Morphological Characteristics of Young and Old Murine Hematopoietic Stem Cell Niches, as Modeled In Vitro

Abstract

The hematopoietic stem cell (HSC) niche undergoes detrimental changes with age. The molecular differences between young and old niches are well studied and understood; however, young and old niches have not yet been extensively characterized in terms of morphology. In the present work, a 2D stromal model of young and old HSC niches isolated from bone marrow was investigated using light and scanning electron microscopy (SEM) to characterize cell density after one, two, or three weeks of culturing, cell shape, and cell surface morphological features. Our work is aimed at identifying morphological differences between young and old niche cells that could be used to discriminate between their respective murine HSC niches. The results show several age-specific morphological characteristics. The old niches differ from the young ones in terms of lower cell proliferating capacity, increased cell size with a flattened appearance, increased number of adipocytes, and the presence of tunneling nanotubes. In addition, proliferating cell clusters are present in the young niches but not in the old niches. Together, these characteristics could be used as a relatively simple and reliable tool to discriminate between young and old murine HSC niches and as a complementary approach to imaging with specific cellular markers.

Figure 1

Components of hematopoietic stem cell (HSC) niches (“HSC synapse”). Abbreviations: MSC: mesenchymal stem cells; Ecs: endothelial cells; HSCs: hematopoietic stem cells. Provided courtesy of P. Rožman.

Figure 2

Preparation of an artificial hematopoietic stem cell (HSC) niche. First, the bone crush method is used to isolate BM cells from young and old mice. The cells are then separately cultivated in a commercial MyeloCult medium for 1-3 weeks and regularly checked with light microscopy and SEM.

Figure 3

Light micrographs of representative cell types of a hematopoietic stem cell (HSC) niche investigated using a phase-contrast light microscope (200x) after four weeks of culturing. Adipocytes with lipid droplets, seen as round structures of different sizes, are indicated with a red arrow (a); fibroblast and mesenchymal stromal cells (MSCs) are characterized by a flattened and elongated shape (b); endothelial cells growing in lines are indicated with a blue arrow (c).

Figure 4

Cell density in young and old murine hematopoietic stem cell (HSC) niches after one, two, and three weeks of culturing. Data are presented as the mean number of cells per mm² (+SD). An asterisk indicates a significant difference between young and old niches (^∗ equals p < 0.05; ANOVA with Bonferroni's post hoc test).

Figure 5

SEM micrographs of young and old bone marrow (BM) niches after one week (a), two weeks (b), and three weeks of culturing (c). The proliferating cell clusters are seen in images (b) and (c) of the young niche (see detail in Figure 6).

Figure 6

Bone marrow cells from young mice after one week of culturing. The red square indicates a proliferating cluster. An example of a flattened cell is indicated with a yellow arrow, while an example of a flattened cell is indicated with a yellow asterisk.

Figure 7

SEM micrographs of different, morphologically distinct cell types in young and old hematopoietic stem cell (HSC) niches. (a–c) Flattened cells with a folded central area ((a, c) round; (b) oval) ((a, c) cells with a low number of short filopodia; (b) cell with a large number of filopodia) and differing cell surface roughness ((a, b) cells with rough membrane surfaces; (c) cell with smooth membrane surface). (d–f) Round-shaped cells with a large number of filopodia (d), extended lamellipodium (e), and visible cellular protrusions (f). (g) Adipocyte filled with numerous lipid droplets. (h) elongated cell. Scale bar = 10 μm.

Figure 8

Relative distribution of different morphological cell types in young and old hematopoietic stem cell (HSC) niches. Data are presented as the mean percentage of all cells (+SD).

Figure 9

The average size of flattened cells in young and old hematopoietic stem cell (HSC) niches ± standard deviations (SD). Asterisk presents a significant difference between young and old niches (^∗ equals p < 0.05; Mann–Whitney U test).

Figure 10

Adipocytes in an old niche are indicated with red arrows. A morphological characteristic of adipocytes is the presence of a large number of round intracellular vesicles (lipid droplets).

Figure 11

Multiple lipid droplets in adipocytes. Lipid droplets are intracellular organelles of varying sizes (from submicron dimensions to several microns) that can be seen as round-shaped structures beneath the cell surface in SEM imaging.

Figure 12

Tunneling nanotubes (TNTs) observed in an old hematopoietic stem cell (HSC) niche, indicated with a red arrow. TNTs were observed only in the old HSC niches.