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A key step in the clinical production of CAR-T cells is the expansion of engineered T cells. To generate enough cells for a therapeutic product, cells must be chronically stimulated, which raises the risk of inducing T-cell exhaustion and reducing therapeutic efficacy. As protocols for T-cell expansion are being developed to optimize CAR T cell yield, function and persistence, fundamental questions about the impact of in vitro manipulation on T-cell identity are important to answer. Namely: 1) what types of cells are generated during chronic stimulation? 2) how many unique cell states can be defined during chronic stimulation? We sought to answer these fundamental questions by performing single-cell multiomic analysis to simultaneously measure expression of 39 proteins and 399 genes in human T cells expanded in vitro. This approach allowed us to study – with unprecedented depth - how T cells change over the course of chronic stimulation. Comprehensive immunophenotypic and transcriptomic analysis at day 0 enabled a refined characterization of T-cell maturational states (from naïve to TEMRA cells) and the identification of a donor-specific subset of terminally differentiated T-cells that would have been otherwise overlooked using canonical cell classification schema. As expected, T-cell activation induced downregulation of naïve-associated markers and upregulation of effector molecules, proliferation regulators, co-inhibitory and co-stimulatory receptors. Our deep kinetic analysis further revealed clusters of proteins and genes identifying unique states of activation defined by markers temporarily expressed upon 3 days of stimulation (PD-1, CD69, LTA), markers constitutively expressed throughout chronic activation (CD25, GITR, LGALS1), and markers uniquely up-regulated upon 14 days of stimulation (CD39, ENTPD1, TNFDF10). Notably, different ratios of cells expressing activation or exhaustion markers were measured at each time point. These data indicate high heterogeneity and plasticity of chronically stimulated T cells in response to different kinetics of activation. In this study, we demonstrate the power of a single-cell multiomic approach to comprehensively characterize T cells and to precisely monitor changes in differentiation, activation and exhaustion signatures in response to different activation protocols.

Advancements in flow cytometry have led to a comprehensive characterization of T cells. While many markers can be used for a detailed immunophenotypic or functional analysis, design of a large multicolor flow cytometry panel is still hampered by the selection of optimal fluorochrome combinations. For example, expansion of existing flow cytometry panels with new markers of interest can result is suboptimal resolution and, in some cases, the need to design a new panel, impacting cost and increasing time to insight. To minimize these challenges and to provide increased flexibility, we have developed a human T cell backbone panel strategically designed to be complemented with 5-8 drop-in markers of choice, depending on instrument configuration. The backbone panel contains five T cell markers (CD3, CD4, CD8, CD45RA, CCR7) conventionally used to identify different maturational states of CD4+ and CD8+ T cells (naïve, central memory, effector memory, effector memory RA). We will show how this backbone panel meets four fundamental requirements: i) clear resolution of major T cell subsets; ii) the fluorochromes used in the backbone panel have minimal resolution impact on the detectors allocated for drop-ins; iii) the fluorochromes assigned to drop-ins have minimal impact on the resolution of the backbone panel; iv) the fluorochromes assigned to drop-ins do not impact each other. We will provide examples of different drop-in combinations for the study of T cell and regulatory T cell biology. Performance of the backbone panel on different instruments will be shown to demonstrate assay consistency across platforms. The compatibility of the backbone panel with intracellular cytokine stain and transcription factor analysis will be further demonstrated. This pre-optimized backbone panel represents a unique tool for researchers who need to study different aspects of T cell biology and want to easily increase their assay capabilities, while minimizing panel design challenges and impact to population resolution. For Research Use Only. Not for use in diagnostic or therapeutic procedures. Citation Format: Mirko Corselli, Chad Sisouvanthong, Nihan Kara, Stephanie Widmann. Development of a human T cell backbone flow cytometry panel enabling flexibility in reagent choice while minimizing panel design challenges [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1392.

