utilizing-low-oxygen-tension-to-reduce-hematopoietic-cells-murine

Utilizing Low Oxygen Tension to Reduce Hematopoietic Cells in Murine Bone Marrow Stromal Cell Cultures

Currently, there remains a lack of universally accepted markers to prospectively isolate a homogeneous population of skeletal stem cells (SSCs). For this ...

Duke University School of Medicine

Oxygen serves as a critical environmental factor essential for maintaining the physiological state of a tissue. Hypoxia, or low oxygen, triggers a cascade of events which allows for cells to adapt to low oxygen tensions and to facilitate oxygen delivery required to maintain tissue homeostasis. In the bone microenvironment (BME), vascular heterogeneity, poor perfusion rates of blood vessels, and high metabolic activity of hematopoietic cells result in the generation of a unique hypoxic landscape. Importantly, in this region, hypoxia and its downstream effectors are associated with establishing stem cell niches and regulating the differentiation of committed progenitors. Given the functional importance of the hypoxic bone niche, visualizing regions of hypoxia may provide valuable insights into the mechanisms that regulate tissue homeostasis. Here, we describe the utilization of the nitroimidazole derivative, pimonidazole, to detect hypoxic regions within the BME.

Detection of Hypoxic Regions in the Bone Microenvironment.

Culture expanded bone marrow stromal cells (BMSCs) are easily isolated, can be grown rapidly en masse, and contain both skeletal stem cells (SSCs) and multipotent mesenchymal progenitors (MMPs). Despite this functional heterogeneity, BMSCs continue to be utilized for many applications due to the lack of definitive and universally accepted markers to prospectively identify and purify SSCs. Isolation is widely based on adherence to tissue culture plastic; however, high hematopoietic contamination is a significant impediment in murine models. Remarkably, when cultured at a physiological oxygen tension of 1% O, a 10-fold reduction in CD45 hematopoietic cells associated with a concomitant increase in PDGFRα stromal cells occur. This is due, in part, to a differential response of the two populations to hypoxia. In standard tissue culture conditions of 21% O, CD45 cells showed increased proliferation coupled with no changes in cell death compared to their counterparts grown at 1% O. In contrast, PDGFR α stromal cells responded to hypoxia by increasing proliferation and exhibiting a 10-fold decrease in cell death. In summary, we describe a simple and reliable method exploiting the divergent biological response of hematopoietic and stromal cells to hypoxia to significantly increase the PDGFR α stromal cell population in murine BMSC cultures.

Hypoxia depletes contaminating CD45 hematopoietic cells from murine bone marrow stromal cell (BMSC) cultures: Methods for BMSC culture purification.

Hypertrophic chondrocytes give rise to osteoblasts during skeletal development; however, the process by which these non-mitotic cells make this transition is not well understood. Prior studies have also suggested that skeletal stem and progenitor cells (SSPCs) localize to the surrounding periosteum and serve as a major source of marrow-associated SSPCs, osteoblasts, osteocytes, and adipocytes during skeletal development. To further understand the cell transition process by which hypertrophic chondrocytes contribute to osteoblasts or other marrow associated cells, we utilized inducible and constitutive hypertrophic chondrocyte lineage tracing and reporter mouse models ( and ) in combination with a  transgenic line, single-cell RNA-sequencing, bulk RNA-sequencing, immunofluorescence staining, and cell transplantation assays. Our data demonstrate that hypertrophic chondrocytes undergo a process of dedifferentiation to generate marrow-associated SSPCs that serve as a primary source of osteoblasts during skeletal development. These hypertrophic chondrocyte-derived SSPCs commit to a CXCL12-abundant reticular (CAR) cell phenotype during skeletal development and demonstrate unique abilities to recruit vasculature and promote bone marrow establishment, while also contributing to the adipogenic lineage.

Hypertrophic chondrocytes serve as a reservoir for marrow-associated skeletal stem and progenitor cells, osteoblasts, and adipocytes during skeletal development.

Article	Year
Detection of Hypoxic Regions in the Bone Microenvironment. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 33197024	2021
Hypoxia depletes contaminating CD45 hematopoietic cells from murine bone marrow stromal cell (BMSC) cultures: Methods for BMSC culture purification. Stem cell research\| PubMed ID: 33848794	2021
Hypertrophic chondrocytes serve as a reservoir for marrow-associated skeletal stem and progenitor cells, osteoblasts, and adipocytes during skeletal development. eLife\| PubMed ID: 35179487	2022

Wendi Guo

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