Spectral Flow Cytometry Methods and Pipelines for Comprehensive Immunoprofiling of Human Peripheral Blood and Bone Marrow.

Profiling hematopoietic and immune cells provides important information about disease risk, disease status, and therapeutic responses. Spectral flow cytometry enables high-dimensional single-cell evaluation of large cohorts in a high-throughput manner. Here, we designed, optimized, and implemented new methods for deep immunophenotyping of human peripheral blood and bone marrow by spectral flow cytometry. Two blood antibody panels capture 48 cell-surface markers to assess more than 58 cell phenotypes, including subsets of T cells, B cells, monocytes, natural killer (NK) cells, and dendritic cells, and their respective markers of exhaustion, activation, and differentiation in less than 2 mL of blood. A bone marrow antibody panel captures 32 markers for 35 cell phenotypes, including stem/progenitor populations, T-cell subsets, dendritic cells, NK cells, and myeloid cells in a single tube. We adapted and developed innovative flow cytometric analysis algorithms, originally developed for single-cell genomics, to improve data integration and visualization. We also highlight technical considerations for users to ensure data fidelity. Our protocol and analysis pipeline accurately identifies rare cell types, discerns differences in cell abundance and phenotype across donors, and shows concordant immune landscape trends in patients with known hematologic malignancy. SIGNIFICANCE: This study introduces optimized methods and analysis algorithms that enhance capabilities in comprehensive immunophenotyping of human blood and bone marrow using spectral flow cytometry. This approach facilitates detection of rare cell types, enables measurement of cell variations across donors, and provides proof-of-concept in identifying known hematologic malignancies. By unlocking complexities of hematopoietic and immune landscapes at the single-cell level, this advancement holds potential for understanding disease states and therapeutic responses.

Temporal responsiveness of adipose-derived stem/stromal cell immune plasticity.

We determined the role of time in adipose-derived stem/stromal cell (ASC) response to a model inflammatory environment. ASCs and other mesenchymal stem/stromal cells exhibit immune plasticity. We evaluated the persistence of pro- and anti-inflammatory phenotypes for ASCs exposed to a sustained or pulse inflammatory stimulus. Using qPCR, flow cytometry, and immunocytochemistry, we monitored the temporal expression and up-regulation patterns of a pro-inflammatory gene (caspase 1), a pleiotropic gene/protein (interleukin 6, IL-6), and an anti-inflammatory gene/protein (indoleamine 2, 3-dioxygenase, IDO1) after exposing ASCs to the cytokines tumor necrosis factor-α and interferon-γ. In response to sustained cytokine stimulation, we discovered that time played a role in the balance of pro- and anti-inflammatory ASC phenotypes. IL-6 was present at all time points for both cytokine-stimulated and non-stimulated conditions, whereas IDO1 was heterogeneously up-regulated in stimulated conditions at later time points. After a pulse stimulus, ASC immunoresponse remained consistent for 96-168 h. As a final measure of immune plasticity, we cultured cytokine-stimulated ASCs with blood-derived macrophages to observe macrophage polarization. While the presence of ASCs altered macrophage phenotype, there was no dependency on the length of ASC cytokine exposure time.

Regenerative Potential and Inflammation-Induced Secretion Profile of Human Adipose-Derived Stromal Vascular Cells Are Influenced by Donor Variability and Prior Breast Cancer Diagnosis.

Adipose tissue contains a heterogeneous population of stromal vascular fraction (SVF) cells that work synergistically with resident cell types to enhance tissue healing. Ease of access and processing paired with therapeutic promise make SVF cells an attractive option for autologous applications in regenerative medicine. However, inherent variability in SVF cell therapeutic potential from one patient to another hinders prognosis determination for any one person. This study investigated the regenerative properties and inflammation responses of thirteen, medically diverse human donors. Using non-expanded primary lipoaspirate samples, SVF cells were assessed for robustness of several parameters integral to tissue regeneration, including yield, viability, self-renewal capacity, proliferation, differentiation potential, and immunomodulatory cytokine secretion. Each parameter was selected either for its role in regenerative potential, defined here as the ability to heal tissues through stem cell repopulation and subsequent multipotent differentiation, or for its potential role in wound healing through trophic immunomodulatory activity. These data were then analyzed for consistent and predictable patterns between and across measurements, while also investigating the influence of the donors' relevant medical histories, particularly if the donor was in remission following breast cancer treatment. Analyses identified positive correlations among the expression of three cytokines: interleukin (IL)-6, IL-8, and monocyte chemoattractant protein (MCP)-1. The expression of these cytokines also positively related to self-renewal capacity. These results are potentially relevant for establishing expectations in both preclinical experiments and targeted clinical treatment strategies that use stem cells from patients with diverse medical histories.

Functionally deficient mesenchymal stem cells reside in the bone marrow niche with M2-macrophages and amyloid-ß protein adjacent to loose total joint implants.

We sought to demonstrate whether there is a difference in the local mesenchymal stem cells (MSC) niche obtained from patients undergoing their first total joint replacement surgery versus those patients undergoing a revision surgery for an failing total joint implant. Bone marrow aspirates collected from patients undergoing revision total joint arthroplasty were observed to be less clonal and the expression of PDGFRα, CD51, ALCAM, endoglin, CXCL12, nestin, and nucleostemin were decreased. Revision MSC were also less able to commit to an osteoblast-lineage or an adipocyte-lineage. Further, in revision MSC, OPG, and IL6 expression were increased. Monocytes, derived from revision whole marrow aspirates, were less capable of differentiating into osteoclasts, the cells implicated in the pathologic degradation of bone. Osteoclasts were also not observed in tissue samples collected adjacent to the implants of revision patients; however, the alternatatively activated M2-macrophage phenotype was observed in parallel with pathologic accumulations of amyloid-ß, τ-protien and 3-nitrotyrosine. Despite the limited numbers of patients examined, our data suggest that nucleostemin may be a useful functional marker for MSC while the observation of M2-macrophage infiltration around the implant lays the foundation for future investigation into a novel mechanism that we propose is associated with loose total joint implants.