile of hematopoietic stem cells.

Reference

Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment. Nombela-Arrieta C, Pivarnik G, Winkel B, Canty KJ, Harley B, Mahoney JE, Park SY, Lu J, Protopopov A, Silberstein LE. Nat Cell Biol. 2013 May;15(5):533-43. doi: 10.1038/ncb2730. Epub 2013 Apr 28.

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