Fig. 2

Enhanced proliferation of HSCs under simulated microgravity conditions. (A) Representative H&E staining images of femurs and tibias from control (CON) and hindlimb unloading (HU) mice are shown, along with quantification scores of bone marrow cellularity. (B) Representative H&E staining images of spleen tissue. Scoring for hematopoiesis was conducted based on the distribution and density of hematopoietic cells. The white pulp (WP) is delineated by black dashed circles, while red pulp (RP) corresponds to the areas outside the WP. (C) Flow cytometric analysis of HSC populations in BM. Upper panel: Representative images and frequency statistics of LSK (Lineage⁻Sca-1⁺c-Kit⁺) cells in BM. Lower panel: Representative images and proportion statistics for LT-HSCs (long-term HSCs), ST-HSCs (short-term HSCs), and MPP (multipotent progenitors) within the LSK population. (D) Representative images and quantification of apoptotic HSCs from flow cytometric analysis. (E) cell cycle distribution and statistical analysis of HSCs between CON and HU groups. (F) Proliferative activity of HSCs: Ki67 + cells and statistical comparison between CON and HU groups. (G) RT-PCR was performed to assess the expression of key proliferation-related genes in LSK cells isolated from BM. Expression levels of target genes were normalized and compared between CON and HU groups. Data are presented as mean ± SEM (A-F: n = 6; G: n = 3). Statistical significance was assessed using two-tailed Student’s t-tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns (not significant) P ≥ 0.05