Exhaustive expansion: A novel technique for analyzing complex data generated by higher-order polychromatic flow cytometry experiments

<p>Abstract</p> <p>Background</p> <p>The complex data sets generated by higher-order polychromatic flow cytometry experiments are a challenge to analyze. Here we describe Exhaustive Expansion, a data analysis approach for deriving hundreds to thousands of cell phenotypes from raw data, and for interrogating these phenotypes to identify populations of biological interest given the experimental context.</p> <p>Methods</p> <p>We apply this approach to two studies, illustrating its broad applicability. The first examines the longitudinal changes in circulating human memory T cell populations within individual patients in response to a melanoma peptide (gp100_209-2M) cancer vaccine, using 5 monoclonal antibodies (mAbs) to delineate subpopulations of viable, gp100-specific, CD8+ T cells. The second study measures the mobilization of stem cells in porcine bone marrow that may be associated with wound healing, and uses 5 different staining panels consisting of 8 mAbs each.</p> <p>Results</p> <p>In the first study, our analysis suggests that the cell surface markers CD45RA, CD27 and CD28, commonly used in historical lower order (2-4 color) flow cytometry analysis to distinguish memory from naïve and effector T cells, may not be obligate parameters in defining central memory T cells (T_CM). In the second study, we identify novel phenotypes such as CD29+CD31+CD56+CXCR4+CD90+Sca1-CD44+, which may characterize progenitor cells that are significantly increased in wounded animals as compared to controls.</p> <p>Conclusions</p> <p>Taken together, these results demonstrate that Exhaustive Expansion supports thorough interrogation of complex higher-order flow cytometry data sets and aids in the identification of potentially clinically relevant findings.</p

Reduced Satellite Cell Numbers and Myogenic Capacity in Aging Can Be Alleviated by Endurance Exercise

Background: Muscle regeneration depends on satellite cells, myogenic stem cells that reside on the myofiber surface. Reduced numbers and/or decreased myogenic aptitude of these cells may impede proper maintenance and contribute to the age-associated decline in muscle mass and repair capacity. Endurance exercise was shown to improve muscle performance; however, the direct impact on satellite cells in aging was not yet thoroughly determined. Here, we focused on characterizing the effect of moderate-intensity endurance exercise on satellite cell, as possible means to attenuate adverse effects of aging. Young and old rats of both genders underwent 13 weeks of treadmill-running or remained sedentary. Methodology: Gastrocnemius muscles were assessed for the effect of age, gender and exercise on satellite-cell numbers and myogenic capacity. Satellite cells were identified in freshly isolated myofibers based on Pax7 immunostaining (i.e., exvivo). The capacity of individual myofiber-associated cells to produce myogenic progeny was determined in clonal assays (in-vitro). We show an age-associated decrease in satellite-cell numbers and in the percent of myogenic clones in old sedentary rats. Upon exercise, there was an increase in myofibers that contain higher numbers of satellite cells in both young and old rats, and an increase in the percent of myogenic clones derived from old rats. Changes at the satellite cell level in old rats were accompanied with positive effects on the lean-to-fat Gast muscle composition and on spontaneous locomotion levels. The significance of these data is that they suggest that the endurance exercise-mediated boost in bot

Muscle-Bound Primordial Stem Cells Give Rise to Myofiber-Associated Myogenic and Non-Myogenic Progenitors

Myofiber cultures give rise to myogenic as well as to non-myogenic cells. Whether these myofiber-associated non-myogenic cells develop from resident stem cells that possess mesenchymal plasticity or from other stem cells such as mesenchymal stem cells (MSCs) remain unsolved. To address this question, we applied a method for reconstructing cell lineage trees from somatic mutations to MSCs and myogenic and non-myogenic cells from individual myofibers that were cultured at clonal density

Plasticity of the Muscle Stem Cell Microenvironment

Satellite cells (SCs) are adult muscle stem cells capable of repairing damaged and creating new muscle tissue throughout life. Their functionality is tightly controlled by a microenvironment composed of a wide variety of factors, such as numerous secreted molecules and different cell types, including blood vessels, oxygen, hormones, motor neurons, immune cells, cytokines, fibroblasts, growth factors, myofibers, myofiber metabolism, the extracellular matrix and tissue stiffness. This complex niche controls SC biology-quiescence, activation, proliferation, differentiation or renewal and return to quiescence. In this review, we attempt to give a brief overview of the most important players in the niche and their mutual interaction with SCs. We address the importance of the niche to SC behavior under physiological and pathological conditions, and finally survey the significance of an artificial niche both for basic and translational research purposes

The Bacterial Fimbrial Tip Acts as a Mechanical Force Sensor

The subunits that constitute the bacterial adhesive complex located at the tip of the fimbria form a hook-chain that acts as a rapid force-sensitive anchor at high flow

Interplay of Nkx3.2, Sox9 and Pax3 Regulates Chondrogenic Differentiation of Muscle Progenitor Cells

Muscle satellite cells make up a stem cell population that is capable of differentiating into myocytes and contributing to muscle regeneration upon injury. In this work we investigate the mechanism by which these muscle progenitor cells adopt an alternative cell fate, the cartilage fate. We show that chick muscle satellite cells that normally would undergo myogenesis can be converted to express cartilage matrix proteins in vitro when cultured in chondrogenic medium containing TGFß3 or BMP2. In the meantime, the myogenic program is repressed, suggesting that muscle satellite cells have undergone chondrogenic differentiation. Furthermore, ectopic expression of the myogenic factor Pax3 prevents chondrogenesis in these cells, while chondrogenic factors Nkx3.2 and Sox9 act downstream of TGFß or BMP2 to promote this cell fate transition. We found that Nkx3.2 and Sox9 repress the activity of the Pax3 promoter and that Nkx3.2 acts as a transcriptional repressor in this process. Importantly, a reverse function mutant of Nkx3.2 blocks the ability of Sox9 to both inhibit myogenesis and induce chondrogenesis, suggesting that Nkx3.2 is required for Sox9 to promote chondrogenic differentiation in satellite cells. Finally, we found that in an in vivo mouse model of fracture healing where muscle progenitor cells were lineage-traced, Nkx3.2 and Sox9 are significantly upregulated while Pax3 is significantly downregulated in the muscle progenitor cells that give rise to chondrocytes during fracture repair. Thus our in vitro and in vivo analyses suggest that the balance of Pax3, Nkx3.2 and Sox9 may act as a molecular switch during the chondrogenic differentiation of muscle progenitor cells, which may be important for fracture healing