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X. Zhu et al.
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Figure 2. Altered CXCL12 gradients and megakaryocyte marrow niche occupancy in immune thrombocytopenia patients with complement deposition on their mes- enchymal stem cells. (A-C) Representative images of radioactive in situ hybridization with CXCL12 antisense probe [CXCL12 transcripts: brown pseudocolor; 4′,6- diamidino-2-phenylindole (DAPI): blue] on bone marrow sections from the three groups [MSC-control: mesenchymal stem cells (MSC) from healthy subjects; MSC- ITP-C-: MSC from patients with immune thrombocytopenia (ITP) without complement deposition on MSC; MSC-ITP-C+: MSC from patients with ITP with complement deposition on MSC]. Scale bar: 100 μm. (D) Representative images of marrow immunohistochemistry for CD41 (megakaryocytes, green) and CD31 (vascular endothe- lium, red) from the MSC-control, MSC-ITP-C- and MSC-ITP-C+ groups (left: bone-associated marrow; right: central marrow). Scale bar: 100 μm. (E) Ratio of CXCL12 transcript area in the bone-associated region (between 0-100 μm from the endosteal surface within the diaphysis) compared to an immediately adjacent region of the same size (between 100-200 μm from the endosteal surface) for ITP patients (MSC-ITP-C- and MSC-ITP-C+) and healthy volunteers (MSC-control). (F) Quantification of CD41+ megakaryocytes (MKs) physically associated with CD31+ vessels (between 0-10 μm from the sinusoidal endothelium within the diaphysis) by immunohistochemistry. (G) Quantification of CD41+ megakaryocytes in the bone-associated region (within 100 μm of the endosteal surface within the diaphysis) by immunohistochemistry. (H) Quantification of vessel densities by immunofluorescence staining for CD31. (I) Quantification of CD41+ megakaryocytes per section. (E- I) MSC-control, n=36; MSC-ITP-C-, n=36; MSC-ITP-C+, n=36; one-way analysis of variance).
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