Fig. 2: Human MF BM cells engraft in NSG mice and give rise to GLI1⁺ fibrocytes in vivo.

A Diagram of a MF xenograft mouse model was generated to study GLI1 expression in MF fibrocytes. BM LDCs from MF patients (n = 2) or NCs (n = 2) were transplanted into NSG mice (n = 5 per group) at 6–8 weeks of age. Following successful engraftment assessed by flow cytometry, sternum and spleen were harvested, and tissue sections were analyzed using mfIHC. B, C Representative micrographs of GLI1⁺ human fibrocytes in the BM (B) and spleen (C) of MF xenograft mice. In addition to GLI1 (Opal 480, pseudo-colored red), tissues were co-stained using HLA-ABC (Opal 780), CD45 (Opal 570), CD68 (Opal 520), and procollagen-I (Opal 690) antibodies (pseudo-colored green). Shown are composites of each phenotype marker along with GLI1 for reference. Large clusters of GLI1⁺ human fibrocytes, defined as HLA-ABC⁺/CD45⁺/CD68⁺/procollagen-I⁺ cells, were found in the BM and spleen tissue of NSG mice transplanted with MF BM LDCs, but only scattered GLI1⁺ fibrocytes were detected in the NC-transplanted mice. DAPI (pseudo-colored blue) was used as a nuclear counterstain. D Quantitation of GLI1⁺ human fibrocytes in the BM and spleen of xenograft mice (n = 5 per group) engrafted with human MF or NC BM LDCs. Whole tissue sections were imaged at ×400 resolution, deconvoluted, and analyzed using an unsupervised segmentation and phenotyping algorithm. Numbers of GLI1⁺/HLA-ABC⁺/CD45⁺/CD68⁺/procollagen-I⁺ cells were calculated per unit area using unbiased counting frames. Horizontal lines denote median. The significance of the difference between MF- and NC-xenografts was assessed using the Mann–Whitney test. E Representative micrographs of reticulin fibrosis in the BM tissue section of the xenograft mice analyzed using Gomori’s silver stain method. Bundles of reticulin fibers were detected in the BM of mice transplanted with BM LDCs from MF patients but not in mice transplanted with NC BM cells. Nuclear fast red was used as a counterstain. All micrographs were adjusted for brightness and contrast linearly and equally between groups. Scale bars represent 100 µm. *P < 0.05; **P < 0.01, respectively.