Automated Quantification of Hematopoietic Cell – Stromal Cell Interactions in Histological Images of Undecalcified Bone

Abstract

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of highresolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data. Video LinkThe video component of this article can be found at

Factors Affecting Early Antibody Secreting Cell Maturation Into Long-Lived Plasma Cells

“…Evidence shows that this support is mediated through cell-cell contacts and/or soluble signals (62, 94–145). BM ASCs are in direct contact with MSCs (126, 146). This interaction is thought to be mediated mainly via ASC adhesion receptors, VLA-4 and LFA-1 (140, 145, 147, 148) and their natural ligands, VCAM-1 and ICAM-1, respectively, on MSCs, and are thought to anchor them to the extracellular matrix (ECM) (149).…”

Multiplexed fluorescence microscopy reveals heterogeneity among stromal cells in mouse bone marrow sections

“…In the present study, multi epitope ligand cartography (MELC) was established to elucidate the heterogeneity among stromal cells, which was underappreciated for a long time (29,30). We use the MELC approach for the first time in the analysis as of bone sections mounted on tapes (49,58) for greater than 30 parameters and demonstrate this to be a robust method, as far as signal quality over time is concerned (Fig. 2, Supporting Information Fig.…”

“…Femurs were prepared, fixed and stained as already published (9,49). Briefly, femurs were paraformaldehyde (PFA)fixed and cut in 7 μm sections using the Kawamoto tape method.…”

“…This difference could be explained by the fact that stromal processes form very fine, faintly stained networks, which are hard to detect without amplification, such as the use of anti GFP antibody. While stromal cells seem to play an important role for plasma cell niches (9,49), it may well be that CXCL12 + stromal cells are indispensable for the formation of the niches. 4B).…”

“…CXCL12 is the major chemokine directing plasma blasts into the bone marrow (17,18), however, its role as a survival factor promoting mPC survival is controversially discussed (19,20). While stromal cells seem to play an important role for plasma cell niches (9,49), it may well be that CXCL12 + stromal cells are indispensable for the formation of the niches. Other subsets may account for the persistence of mPCs in these microenvironments, further supporting the idea of heterogeneity among stromal subsets in the bone marrow.…”