Human culture-expanded mesenchymal stromal cells (MSC) are being considered for multiple therapeutic applications because of their regenerative and anti-inflammatory properties. Although a large number of MSC can be propagated from a small initial sample, several lines of evidence indicate that MSC lose their immunosuppressive and regenerative potency after multiple passages. In this report we use the FACSCAP Lyoplate proteomic analysis system to detect changes in cell surface protein expression of CD45−/CD31−/CD34−/CD73+/CD105+ stromal cells in unpassaged bone marrow and through 10 serial culture passages. We provide for the first time a detailed characterization of native unpassaged bone marrow MSC (0.08% of bone marrow mononuclear cells) as well as the changes that occur during the initial expansion. Adipogenic and osteogenic differentiative potential was determined though the serial passages and correlated with immunophenotypic changes and senescence. Among the most prominent were striking decreases in Fas ligand, CD98, CD205 and CD106, accompanied by a gain in the expression of CD49c, CD63, CD98, and class 1 and class 2 major histocompatibility complex (MHC) molecules. Other molecules that are down-modulated with later passage include CD24, CD54, CD59, CD243/P-glycoprotein, and CD273/PD-L2. Early senescence, as defined by loss of replicative capacity occurring with loss of differentiative capacity, increase in CDKN2A p16 and increased time to confluence, was accompanied by loss of the motility-associated metalloproteinase CD10 and the proliferation-associated transferrin receptor CD71. Among the strongest statistical associations were loss of MAC-inhibitory protein/CD59, loss of ICAM-1/CD54, and increase in CDKN2A as a function of increasing passage, as well as increased CD10 expression with adipogenic and osteogenic capacity. The data provide clear set of markers that can be used to assess MSC quality. We suggest that clinically relevant numbers of highly functional low passage MSC can be manufactured starting with large quantities of bone marrow, which are readily available from cadaveric organ donors.

Summary: To facilitate understanding of human cardiomyocyte (CM) subtype specification, and the study of ventricular CM biology in particular, we developed a broadly applicable strategy for enrichment of ventricular cardiomyocytes (VCMs) derived from human embryonic stem cells (hESCs). A bacterial artificial chromosome transgenic H9 hESC line in which GFP expression was driven by the human ventricular-specific myosin light chain 2 (MYL2) promoter was generated, and screened to identify cell-surface markers specific for MYL2-GFP-expressing VCMs. A CD77+/CD200− cell-surface signature facilitated isolation of >97% cardiac troponin I-positive cells from H9 hESC differentiation cultures, with 65% expressing MYL2-GFP. This study provides a tool for VCM enrichment when using some, but not all, human pluripotent stem cell lines. Tools generated in this study can be utilized toward understanding CM subtype specification, and enriching for VCMs for therapeutic applications. : In this article, Evans and colleagues generated an H9 BAC transgenic reporter cell line and performed a flow cytometry screen to identify a cell-surface signature specific for MYL2-GFP-expressing VCMs. The cell-surface signature, CD77+/CD200−, facilitated isolation of a nearly pure hESC-derived CM population, enriched for VCMs. VCM enrichment was achieved when using some, but not all, human pluripotent stem cell lines. Tools generated in this study serve to advance our understanding of CM subtype specification, commitment, and maturation. Keywords: cardiac differentiation, human embryonic stem cells, ventricular cardiomyocytes, cell-surface marker signature

Adipose tissue is a rich source of multipotent mesenchymal stem-like cells, located in the perivascular niche. Based on their surface markers, these have been assigned to two main categories: CD31−/CD45−/CD34+/CD146− cells (adventitial stromal/stem cells [ASCs]) and CD31−/CD45−/CD34−/CD146+ cells (pericytes [PCs]). These populations display heterogeneity of unknown significance. We hypothesized that aldehyde dehydrogenase (ALDH) activity, a functional marker of primitivity, could help to better define ASC and PC subclasses. To this end, the stromal vascular fraction from a human lipoaspirate was simultaneously stained with fluorescent antibodies to CD31, CD45, CD34, and CD146 antigens and the ALDH substrate Aldefluor, then sorted by fluorescence-activated cell sorting. Individual ASCs (n = 67) and PCs (n = 73) selected from the extremities of the ALDH-staining spectrum were transcriptionally profiled by Fluidigm single-cell quantitative polymerase chain reaction for a predefined set (n = 429) of marker genes. To these single-cell data, we applied differential expression and principal component and clustering analysis, as well as an original gene coexpression network reconstruction algorithm. Despite the stochasticity at the single-cell level, covariation of gene expression analysis yielded multiple network connectivity parameters suggesting that these perivascular progenitor cell subclasses possess the following order of maturity: (a) ALDHbrASC (most primitive); (b) ALDHdimASC; (c) ALDHbrPC; (d) ALDHdimPC (least primitive). This order was independently supported by specific combinations of class-specific expressed genes and further confirmed by the analysis of associated signaling pathways. In conclusion, single-cell transcriptional analysis of four populations isolated from fat by surface markers and enzyme activity suggests a developmental hierarchy among perivascular mesenchymal stem cells supported by markers and coexpression networks.

