Fig. 1
Artificial Marrow Organoid (AMO) formation and observation. AMO can be formed by concentrating h-MSCs and other cell types and depositing a drop of cell concentrate in a membrane placed over cell-culture medium. (A) CD34 + hematopoietic stem and progenitor cells isolated from cord blood were differentiated into myeloid and NK cells in AMO using an appropriate cocktail of cytokines. Direct optical microscopic observations of AMO with CD34 + HSPCs after 7 and 14 days of culture are shown. Scale bar 100 μm. (B) Violin plot of the AMO diameters on day 3 of culture (n = 14, 3 different h-MSCs are indicated with different shape: MSC#1 square, MSC#2 triangle and MSC#3 circle. N= 5, 5 and 4 replicates respectively). Data are represented as mean ± SD. (C) Analysis of a representative whole mount organoid stained with Hoechst (nuclei, blue), Phalloidin-FITC (stromal counterpart, green) and CD45-AF647 (hematopoietic counterpart cell membrane, red). Scale bar 100 μm. One representative experiment of three is presented. (D-G) Characterization of FACS-sorted h-MSCs after 2 weeks of AMO co-culture, compared to paired ex vivo h-MSCs. (D) Histograms showing expression of 3 mesenchymal lineage markers CD73, CD90 and CD105 assessed by flow cytometry compared to unstained cells (negative control, in grey). H-MSCs are gated on forward scatter. One representative experiment of three is presented. (E) Colony Forming Unit Fibroblast (CFU-F) assay of h-MSCs after crystal violet staining. The 2 different h-MSCs used are indicated with different shape: MSC#1 square, MSC#2 circle. A triplicate for each h-MSC has been performed. (F) Osteogenic differentiation of h-MSCs stained with red alizarin. Scale bar 200 μm. (G) Adipogenic differentiation of h-MSCs observed after oil red staining. Scale bar 50 μm. One representative experiment of three is presented.
