Red blood cells and their releasates compromise bone marrow-derived human mesenchymal stem/stromal cell survival in vitro

Abstract

Purpose: The use of bone marrow aspirate (BMA) and bone marrow aspirate concentrate (BMC) in the treatment of inflammatory orthopedic conditions has become a common practice. The therapeutic effect of BMA/BMC is thought to revolve primarily around the mesenchymal stem/stromal cell (MSC) population residing within the nucleated cell fraction. MSCs have the unique ability to respond to site of injury via the secretion of immunomodulating factors, resolving inflammation in diseased joints. Recently, the importance of hematocrit (HCT) in BMC has been debated, as the potential impact on MSC function is unknown. In the present study, we investigate MSC health over a short time-course following exposure to a range of HCT and red blood cell releasate (RBC_rel) conditions.

Methods: Bone marrow-derived human MSCs in early passage were grown under conditions of 0%, 2.5%, 5%, 10%, 20% and 40% HCT and RBC_rel conditions for 3 days. At each day, the percentage of viable, apoptotic and necrotic MSCs was determined via flow cytometry. Relative viable MSC counts in each condition was determined to account for dynamic changes in overall MSC densities over the time-course. Statistical analysis was performed using a one-way ANOVA comparing test conditions to the control followed by a Dunnett's multiple comparison test.

Results: Significant reductions in viable MSCs concurrent with an increase in necrotic MSCs in high HCT and RBC_rel conditions was observed within 24 h. At each successive timepoint, the percent and relative number of viable MSCs were reduced, becoming significant in multiple HCT and RBC_rel conditions by Day 3. Necrosis appears to be the initial mode of MSC death following exposure to HCT and RBC_rel, followed by apoptosis in surviving MSC fractions.

Conclusion: Various levels of HCT and RBC_rel severely compromise MSC health within 3 days and HCT should be controlled in the preparation of BMC products. Further, HCT of BMCs should be routinely recorded and tracked with patient outcomes along with routine metrics (e.g. nucleated cell counts, fibroblast-colony forming units). Differences in HCT may account for the inconsistencies in the efficacy of BMC reported when treating orthopedic conditions.

Keywords: Apoptosis; Bone marrow aspirate concentrate; Hematocrit; Mesenchymal stem cells; Necrosis; Red blood cells; Viability.

Fig. 1

Fluorescent imaging of MSCs in HCTs at Day 1 (A–F), Day 3 (G–L) and quantitation of MSC viability Day 1–3 for HCT (M–O) and RBC_rel (P–R). MSCs in culture were stained with calcein-AM (green, live) and DAPI (blue, dead) and imaged prior to harvesting for viability analysis. Day 1 (A). 0% HCT control (B). 2.5% HCT (C). 5% HCT (D). 10% HCT (E). 20% HCT, (E)(i). 20% HCT with fragmentation of nuclei (subpanel is enlarged, not to scale) (F). 40% HCT (arrow shows microparticle formation from MSC). Day 3 (G). 0% HCT control (H). 2.5% HCT (I). 5% HCT (J). 10% HCT (K). 20% HCT (L). 40% HCT, (L)(i). 40% HCT with RBC aggregate containing non-viable MSCs (subpanel is enlarged, not to scale). For all micrographs: Scale bar = 860 microns; Total magnification = 104x (excluding E)(i). and (L)(i).). Quantitation of viable MSCs via fluorescent microscopy in HCT and RBC_rel conditions. Viable MSCs in HCT conditions at (M) Day 1, (N) Day 2 and (O) Day 3. Viable MSCs in RBC_rel conditions at (P) Day 1, (Q) Day 2 and (R) Day 3. Error bars represent the standard deviation of the data set. Levels of significance: *p < 0.05, **p < 0.01

Fig. 2

Staining, gating and analysis of MSCs via CD90, AV and 7AAD. a Controls for apoptosis and necrosis via H₂O₂ and doxorubicin, respectively. b Untreated MSCs gated via FITC-isotype control for CD90 gating (histogram) and unlabeled for AV and 7AAD (complimenting the axes in “a”) for the following HCT and RBC_rel data sets. c CD90-labeled MSC health status in HCT conditions Day 1–3. d CD90-labeled MSC health status in RBC_rel conditions Day 1–3. Dot plot quadrants for c and d are identical to the labeled controls in (a) and are as follows: viable (AV-/7AAD-) are in the lower left quadrant, AV + /7AAD- are upper left quadrant, AV + /7AAD + upper right and AV-/7AAD + are lower right quadrant

Fig. 3

Percentage of viable MSCs via flow cytometry in HCT and RBC_rel conditions. MSCs stained with CD90, annexin-V (AV) and 7AAD where CD90 + /AV-/7AAD- are viable MSCs. Viable MSCs in HCT conditions at a Day 1, b Day 2 and c Day 3. Viable MSCs in RBC_rel conditions at d Day 1, e Day 2 and f Day 3. Error bars represent the standard deviation of the data set. Levels of significance: *p < 0.05, **p < 0.01

Fig. 4

Percentage of necrotic MSCs via flow cytometry in HCT and RBC_rel conditions. MSCs stained with CD90, annexin-V (AV) and 7AAD where CD90 + /AV-/7AAD + are necrotic MSCs. Necrotic MSCs in HCT conditions at (a) Day 1, (b) Day 2 and (c) Day 3. Necrotic MSCs in RBC_rel conditions at (d) Day 1, (e) Day 2 and (f) Day 3. Error bars represent the standard deviation of the data set. Levels of significance: *p < 0.05, **p < 0.01

Fig. 5

Percentage of apoptotic MSCs via flow cytometry in HCT and RBC_rel conditions. MSCs stained with CD90, annexin-V (AV) and 7AAD where CD90 + AV + / 7AAD ± are apoptotic MSCs. Apoptotic MSCs in HCT conditions at (a). Day 1 (b). Day 2 and (c). Day 3. Apoptotic MSCs in RBC_rel conditions at (d). Day 1 (e). Day 2 and (f). Day 3. Error bars represent the standard deviation of the data set. Levels of significance: *p < 0.05, **p < 0.01

Fig. 6

Normalized quantitative MSC viability via flow cytometry in HCT and RBC_rel conditions. MSCs stained with CD90, annexin-V (AV) and 7AAD where CD90 + /AV-/7AAD- are viable; percentages derived from viable counts in each condition relative to the control. Viable MSCs in HCT conditions at a Day 1, b Day 2 and c Day 3. Viable MSCs in RBC_rel conditions at d Day 1, e Day 2 and f Day 3. Error bars represent the standard deviation of the data set. Levels of significance: *p < 0.05, **p < 0.01

Fig. 7

Fluorescent imaging of lipid peroxidation in MSCs exposed to high HCTs. a Unstained control, b negative control staining for normal lipids (no peroxidation, red), c positive lipid peroxidation control, MSCs treated for 2 h with cumene hydroperoxide (green signal). MSCs incubated for 48 h prior to staining with d 10% HCT, e 20% HCT and f 40% HCT show evidence of lipid peroxidation (green) and cell detachment (rounded signals) and dampening of red fluorescence. Unstained cells in d–f are residual RBCs post-washing. Scale bar = 200 microns. For all micrographs: total magnification = 520×