A key step in the clinical production of CAR-T cells is the expansion of engineered T cells. To generate enough cells for a therapeutic product, cells must be chronically stimulated, which raises the risk of inducing T-cell exhaustion and reducing therapeutic efficacy. As protocols for T-cell expansion are being developed to optimize CAR T cell yield, function and persistence, fundamental questions about the impact of in vitro manipulation on T-cell identity are important to answer. Namely: 1) what types of cells are generated during chronic stimulation? 2) how many unique cell states can be defined during chronic stimulation? We sought to answer these fundamental questions by performing single-cell multiomic analysis to simultaneously measure expression of 39 proteins and 399 genes in human T cells expanded in vitro. This approach allowed us to study – with unprecedented depth - how T cells change over the course of chronic stimulation. Comprehensive immunophenotypic and transcriptomic analysis at day 0 enabled a refined characterization of T-cell maturational states (from naive to TEMRA cells) and the identification of a donor-specific subset of terminally differentiated T-cells that would have been otherwise overlooked using canonical cell classification schema. As expected, T-cell activation induced downregulation of naive-associated markers and upregulation of effector molecules, proliferation regulators, co-inhibitory and costimulatory receptors. Our deep kinetic analysis further revealed clusters of proteins and genes identifying unique states of activation defined by markers temporarily expressed upon 3 days of stimulation (PD-1, CD69, LTA), markers constitutively expressed throughout chronic activation (CD25, GITR, LGALS1), and markers uniquely upregulated upon 14 days of stimulation (CD39, ENTPD1, TNFDF10). Notably, different ratios of cells expressing activation or exhaustion markers were measured at each time point. These data indicate high heterogeneity and plasticity of chronically stimulated T cells in response to different kinetics of activation. In this study, we demonstrate the power of a single-cell multiomic approach to comprehensively characterize T cells and to precisely monitor changes in differentiation, activation and exhaustion signatures in response to different activation protocols.

A key step in the clinical production of CAR T cells is the expansion of engineered T cells. To generate enough cells for viable adoptive cell therapy, cells must be robustly stimulated, which raises the risk of inducing T-cell exhaustion and reducing therapeutic efficacy. As protocols for T-cell expansion are being developed to optimize CAR T-cell yield, function and persistence, fundamental questions about the impact of in vitro manipulation on T-cell identity are important to answer. Namely: 1) what types of cells are generated during chronic stimulation? 2) how many unique cell states can be defined during chronic stimulation? We sought to answer these fundamental questions by performing single-cell multiomic analysis using BD® AbSeq and BD Rhapsody™ Single-Cell Analysis system to simultaneously measure expression of 38 proteins and 399 genes in human T cells expanded in vitro. This approach allowed us to study - with unprecedented depth - how T cells change throughout chronic stimulation. Human PBMCs from three healthy donors were stimulated for 14 days in the presence of CD3/CD28 antibody-coated beads and recombinant human IL-2. This model system resembles culture conditions that may be used for CAR T-cell expansion. Cells were collected at different time points (day 0, 3, 7 and 14) prior to downstream analysis. Comprehensive immunophenotypic and transcriptomic analysis at day 0 enabled a refined characterization of T-cell maturational states (from naïve to TEMRA cells) and the identification of a donor-specific subset of terminally differentiated T cells that would have been otherwise overlooked using canonical cell classification schema. As expected, T-cell activation induced downregulation of naïve-associated markers and upregulation of effector molecules, proliferation regulators, co-inhibitory and co-stimulatory receptors. Our deep kinetic analysis further revealed clusters of proteins and genes identifying unique states of activation defined by markers temporarily expressed upon 3 days of stimulation (PD-1, CD69, LTA), markers constitutively expressed throughout chronic activation (CD25, GITR, LGALS1), and markers uniquely up-regulated upon 14 days of stimulation (CD39, ENTPD1, TNFSF10). Notably, different ratios of cells expressing activation or exhaustion markers were measured at each time point. These data indicate heterogeneity and plasticity of chronically stimulated T cells in response to different kinetics of activation. We demonstrate the power of a single-cell multiomic approach to comprehensively characterize T cells and to precisely monitor changes in differentiation, activation and exhaustion signatures in response to different activation protocols. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved. Citation Format: Mirko Corselli, Suraj Saksena, Margaret Nakamoto, Woodrow E. Lomas, Ian Taylor, Pratip K. Chattopadhyay. Deep characterization of in vitro chronically stimulated T cells via single-cell multiomic analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2166.

