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Isolation of Human Bone Marrow Non-hematopoietic Cells for Single-cell RNA Sequencing

Li, Hongzhe LU ; Bräunig, Sandro LU and Scheding, Stefan LU (2024) In Bio-protocol 14(12). p.1-10
Abstract

The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME's intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive... (More)

The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME's intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal bona fide stromal cell population with high precision. The outlined step-by-step protocol provides a robust tool for isolating and characterizing non-hematopoietic cells, including stromal cells, from human bone marrow preparations. This approach thus contributes valuable information to promote research in a field that is marked by a scarcity of studies and helps to conduct important experimentation that will deepen our understanding of the intricate cellular interactions within the bone marrow niche. Key features • Isolation of high-quality human non-hematopoietic bone marrow cells for scRNAseq • Targeted strategy for enriching low-frequency stromal cells.

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author
; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Bio-protocol
volume
14
issue
12
article number
e5020
pages
1 - 10
publisher
Bio-protocol LLC
external identifiers
  • scopus:85196484857
  • pmid:38948257
ISSN
2331-8325
DOI
10.21769/BioProtoc.5020
language
English
LU publication?
yes
additional info
©Copyright : © 2024 The Authors; This is an open access article under the CC BY license.
id
2d6cc5cc-e0ae-4713-9aa5-14ce048bf512
date added to LUP
2024-09-12 15:26:29
date last changed
2024-09-13 07:17:01
@article{2d6cc5cc-e0ae-4713-9aa5-14ce048bf512,
  abstract     = {{<p>The intricate composition, heterogeneity, and hierarchical organization of the human bone marrow hematopoietic microenvironment (HME) present challenges for experimentation, which is primarily due to the scarcity of HME-forming cells, notably bone marrow stromal cells (BMSCs). The limited understanding of non-hematopoietic cell phenotypes complicates the unraveling of the HME's intricacies and necessitates a precise isolation protocol for systematic studies. The protocol presented herein puts special emphasis on the accuracy and high quality of BMSCs obtained for downstream sequencing analysis. Utilizing CD45 and CD235a as negative markers ensures sufficient enrichment of non-hematopoietic cells within the HME. By adding positive selection based on CD271 expression, this protocol allows for selectively isolating the rare and pivotal bona fide stromal cell population with high precision. The outlined step-by-step protocol provides a robust tool for isolating and characterizing non-hematopoietic cells, including stromal cells, from human bone marrow preparations. This approach thus contributes valuable information to promote research in a field that is marked by a scarcity of studies and helps to conduct important experimentation that will deepen our understanding of the intricate cellular interactions within the bone marrow niche. Key features • Isolation of high-quality human non-hematopoietic bone marrow cells for scRNAseq • Targeted strategy for enriching low-frequency stromal cells.</p>}},
  author       = {{Li, Hongzhe and Bräunig, Sandro and Scheding, Stefan}},
  issn         = {{2331-8325}},
  language     = {{eng}},
  month        = {{06}},
  number       = {{12}},
  pages        = {{1--10}},
  publisher    = {{Bio-protocol LLC}},
  series       = {{Bio-protocol}},
  title        = {{Isolation of Human Bone Marrow Non-hematopoietic Cells for Single-cell RNA Sequencing}},
  url          = {{http://dx.doi.org/10.21769/BioProtoc.5020}},
  doi          = {{10.21769/BioProtoc.5020}},
  volume       = {{14}},
  year         = {{2024}},
}