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

Summary

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

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 high-resolution 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.

Introduction

Due to recent rapid technological developments in microscopy, including optical imaging, the analysis of cells within the context of the whole tissue has become increasingly accessible for immunologists. The characterization of single cells in suspension represents a valuable and indispensable method to understand cellular and molecular function. However, the analysis of the cells within their (micro)-anatomical environment is essential for understanding the interactions between various cell types that collaborate in complex processes such as the development of immune responses.

While it is relatively easy for microscopists to obtain qualit....

Protocol

The animal experiments were approved by the appropriate state committees for animal welfare (Landesamt für Gesundheit und Soziales, Berlin) and were performed in accordance with current guidelines and regulations (animal experiment license G0194/11).

1. Generation of Fluorescent Bone Marrow Chimeric Mice

NOTE: The generation of fluorescent bone marrow chimeric mice to visualize bone marrow stromal cells is performed as described before⁹.

1. Start treating Del-Cre x ROSA-tdRFP mice (mice expressing tandem red fluorescent protein (tdRFP) ubiquitously^11-13) to prepare them for irr....

Results

Cutting cryosections of undecalcified bone with the Kawamoto tape method allows the whole bone to be cut as an intact section, with the bone marrow of the endosteal region still attached to the mineralized bone, both in the diaphysis as well as in the epiphyseal areas with their high density of trabecular bone (Figure 1). Nuclear staining of the sections reveals that although small cracks in the preparation cannot be fully avoided, the structure of the sinusoids and arteries as well as the reticular netw.......

Discussion

Despite the progress in modern optical imaging methods, the analysis of histological data is still often hindered by the lack of proper quantification tools and methods, or by biased analyses that focus on a small area of interest. The synergistic approach presented here combines image analysis covering the entire bone marrow region, automated segmentation and object recognition of various hematopoietic and stromal cell types, co-localization analysis, and finally a validation tool of non-randomly occurring contacts prov.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Neomycin	Sigma	N6386 SIGMA	Neomycin trisulfate salt hydrate, EU hazard code: GHS08
Ursovit AD3EC	Serumwerke Bernburg		1 ml contains: 50.000 I.E. retinyl palmitate, 5.000 I.E. cholecalciferol, 30 mg tocopheryl acetate, 100 mg ascorbic acid, 1 mg sorbic acid, 200 mg polyoxyl 35 castor oil, 0,5 mg propyl gallate
Transfer buffer (100 ml PBS, 1 ml 1 M HEPES, 50 U/ml penicillin/streptomycin)	Sigma	P4333, H3375
4-Hydroxy-3-nitrophenylacetyl hapten conjugated to chicken gamma globulin
Chicken gamma globulin (CGG) 100 mg	Rockland	D602-0100
20% Paraformaldehyde solution (EM-grade)	Science Services	15713	EU hazard codes: GHS02, GHS05, GHS07, GHS08
D(+)-sucrose	Carl Roth	4621.1
Dry ice
Acetone	Sigma-Aldrich	179124 SIGMA-ALDRICH	EU hazard codes: GHS02, GHS07
Hexane	Sigma-Aldrich	208752 SIGMA-ALDRICH	EU hazard codes: GHS02, GHS07, GHS08, GHS09
Tissue-Tek cryomolds (standard)	Sakura	4557	25 x 20 x 5 mm
Tissue-Tek O.C.T.	Sakura	4583
Kawamoto's SCEM embedding medium	Section-Lab, JP
Kawamoto's cryosection preparation kit	Section-Lab, JP
Kawamoto's cryofilm type 2C(9)	Section-Lab, JP
Microtome blade MX35 premier plus, low profile	Thermo Scientific	3052835	L X W: 80 x 8 mm (31.5 x 3.13"), thickness: 0.25 mm (0.01")
Polyclonal rabbit anti-RFP antibody, biotinylated	Rockland	600-406-379
Alexa Fluor 555 streptavidin	Life Technologies	S-32355
Rat anti-MBP	J. and N. Lee		available from: J. and N. Lee, Mayo Clinic, Scottsdale, AZ , U.S.A., clone MT-14.7
Goat anti-rat-Alexa Fluor 647	Life Technologies	A-21247
Rat anti-B220 - Alexa Fluor 594			produced and coupled in-house (DRFZ), clone RA3.6B2, Alexa Fluor 594 from Life Technologies
Mouse anti-l1 light chain -FITC			produced and coupled in-house (DRFZ), clone LS136
Rat anti-k light chain - FITC			produced and coupled in-house (DRFZ), clone 187.1
DAPI (4′,6-diamidino-2-phenylindole dihydrochloride)	Sigma	D9542 SIGMA
Fluorescent mounting medium	DAKO	S3023
Cover slips (24 x 24 x 0.13-0.16 mm)	Carl Roth	H875.2
Superfrost slides glasses (75 x 25 mm)	VWR	48311-703
Laser scanning confocal microscope			equipped with laser lines of 405, 488, 561, 594, 633 nm and a 20X/0.8 NA air objective lens. We used a Zeiss LSM710 and Zen 2010 Version 6.0 software.
Automated image analysis tools for bone marrow	Wimasis, Munich		The cell contact tool and cell vicinity tool will be made available by Wimasis upon request.
VC2012 runtime	Microsoft		free download
Simulation tool for random cell positioning			available from us, upon request
Image analysis software with image segmentation functions			We used Volocity (Perkin Elmer) for measuring cell size distributions of hematopoietic cell types in bone marrow (Figure 5). Alternatives are Definiens Image Analysis (Definiens) or Cell Profiler (free download)
Fiji image analysis software			free download. Fiji was used by trained raters for manual cell count (Figure 3).