Cancer treatment has been revolutionized with the development of immunomodulatory therapies. These therapies have primarily focused on enhancing T-cell responses: whether it is unleashing T cells through blockade of regulatory checkpoint inhibitors or generation of chimeric antigen receptor (CAR) T cells. However, there has been recent interest in harnessing the immunotherapeutic potential of other cytotoxic cells such as Natural Killer (NK) cells. Similar to NK cells, innate lymphoid cells (ILCs) may offer another target of these immunotherapy approaches. Before the potential of these cells can be realized, there is a need for better understanding of these recently described cell populations. ILCs act as the immune system's first responders and have been shown to play a key role in tissue homeostasis, chronic inflammation and cancer. Three main groups of non-cytotoxic ILCs (ILC1, ILC2 and ILC3) have been broadly defined based on developmental trajectories and function, driven by expression of specific transcription factors. Deeper characterization of these cells through either high parameter protein analysis or single-cell RNA sequencing has revealed a more complex and heterogeneous nature of ILCs across different tissues and donors. Therefore, the identity of ILCs is still elusive and controversial. In this study, we developed a comprehensive approach to further refine the signatures of human circulating ILC subsets. Total ILCs (Lineage- CD127+ cells) were enriched from 4 normal donors by flow sorting using the BD FACSAria™ Fusion cell sorter and processed for downstream single-cell multiomic characterization. BD® AbSeq reagents and a targeted BD Rhapsody™ Immune Response Panel were used to enable simultaneous detection of 42 proteins and 399 genes using the BD Rhapsody™ Single-Cell Analysis System. Differential protein and gene expression analysis in addition to combinatorial expression of CD294 and CD117 confirmed 3 conventional ILC populations as well as the signatures of three distinct subsets within ILC1. This discovery approach provided information about relative expression of a small selection of proteins or surface marker-coding genes that enable the discrimination of these ILC subsets. These data were used to design high-parameter flow cytometry panels for high-throughput analysis of different healthy donors. ILC subsets differentially distributed across donors were detected and defined using unsupervised computational analysis confirming the result of multiomic analysis, while the functional and biological relevance of the identified subsets remains to be assessed. For Research Use Only. Not for use in diagnostic or therapeutic procedures. Class 1 laser product. BD, the BD Logo, FACSymphony and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved. Citation Format: Aaron J. Tyznik, Mirko Corselli, Gisle V. Baracho, Chip Lomas, Silin Sa, Stephanie Widmann, Suraj Saksena, Smita Ghanekar. Resolving the heterogeneity of human circulating innate lymphoid cells via simultaneous, high-dimensional analysis of protein and gene expression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3310.

To generate enough cells for viable adoptive cell therapy, cells must be robustly stimulated, which raises the risk of inducing T-cell exhaustion and reducing therapeutic efficacy. To comprehensively characterize in vitro chronically expanded human T cells, we performed single-cell multiomic analysis using BD® AbSeq and BD Rhapsody™ Single-Cell Analysis system for simultaneous measurement of the expression of 38 proteins and 399 genes. Comprehensive immunophenotypic and transcriptomic analysis at day 0 enabled a refined characterization of T-cell maturational states. T-cell activation induced by anti-CD3/CD28 beads and recombinant human IL-2 led to downregulation of naïve-associated markers and upregulation of effector molecules, proliferation regulators, co-inhibitory and co-stimulatory receptors. Our deep kinetic analysis further revealed clusters of proteins and genes identifying unique states of activation defined by markers temporarily expressed upon 3 days of stimulation, markers constitutively expressed throughout chronic activation, and markers uniquely up-regulated upon 14 days of stimulation. These data indicate heterogeneity and plasticity of chronically stimulated T cells in response to different kinetics of activation. We demonstrate the power of a single-cell multiomic approach to comprehensively characterize T cells and to precisely monitor changes in differentiation, activation and exhaustion signatures in response to different activation protocols. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved.

