Spatial and biochemical interactions between bone marrow adipose tissue and hematopoietic stem and progenitor cells in rhesus macaques

Abstract

Recent developments in in situ microscopy have enabled unparalleled resolution of the architecture of the bone marrow (BM) niche for murine hematopoietic stem and progenitor cells (HSPCs). However, the extent to which these observations can be extrapolated to human BM remains unknown. In humans, adipose tissue occupies a significant portion of the BM medullary cavity, making quantitative immunofluorescent analysis difficult due to lipid-mediated light scattering. In this study, we employed optical clearing, confocal microscopy and nearest neighbor analysis to determine the spatial distribution of CD34+ HSPCs in the BM in a translationally relevant rhesus macaque model. Immunofluorescent analysis revealed that femoral BM adipocytes are associated with the branches of vascular sinusoids, with half of HSPCs localizing in close proximity of the nearest BM adipocyte. Immunofluorescent microscopy and flow cytometric analysis demonstrate that BM adipose tissue exists as a multicellular niche consisted of adipocytes, endothelial cells, granulocytes, and macrophages. Analysis of BM adipose tissue conditioned media using liquid chromatography-tandem mass spectrometry revealed the presence of multiple bioactive proteins involved in regulation of hematopoiesis, inflammation, and bone development, with many predicted to reside inside microvesicles. Pretreatment of purified HSPCs with BM adipose tissue conditioned media, comprising soluble and exosomal/microvesicle-derived factors, led to enhanced proliferation and an increase in granulocyte-monocyte differentiation potential ex vivo. Our work extends extensive studies in murine models, indicating that BM adipose tissue is a central paracrine regulator of hematopoiesis in nonhuman primates and possibly in humans.

Keywords: Bone marrow adipose tissue; Bone marrow niche; Hematopoietic stem and progenitor cells; Microscopy; Nonhuman primates.

Figure 1.. Characterization of the adipocyte niche in the rhesus BM.

(A) Spatial organization of vascular niches and BM adipose tissue in the rhesus macaque femur. Central sinus, centrally located vein; sinusoids, branches of central sinus; artery, central arterial blood vessel (smaller arterioles are not shown). Distal and central parts of the femur are enriched in BM adipose tissue; proximal femur is enriched in hematopoietic cells. (B) Bone sections representing the proximal (p), medial (m), and distal (d) parts of the femur from a male rhesus macaque 2 years and 229 days of age. (C) Representative image of the proximal femur stained with Hoechst (DNA), isolectin GS-IB4 Alexa 488 (blood vessels), and HCS LipidTox Deep Red (adipocytes); arrows, adipocytes associated with blood vessels; CS, central sinus. (D) Low- and (E) high-magnification images of BM stained with antibodies to CD31 and the lipid dye HCS LipidTox Deep Red; E, right panel, BM adipocytes (a) stained with antibodies to perilipin-1 without HCS LipidTox Deep Red. To retain HCS LipidTox Deep Red in the lipid droplets of BM adipocytes, immunohistochemistry experiments presented in this figure were performed without optical clearing.

Figure 2.. Primate CD34+ HSPCs localize in close proximity to BM adipocytes.

(A) Representative confocal image of optically cleared BM from the proximal metaphysis of a 7-year-old male rhesus macaque stained with antibodies to CD34 and CD31; s, sinusoids; arrows, CD31+CD34+ endothelium; a, adipocytes; a representative example of the adipocyte niche is outlined by the dotted line. (B and C) High-magnification confocal images depicting the localization of CD34+ HSPCs (asterisks) next to the BM adipocytes (a). (D) Nearest-neighbor analysis of distances between CD34+ HSPCs vs random dots and BM adipocytes; representative traces from a single animal; “NND,” nearest neighbor distance. Inset depicts the theoretical position of HSPCs (red) or random dots (blue) relative to adipocytes; pm, plasma membrane; ldm, lipid droplet membrane. (E) NNDs between HSPCs and BM adipocytes. Bars are means ± SD; n=3 males of 7–10 years of age; each point represents one animal and was calculated as the average NND derived from 4 nonadjacent confocal slices. (F and G) Digital analysis of anatomical structures in the BM of a 7-year old male rhesus macaque; CD34+ HSPCs (red), BM adipocytes (grey shapes), CD31+CD34+ endothelium (yellow lines), random dots (blues). Anatomical boundaries from the same BM sample: central sinus (F); the bone (G).

Figure 3.. BM occupancy by adipocytes regulate their spatial proximity to HSPCs.

