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

Abstract

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% O₂, 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% O₂, CD45⁺ cells showed increased proliferation coupled with no changes in cell death compared to their counterparts grown at 1% O₂. 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.

FIG 1.. CD45⁺ cells constitute the majority of cells in BMSC cultures.

A) Phase contrast and overlay of immunofluorescent images of BMSCs isolated from LepRCre;Rosa26^tdTm/+ mice. Red depicts cells in which CRE recombinase is active. Representative flow analysis plots and quantification of the percentage of CD45⁺, tdTomato⁺ (Td/Tm), and PDGFRα⁺;tdTomato⁺ double positive cells isolated from LepRCre;Rosa26^tdTm/+ mice. B) Schematic representation of isolation methods. C) Phase contrast images of passage 0 BMSCs after 7 days in culture. Representative flow analysis plots and quantification of the percentage C) CD45⁺, D) PDGFRα⁺, and PDGFRα⁺;CD45⁺ double positive cells in WT BMSCs. Each graph represents an individual trial using BMSCs isolated from 2–3 month male and female mice. n≥3, unpaired two tailed t-tests, significance p < 0.05.

FIG 2.. Hypoxia depletes CD45⁺ cells in BMSC cultures.

A) Phase contrast images of passage 0 BMSCs after 7 days in either 21% O₂ (normoxia, Nx) or 1% O₂ (hypoxia, Hx). Representative flow analysis plots and quantification of the percentage of CD45⁺, PDGFRα⁺ and PDGFRα⁺;tdTomato⁺ double positive cells in B) WT or C) LepRCre;Rosa26^tdTm/+. Each graph represents an individual trial using BMSCs isolated from 2–3M male mice were used for analysis, n≥3, unpaired two tailed t-tests, significance p < 0.05

FIG 3.. Hypoxic depletion of CD45⁺ cells is maintained over time and passage number.

A) Quantification of flow analysis showing the percentage of CD45⁺ and PDGFRα cells over 7 days in either normoxia (grey) or hypoxia (red). B) Quantification of the percentage of CD45⁺, PDGFRα⁺, and CD45⁺;PDGFRα⁺ double positive cells after two passages. C) Crystal violet staining and quantification of BMSCs plated at clonogenic density (left) and representative flow analysis plots and quantification of the percentage of CD45⁺ and PDGFRα⁺ (right). Each graph represents an individual trial using BMSCs isolated from 2–3M male mice were used for analysis, n≥3, unpaired two tailed t-tests, significance p < 0.05

Figure 4. Differential effect of hypoxia on proliferation and apoptosis within CD45 and PDGFRα subsets.

A) Representative flow analysis plots of CD45⁺ cells or B) PDGFRα⁺ and percentage of cells which have incorporated EdU C) Quantification of flow analysis for CD45⁺;EdU⁺ and PDGFRα⁺;EdU⁺ cells. D) Representative flow analysis plots of CD45⁺ cells or E) PDGFRα⁺ and percentage of TUNEL⁺ cells F) Quantification of flow analysis for CD45⁺;TUNEL⁺ and PDGFRα⁺;TUNEL⁺ cells. BMSCs isolated from 2–3M old male and female mice, n≥3 for each condition. One-way ANOVA, post-hoc Tukey’s multiple comparisons test, significance p < 0.05.