Cancer treatment has been revolutionized with the development of immunomodulatory therapies. While these therapies have primarily focused on enhancing T-cell responses, there has been interest in harnessing the potential of other cytotoxic cells such as Natural Killer (NK) cells. Similar to NK cells, innate lymphoid cells (ILCs) may offer another target of these immunotherapy approaches. However, there is a need for better understanding of these recently described cells. In this study, we developed a comprehensive approach to further refine the signatures of human circulating ILC subsets. Total ILCs were enriched using the BD FACSAria™ Fusion cell sorter and processed for downstream single-cell multiomic characterization. BD® AbSeq reagents and a targeted BD Rhapsody™ Immune Response Panel enabled simultaneous detection of 42 proteins and 399 genes using the BD Rhapsody™ Single-Cell Analysis System. Differential protein and gene expression analysis in addition to combinatorial expression of CD294 and CD117 confirmed 3 conventional ILC populations as well as the signatures of 3 distinct subsets within ILC1. This discovery approach provided information about relative expression of a small selection of proteins or surface marker-coding genes that enable the discrimination of these ILC subsets. These data were used to design high-parameter flow cytometry panels for high-throughput analysis. ILC subsets differentially distributed across donors were detected and defined using unsupervised computational analysis confirming the result of multiomic analysis. For Research Use Only. Class 1 laser product. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved.

A key step in the clinical production of CAR T cells is the expansion of engineered T cells. To generate enough cells for a therapeutic product, cells must be robustly stimulated, which raises the risk of inducing T-cell exhaustion and reducing therapeutic efficacy. As protocols for T-cell expansion are being developed to optimize CAR T cell yield, function, and persistence, fundamental questions about the impact of in vitro manipulation on T-cell identity are important to answer. Namely: 1) What types of cells are generated during chronic stimulation? 2) How many unique cell states can be defined during chronic stimulation? We sought to answer these fundamental questions by performing single-cell multiomic analysis to simultaneously measure expression of 38 proteins and 399 genes in human T cells expanded in vitro. This approach allowed us to study—with unprecedented depth—how T cells change over the course of chronic stimulation. Human PBMCs from three healthy donors were continuously stimulated for 14 days in the presence of CD3/CD28 antibody-coated beads and recombinant human IL-2. This model system was developed to resemble culture conditions that may be used for CAR T cell expansion. Cells were collected at different time points (day 0, 3, 7, and 14) prior to downstream analysis. Comprehensive immunophenotypic and transcriptomic analysis at day 0 enabled a refined characterization of T-cell maturational states (from naive to TEMRA cells) and the identification of a donor-specific subset of terminally differentiated T-cells that would have been otherwise overlooked using canonical cell classification schema. As expected, T-cell activation induced downregulation of naive-associated markers and upregulation of effector molecules, proliferation regulators, coinhibitory and costimulatory receptors. Our deep kinetic analysis further revealed clusters of proteins and genes identifying unique states of activation defined by markers temporarily expressed upon 3 days of stimulation (PD-1, CD69, LTA), markers constitutively expressed throughout chronic activation (CD25, GITR, LGALS1), and markers uniquely upregulated upon 14 days of stimulation (CD39, ENTPD1, TNFSF10). Notably, different ratios of cells expressing activation or exhaustion markers were measured at each time point. These data indicate high heterogeneity and plasticity of chronically stimulated T cells in response to different kinetics of activation. In this study, we demonstrate the power of a single-cell multiomic approach to comprehensively characterize T cells and to precisely monitor changes in differentiation, activation, and exhaustion signatures in response to different activation protocols. Citation Format: Mirko Corselli, Margaret Nakamoto, Chip Lomas, Ian Taylor, Suraj Saksena, Tariq Arshad, Pratip K. Chattopadhyay. Deep characterization of in vitro chronically stimulated T cells through single-cell multiomic analysis [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B90.

