Fig. 5: Engineered stromal niches support HSPC maintenance in vivo.

a Experimental strategy for an in vivo synthetic niche. Human HSPCs were injected subcutaneously along with MS5_FS12 in a basement membrane matrix (Matrigel) preparation. b Hematoxylin–eosin staining of subcutaneous organoids at day 12. Arrows show clusters of Matrigel-embedded cells. Scale bar represents 500 μm (left) and 250 μm (right). c Flow cytometry analysis at day 12 of Matrigel organoids containing either MS5_CTRL or MS5_FS12 cells. Absolute number and frequency of human CD45⁺ cells recovered are summarized in bar graphs (n = 13 cord blood donors in 6 independent experiments; **p < 0.01, two-tailed paired Student’s t-test). d Experimental strategy and quantification of human CD45⁺ cells recovered from physically separated plugs containing either MS5_CTRL or MS5_FS12 cells injected in the same recipient (n = 3 cord blood donors in one experiment; two-tailed paired Student’s t-test). e Immunofluorescence staining of plug sections displaying the interaction of GFP⁺ MS5_FS12 (green) with human CD45⁺ cells (red). Human hematopoietic progenitors were also identified as CD45⁺ (red) CD34⁺ (blue) cells in MS5_FS12 plugs. Nuclei were stained with Hoechst (blue). Arrows show interaction of human CD45⁺ leukocytes with GFP⁺ MS5_FS12. Scale bar represents 100 μm (left panel) and 20 μm (right panel). Similar results were observed in n = 5 Matrigel organoids. The presence of GFP⁺ stromal cells in Matrigel organoids at day 12 was further confirmed by flow cytometry (n = 15 independent organoids). f Visualization of mouse CD31⁺ endothelial cells by immunofluorescence. Fixed sections were stained for human CD45 (green) and mouse CD31 (red). Nuclei were stained with Hoechst (blue). Scale bar represents 100 μm (left panel) and 50 μm (right panel). Similar results were observed in n = 5 Matrigel organoids. The presence of mouse CD31⁺ cells was further confirmed by flow cytomery. Data are presented as floating bars ranging from min to max and line represents median (c, d) or scatter plots with mean ± SEM (e).