(A) Representative images of bone sections from a 7-year-old adult (ADT) and a 2-year-old juvenile (JUV) male rhesus macaques. (B) Representative confocal image depicting the localization of CD34+ HSPCs (asterisks) next to BM adipocytes (a) in the femoral BM of a juvenile rhesus macaque; arrows, CD34+ capillaries. (C) Nearest neighbor distances (NNDs) between HSPCs and BM adipocytes. Bars are means ± SD; n=3 nonadjacent confocal slices derived from one juvenile animal (JUV1). (D) The principle of Euclidian NND analysis; algorithm computes the distances between the centroid of each object classified as “HSPC” (3 cells are shown) and the nearest coordinate associated with the outer boundary of the BM adipocyte (a); solid red lines, NNDs; black dotted lines, distances between HSPC#2 and distant adipocytes. (E) Scatter plot showing the distribution of NNDs between HSPCs and BM adipocytes across 4 experimental animals (3 adults and 1 juvenile). Each data column represents one nonadjacent confocal slice; bar-median NND.

Figure 4.. BM adipose tissue is a multicellular niche.

(A) Morphological assessment of isolated BM adipose tissue before (0 h) and after 48-hour ex vivo incubation. Isolated BM adipose tissue was labeled with the cell viability dye calcein red-orange AM, endothelial-specific isolectin GS-IB4, Alexa Fluor^™ 488 conjugate, lipid droplet-specific HCS LipidTox Deep Red, and nuclear Hoechst 33342; BF, fluorescent images overlaid with bright field; max, maximal z-projections; z1-z3, confocal planes; arrows, calcein-positive BM adipocytes. (B and C) Histological evaluation of isolated BM adipose tissue stained with Wright Giemsa (WG) and Hematoxylin-Eosin (HE); arrows, multilobar cells (granulocytes); arrowhead, putative oil released by adipocytes. (D) Isolation of adipocyte-bound (AB) and adipocyte free (AF) fractions. BM was disrupted mechanically and separated by centrifugation. AB fraction was treated with collagenase to release bound leukocytes and AF fraction was cleared of red blood cells (RBC) before flow cytometry analysis. (E) AB and AF fractions were stained with anti-CD45 antibodies (pan-leukocyte) and compared against each cell’s side scatter profile (measure of cell complexity and granularity); % of live cell populations are indicated.

Figure 5.. BM adipose tissue stimulates proliferation and differentiation of HSPCs ex vivo.

(A-B) Dye dilution assay using CD34+ cells isolated from a 2-year old male rhesus macaque. (A) Representative fields of view showing CFSE-labelled CD34+ cells after incubation in suspension for 1, 2, 3, and 5 days in StemSpan media supplemented with 100 ng/ml SCF, TPO and Flt3L in the presence or absence BM adipose tissue conditioned media (CM). At each time point, a cell aliquot was collected and cells were adhered to fibronectin-coated plates. Cells were fixed and stained with Hoechst, and images were recorded by confocal microscopy. (B) Quantification of intracellular CFSE fluorescence of CD34+ cells isolated from a 2–year old male rhesus macaque. Each data point represents an individual CD34+ cell. Mean CFSE fluorescent values are indicated; T-test * p<0.05. CFSE-labeling experiment was repeated twice, using a different animal (Supplementary Figure S7). (C) Immunolabeling of HSPCs; unlabeled CD34+ cells were incubated in the presence or absence BM adipose tissue conditioned media for 5 days, adhered to the imaging plates and stained with Hoechst and antibodies to Ki67 and CD38. (D) Bars are means ± SEM, n=4394 cells; ^** p<0.001. This experiment was repeated twice with similar results. (E) CFU assay; representative examples of granulocyte-monocyte (GM), erythroid (E) and multilineage progenitor (GEMM) colonies recorded on day 10; insets, early hematopoietic colonies recorded on day 7. (F) 2000 cells were plated onto MethoCult™ (H4435 Enriched) supplied with 100 ng/ml SCF, TPO and Flt3L. Bars are means ± SEM, n=3 animals (7-year-old males). * T-test p<0.01. Each biological data point represents the mean of three replicate experiments.

Figure 6.. BM adipose tissue is a rich source of hematopoietic factors and adhesion molecules.

(A) String analysis of the top 200 proteins released by BM adipocytes. The graph represents the String interaction network depicting the KEGG pathway enrichments analysis. Additional analysis is included in Supplementary Table S2. BM adipose tissue was isolated from six 7 to 10-year old rhesus macaque males. BM adipose tissue was incubated at 37°C for 48 hours. Following incubation, media was collected, cleared by ultracentrifugation, dialyzed against PBS, and analyzed by mass spectrometry as described in “Materials and Methods.” (B) KEGG pathways analysis of BM adipose tissue secretome (20 top pathways are shown). (C) Top protein domains identified in BM adipose tissue secretome.