Perivascular mesenchymal precursor cells (i.e., pericytes) reside in skeletal muscle where they contribute to myofiber regeneration; however, the existence of similar microvessel-associated regenerative precursor cells in cardiac muscle has not yet been documented. We tested whether microvascular pericytes within human myocardium exhibit phenotypes and multipotency similar to their anatomically and developmentally distinct counterparts. Fetal and adult human heart pericytes (hHPs) express canonical pericyte markers in situ, including CD146, NG2, platelet-derived growth factor receptor (PDGFR) β, PDGFRα, alpha-smooth muscle actin, and smooth muscle myosin heavy chain, but not CD117, CD133, and desmin, nor endothelial cell (EC) markers. hHPs were prospectively purified to homogeneity from ventricular myocardium by flow cytometry, based on a combination of positive- (CD146) and negative-selection (CD34, CD45, CD56, and CD117) cell lineage markers. Purified hHPs expanded in vitro were phenotypically similar to human skeletal muscle-derived pericytes (hSkMPs). hHPs express mesenchymal stem/stromal cell markers in situ and exhibited osteo-, chondro-, and adipogenic potentials but, importantly, no ability for skeletal myogenesis, diverging from pericytes of all other origins. hHPs supported network formation with/without ECs in Matrigel cultures; hHPs further stimulated angiogenic responses under hypoxia, markedly different from hSkMPs. The cardiomyogenic potential of hHPs was examined following 5-azacytidine treatment and neonatal cardiomyocyte coculture in vitro, and intramyocardial transplantation in vivo. Results indicated cardiomyocytic differentiation in a small fraction of hHPs. In conclusion, human myocardial pericytes share certain phenotypic and developmental similarities with their skeletal muscle homologs, yet exhibit different antigenic, myogenic, and angiogenic properties. This is the first example of an anatomical restriction in the developmental potential of pericytes as native mesenchymal stem cells. Stem Cells 2015;33:557–573

Flow cytometry is often performed on adherent cells or solid tissues that have been released from their growth substrate or disaggregated by enzymatic digestion. Although detection of strongly expressed cell surface proteins following such procedures indicates that many survive treatment with proteolytic enzymes, applications such as cell surface proteomics involve assessment of the expression of more than 200 proteins and it is important to know how to interpret negative results. To address this problem, we performed flow cytometry-based cell surface proteomic analysis on two non-adherent cell lines, THP1 and K562, after mock and authentic trypsin treatment, according to a widely used protocol to remove adherent cells (0.25% trypsin, 2.21 mM EDTA, 37°C, 5 min). In a single screening experiment, we examined the effect of treatment on mean fluorescence intensity and on the percent of positive cells and determined the false negative rate. Of 164 determinations that were ≥20% positive after mock treatment, 13 (7.9%) were20% positive after trypsin treatment. Four proteins were chosen for time-course studies (performed in triplicate), confirming initial sensitivity results but revealing significant variability in the magnitude of the trypsin effect. When trypsin sensitivity of individual proteins was examined as a function of the number of predicted high probability extracellular trypsin cleavage sites, we found that the markers that yielded false negatives all had high numbers of sites (30), but even so, the majority of proteins with high numbers of trypsin sites could still be detected after mild trypsin treatment. We conclude that the great majority of cell surface proteins can be detected after mild trypsin treatment, but that negative results should not be over-interpreted, due to the possibility of false negatives.

T-cell exhaustion is a dysfunctional state caused by persistent antigen stimulation mediated by chronic infections or cancer. Current immunotherapy approaches focus on reinvigorating exhausted T cells through immune-receptor-blocking antibodies. A deeper understanding of the exhausted T-cell state is crucial for identification of new therapeutic targets. Single cell RNA-sequencing (scRNA-seq) is a powerful tool for resolving heterogeneity in complex systems like these; however the lack of protein information for the same cells can make it difficult to identify cells of interest or detect cellular responses. To resolve both mRNA and protein information in the same assay, we utilized DNA-conjugated BD™ AbSeq antibodies to profile protein and mRNA expression, using high-throughput sequencing analysis at the single cell level. Sorted T cells that had been transiently and chronically stimulated were multiplexed using the BD™ Single-Cell Multiplexing Kit and pooled before undergoing cell partitioning and cDNA capture on the BD Rhapsody™ system; this minimized batch effects and saved time and reagents. A panel of 400 mRNA targets alongside the 39-plex antibody panel was used for the study. We showed that cell-type resolution increased with higher protein plex, and the addition of mRNA information alongside protein markers revealed candidate protein markers for specific cell states. This study showcased the power of multiomics in analyzing heterogenous cell populations and cell states and answering complex biological questions. Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company. © 2019 BD and its subsidiaries. All rights reserved.

