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

Cytometry A. 2018 Jul;93(9):876-888. doi: 10.1002/cyto.a.23526. Epub 2018 Aug 14.


The bone marrow (BM) consists of multiple, structured micro-environmental entities-the so called niches, which contain hematopoietic cells as well as stromal cells. These niches fulfill a variety of functions, such as control of the hematopoietic stem cell pool, differentiation of hematopoietic cells, and maintenance of immunological memory. However, due to the molecular and cellular complexity and a lack of suitable histological multiplexing methods, the composition of the various BM niches is still elusive. In this study, we apply multiepitope-ligand-cartography (MELC) on bone sections from mice. We combine multiplexed immunofluorescence histology data with various object-based segmentation approaches in order to define irregularly shaped, net-like structures of stromal cells. We confirm MELC as a robust histological method and validate our automated segmentation algorithms using flow cytometry and manual evaluation. By means of MELC multiplexing, we reveal heterogeneous expression of leptin receptor (LpR), BP-1, and VCAM-1 in the stromal network. Moreover, we demonstrate by quantification a preferential contact of B cell subsets as well as of plasma cells to processes of CXCL12-expressing stromal cells, compared with stromal somata. In summary, our approach is suitable for spatial analysis of complex tissue structures.

Keywords: bone marrow; histology; immunofluorescence; multiplexing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bone Marrow / metabolism
  • Bone Marrow / physiology*
  • Bone Marrow Cells / cytology*
  • Bone Marrow Cells / metabolism
  • Cells, Cultured
  • Chemokine CXCL12 / metabolism
  • Hematopoietic Stem Cells / cytology
  • Hematopoietic Stem Cells / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Microscopy, Fluorescence / methods
  • Receptors, Leptin / metabolism
  • Stromal Cells / cytology*
  • Stromal Cells / metabolism
  • Transcription Factors / metabolism
  • Vascular Cell Adhesion Molecule-1 / metabolism


  • Chemokine CXCL12
  • Receptors, Leptin
  • Transcription Factors
  • Vascular Cell Adhesion Molecule-1