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Three-dimensional spatial mapping of the human hematopoietic microenvironment in healthy and diseased bone marrow

Bräunig, Sandro LU ; Karmhag, Isak LU ; Li, Hongzhe LU ; Enoksson, Jens LU ; Hultquist, Anne LU and Scheding, Stefan LU (2023) In Cytometry Part A 103(10). p.763-776
Abstract

The bone marrow hematopoietic microenvironment (HME) plays a pivotal role in regulating normal and diseased hematopoiesis. However, the spatial organization of the human HME has not been thoroughly investigated yet. Therefore, we developed a three-dimensional (3D) immunofluorescence model to analyze changes in the cellular architecture in control and diseased bone marrows (BMs). BM biopsies from patients with myeloproliferative neoplasms (MPNs) were stained sequentially for CD31, CD34, CD45, and CD271 with repetitive bleaching steps to realize five color images with DAPI as a nuclear stain. Hematopoietically normal age-matched BM biopsies served as controls. Twelve subsequent slides per sample were stacked to create three-dimensional... (More)

The bone marrow hematopoietic microenvironment (HME) plays a pivotal role in regulating normal and diseased hematopoiesis. However, the spatial organization of the human HME has not been thoroughly investigated yet. Therefore, we developed a three-dimensional (3D) immunofluorescence model to analyze changes in the cellular architecture in control and diseased bone marrows (BMs). BM biopsies from patients with myeloproliferative neoplasms (MPNs) were stained sequentially for CD31, CD34, CD45, and CD271 with repetitive bleaching steps to realize five color images with DAPI as a nuclear stain. Hematopoietically normal age-matched BM biopsies served as controls. Twelve subsequent slides per sample were stacked to create three-dimensional bone marrow reconstructions with the imaging program Arivis Visions 4D. Iso-surfaces for niche cells and structures were created and exported as mesh objects for spatial distribution analysis in the 3D creation suite Blender. We recapitulated the bone marrow architecture using this approach and produced comprehensive 3D models of endosteal and perivascular BM niches. MPN bone marrows displayed apparent differences compared to the controls, especially concerning CD271 staining density, megakaryocyte (MK) morphology, and distribution. Furthermore, measurements of the spatial relationships of MKs and hematopoietic stem and progenitor cells with vessels and bone structures in their corresponding niche environments revealed the most pronounced differences in the vascular nice in polycythemia vera. Taken together, using a repetitive staining and bleaching approach allowed us to establish a 5-color analysis of human BM biopsies, which is difficult to achieve with conventional staining approaches. Based on this, we generated 3D BM models which recapitulated key pathological features and, importantly, allowed us to define the spatial relationships between different bone marrow cell types. We, therefore, believe that our method can provide new and valuable insights into bone marrow cellular interaction research.

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author
; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
hematopoietic microenvironment, imaging, immunofluorescence, myeloproliferative neoplasms, spatial relationships, three-dimensional
in
Cytometry Part A
volume
103
issue
10
pages
763 - 776
publisher
John Wiley & Sons Inc.
external identifiers
  • pmid:37421296
  • scopus:85165425272
ISSN
1552-4922
DOI
10.1002/cyto.a.24775
language
English
LU publication?
yes
id
f214580f-e3c8-471f-99cd-db178786500f
date added to LUP
2023-09-22 13:15:38
date last changed
2024-11-01 21:13:53
@article{f214580f-e3c8-471f-99cd-db178786500f,
  abstract     = {{<p>The bone marrow hematopoietic microenvironment (HME) plays a pivotal role in regulating normal and diseased hematopoiesis. However, the spatial organization of the human HME has not been thoroughly investigated yet. Therefore, we developed a three-dimensional (3D) immunofluorescence model to analyze changes in the cellular architecture in control and diseased bone marrows (BMs). BM biopsies from patients with myeloproliferative neoplasms (MPNs) were stained sequentially for CD31, CD34, CD45, and CD271 with repetitive bleaching steps to realize five color images with DAPI as a nuclear stain. Hematopoietically normal age-matched BM biopsies served as controls. Twelve subsequent slides per sample were stacked to create three-dimensional bone marrow reconstructions with the imaging program Arivis Visions 4D. Iso-surfaces for niche cells and structures were created and exported as mesh objects for spatial distribution analysis in the 3D creation suite Blender. We recapitulated the bone marrow architecture using this approach and produced comprehensive 3D models of endosteal and perivascular BM niches. MPN bone marrows displayed apparent differences compared to the controls, especially concerning CD271 staining density, megakaryocyte (MK) morphology, and distribution. Furthermore, measurements of the spatial relationships of MKs and hematopoietic stem and progenitor cells with vessels and bone structures in their corresponding niche environments revealed the most pronounced differences in the vascular nice in polycythemia vera. Taken together, using a repetitive staining and bleaching approach allowed us to establish a 5-color analysis of human BM biopsies, which is difficult to achieve with conventional staining approaches. Based on this, we generated 3D BM models which recapitulated key pathological features and, importantly, allowed us to define the spatial relationships between different bone marrow cell types. We, therefore, believe that our method can provide new and valuable insights into bone marrow cellular interaction research.</p>}},
  author       = {{Bräunig, Sandro and Karmhag, Isak and Li, Hongzhe and Enoksson, Jens and Hultquist, Anne and Scheding, Stefan}},
  issn         = {{1552-4922}},
  keywords     = {{hematopoietic microenvironment; imaging; immunofluorescence; myeloproliferative neoplasms; spatial relationships; three-dimensional}},
  language     = {{eng}},
  number       = {{10}},
  pages        = {{763--776}},
  publisher    = {{John Wiley & Sons Inc.}},
  series       = {{Cytometry Part A}},
  title        = {{Three-dimensional spatial mapping of the human hematopoietic microenvironment in healthy and diseased bone marrow}},
  url          = {{http://dx.doi.org/10.1002/cyto.a.24775}},
  doi          = {{10.1002/cyto.a.24775}},
  volume       = {{103}},
  year         = {{2023}},
}