Mesenchymal stem/stromal cells (MSCs) are adult multipotent progenitors of great promise for cell therapy. MSCs can mediate tissue regeneration, immunomodulation, and hematopoiesis support. Despite the unique properties of MSCs and their broad range of potential clinical applications, the very nature of these cells has been uncertain. Furthermore, MSCs are heterogeneous and only defined subpopulations of these are endowed with the particular abilities to sustain hematopoietic stem cells, regulate immune responses, or differentiate into mesodermal cell lineages. It is becoming evident that current criteria used to define cultured polyclonal MSCs (expression of nonspecific markers and in vitro mesodermal differentiation) are not sufficient to fully understand and exploit the potential of these cells. Here, we describe how flow cytometry has been used to reveal a perivascular origin of MSCs. As a result, the prospective purification of MSCs and specialized subsets thereof is now possible, and the clinical use of purified autologous MSCs is now within reach.

Hematopoietic stem and progenitor cells (HSPCs) emerge and develop adjacent to blood vessel walls in the yolk sac, aorta-gonad-mesonephros region, embryonic liver, and fetal bone marrow. In adult mouse bone marrow, perivascular cells shape a "niche" for HSPCs. Mesenchymal stem/stromal cells (MSCs), which support hematopoiesis in culture, are themselves derived in part from perivascular cells. In order to define their direct role in hematopoiesis, we tested the ability of purified human CD146(+) perivascular cells, as compared with unfractionated MSCs and CD146(-) cells, to sustain human HSPCs in coculture. CD146(+) perivascular cells support the long-term persistence, through cell-to-cell contact and at least partly via Notch activation, of human myelolymphoid HSPCs able to engraft primary and secondary immunodeficient mice. Conversely, unfractionated MSCs and CD146(-) cells induce differentiation and compromise ex vivo maintenance of HSPCs. Moreover, CD146(+) perivascular cells express, natively and in culture, molecular markers of the vascular hematopoietic niche. Unexpectedly, this dramatic, previously undocumented ability to support hematopoietic stem cells is present in CD146(+) perivascular cells extracted from the nonhematopoietic adipose tissue.

Adipose tissue is an ideal mesenchymal stem cell (MSC) source, as it is dispensable and accessible with minimal morbidity. However, the stromal vascular fraction (SVF) of adipose tissue is a heterogeneous cell population, which has disadvantages for tissue regeneration. In the present study, we prospectively purified human perivascular stem cells (PSCs) from n = 60 samples of human lipoaspirate and documented their frequency, viability, and variation with patient demographics. PSCs are a fluorescence-activated cell sorting-sorted population composed of pericytes (CD45−, CD146+, CD34−) and adventitial cells (CD45−, CD146−, CD34+), each of which we have previously reported to have properties of MSCs. Here, we found that PSCs make up, on average, 43.2% of SVF from human lipoaspirate (19.5% pericytes and 23.8% adventitial cells). These numbers were minimally changed by age, gender, or body mass index of the patient or by length of refrigerated storage time between liposuction and processing. In a previous publication, we observed that human PSCs (hPSCs) formed significantly more bone in vivo in comparison with unsorted human SVF (hSVF) in an intramuscular implantation model. We now extend this finding to a bone injury model, observing that purified hPSCs led to significantly greater healing of mouse critical-size calvarial defects than hSVF (60.9% healing as opposed to 15.4% healing at 2 weeks postoperative by microcomputed tomography analysis). These studies suggest that adipose-derived hPSCs are a new cell source for future efforts in skeletal regenerative medicine. Moreover, hPSCs are a stem cell-based therapeutic that is readily approvable by the U.S. Food and Drug Administration, with potentially increased safety, purity, identity, potency, and efficacy.

