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Targeting hematopoietic stem cells

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

New technology uncovered the stem cell niche in the bone marrow

Hematopoietic stem cells (HSCs) are so rare that it’s difficult to comprehensively localize dividing and non-dividing HSCs. Thus, there has controversy about their specific location in the bone marrow. A recent Nature publication reported that the HSCs resides mainly in perisinusoidal niches through out the bone marrow and there are no spatially distinct niches for dividing and non-dividing blood-forming stem cells. This group of researchers at UT Southwestern Medical Center started the generation of a GFP knock-in for the gene Ctnnal1, a generic marker for HSCs in mice (α-catulin^GFP mice) and confirmed that α-catulin-GFP⁺c-kit⁺ cells represent blood-forming HSCs by showing that α-catulin-GFP⁺c-kit⁺ cells gave long term multi-lineage reconstitution of irradiated mice. Using a tissue-clearing technique and deep confocal imaging, they were able to image thousands of α-catulin-GFP⁺c-kit⁺ cells and see their relation to other cells. This publication improved the understanding of the microenvironment of HSCs in the bone marrow, which would significantly improve the safety and effectiveness of bone marrow transplantation.

Melih Acar, etc. (October 2015) Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal. Nature

 

Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal

M Acar, KS. Kocherlakota, MM. Murphy, JG. Peyer, H Oguro, CN. Inra, C Jaiyeola, Z Zhao, K Luby-Phelps & Sean J. Morrison
Nature526,126–130(01 October 2015)           doi:10.1038/nature15250

 

Haematopoietic stem cells (HSCs) reside in a perivascular niche but the specific location of this niche remains controversial¹. HSCs are rare and few can be found in thin tissue sections^{2, 3} or upon live imaging⁴, making it difficult to comprehensively localize dividing and non-dividing HSCs. Here, using a green fluorescent protein (GFP) knock-in for the gene Ctnnal1 in mice (hereafter denoted as α–catulin^GFP), we discover that α–catulin^GFP is expressed by only 0.02% of bone marrow haematopoietic cells, including almost all HSCs. We find that approximately 30% of α–catulin−GFP⁺c-kit⁺ cells give long-term multilineage reconstitution of irradiated mice, indicating thatα–catulin−GFP⁺c-kit⁺ cells are comparable in HSC purity to cells obtained using the best markers currently available. We optically cleared the bone marrow to perform deep confocal imaging, allowing us to image thousands of α–catulin–GFP⁺c-kit⁺ cells and to digitally reconstruct large segments of bone marrow. The distribution of α–catulin–GFP⁺c-kit⁺ cells indicated that HSCs were more common in central marrow than near bone surfaces, and in the diaphysis relative to the metaphysis. Nearly all HSCs contacted leptin receptor positive (Lepr⁺) and Cxcl12^high niche cells, and approximately 85% of HSCs were within 10 μm of a sinusoidal blood vessel. Most HSCs, both dividing (Ki-67⁺) and non-dividing (Ki-67⁻), were distant from arterioles, transition zone vessels, and bone surfaces. Dividing and non-dividing HSCs thus reside mainly in perisinusoidal niches with Lepr⁺Cxcl12^high cells throughout the bone marrow.

 

Figure 1: Deep imaging of α–catulin−GFP⁺ HSCs in digitally reconstructed bone marrow.close

 

 

Extended Data Figure 3: α–catulin−GFP expression among haematopoietic cells is highly restricted to HSCs.

 

 

a, The frequency of α–catulin−GFP⁺ bone marrow cells in negative control α–catulin^+/+ (WT) mice and α-catulin^GFP/+ mice (n = 14 mice per genotype in 11 independent experiments). In all cases in this figure, percentages refer to the frequency of each population as a percentage of WBM cells. b, α–catulin−GFP⁺c-kit⁺ cells from Fig. 1b are shown (blue dots) along with all other bone marrow cells in the same sample (red dots). c, CD150⁺CD48⁻LSK HSCs express α–catulin−GFP but CD150⁻CD48⁻LSK MPPs do not (n = 17 mice in 12 independent experiments). A minority of the α–catulin−GFP⁺c-kit⁺ cells had high forward scatter, lacked reconstituting potential, and were gated out when isolating HSCs by flow cytometry and when identifying HSCs during imaging (see Extended Data Fig. 5for further explanation). d, Lin⁻c-kit^lowSca-1^lowCD127⁺CD135⁺ common lymphoid progenitors (CLPs), Lin⁻c-kit⁺Sca-1⁻CD34⁺CD16/32⁻ common myeloid progenitors (CMPs), Lin⁻c-kit⁺Sca-1⁻CD34⁺CD16/32⁺ granulocyte-macrophage progenitors (GMPs), and Lin⁻c-kit⁺Sca-1⁻CD34⁻CD16/32⁻ megakaryocyte-erythroid progenitors (MEPs) did not express α–catulin−GFP. α–catulin^GFP/+ and control cell populations had similar levels of background GFP signals that accounted for fewer than 1% of the cells in each population (n = 9 mice per genotype in 2 independent experiments).

 

Extended Data Figure 7: HSC density is higher in the diaphysis as compared to the metaphysis.

 

 

Extended Data Figure 9: Bone marrow blood vessel types can be distinguished based on vessel diameter, continuity of basal lamina, morphology, and position; and no difference in the distribution of HSCs in the bone marrow of male and female mice was detected.close