We previously demonstrated that human pericytes, which encircle capillaries and microvessels, give rise in culture to genuine mesenchymal stem cells (MSCs). This raised the question as to whether all MSC are derived from pericytes. Pericytes and other cells defined on differential expression of CD34, CD31, and CD146 were sorted from the stromal vascular fraction of human white adipose tissue. Besides pericytes, CD34+ CD31- CD146- CD45- cells, which reside in the outmost layer of blood vessels, the tunica adventitia, natively expressed MSC markers and gave rise in culture to clonogenic multipotent progenitors identical to standard bone marrow-derived MSC. Despite common MSC features and developmental properties, adventitial cells and pericytes retain distinct phenotypes and genotypes through culture. However, in the presence of growth factors involved in vascular remodeling, adventitial cells acquire a pericytes-like phenotype. In conclusion, we demonstrate the co-existence of 2 separate perivascular MSC progenitors: pericytes in capillaries and microvessels and adventitial cells around larger vessels.

Advancements in cell analysis capabilities have significantly expanded our understanding of the complexity of immunological systems. As the need for deeper analysis has increased, many laboratories have employed a set of lineage markers to define a cell population, followed by the addition of unique markers specific to their biological question. Utilizing regulatory T cells (Treg) as a model population, we describe the design of an 8-color backbone panel on a 12-Color BD FACSLyric™ flow cytometer that can be supplemented with 4 drop-in markers (colors) to characterize two critical facets of Treg biology: activation and homing. Intelligent panel design enabled identification of human naïve and activated Treg cells utilizing established Treg markers (CD3, CD4, CD25, CD127, FoxP3, CD45RA). CD15s and CD161 were included in the 8-color backbone panel to identify functionally suppressive effector and/or pro-inflammatory cytokine secreting Tregs. Importantly, addition of a 4-color drop-in activation panel (PI16, CD147, CD39 and HLA-DR) or a 4-color drop-in homing panel (CCR4, CCR6, CXCR3 and CD31) did not impact resolution of the backbone Treg population(s), while providing new and interesting insights into Treg biology. We highlight the utility of a modular panel design approach that provides an efficient, scalable, and standardized solution for complex analysis of essentially any cell population of interest.

Multi-lineage progenitors, e.g. mesenchymal stem cells, persist in adult developed organs, making a windfall for the cell therapist but an enigma for stem cell biologists. Recent results from our own and other laboratories show that the ancestor of these elusive adult stem cells is likely to be found in the perivascular area, explaining the ubiquitous distribution of these cells in the body. We have prospectively identified and purified vascular pericytes in multiple human organs and shown that these cells are potent mesodermal progenitors that give rise to genuine mesenchymal stem cells in culture. Pericytes can differentiate into diverse cell lineages, but also secrete multiple paracrine growth factors/cytokines, which likely explains in part their robust regenerative potential.

We have identified vascular pericytes in multiple human organs on expression of CD146, NG2, PDGF-Rbeta, and mesenchymal stem cell markers (CD44, CD73, CD90, CD105) and absence of blood, endothelial, and myogenic cell markers. Pericytes purified from all tissues were myogenic in culture and in vivo, sustained long-term culture during which they expressed markers of mesenchymal stem cells, and exhibited, at the clonal level, osteogenic, chondrogenic, and adipogenic potentials. These results suggest that human capillary and microvessel walls all over the organism harbor a reserve of progenitor cells that are at the origin of the elusive mesenchymal stem cells, so far identified only retrospectively in primary tissue cultures.

Background CD1d is a monomorphic antigen presentation molecule expressed in several hematologic malignancies. Alpha-galactosylceramide (α-GalCer) is a glycolipid that can be presented to cytotoxic CD1d-restricted T cells. These reagents represent a potentially powerful tool for cell mediated immunotherapy.Design and Methods We set up an experimental model to evaluate the use of adoptively transferred cytotoxic CD1d-restricted T cells and α-GalCer in the treatment of mice engrafted with CD1d+ lymphoid neoplastic cells. To this end the C1R cell line was transfected with CD1c or CD1d molecules. In addition, upon retroviral infection firefly luciferase was expressed on C1R transfected cell lines allowing the evaluation of tumor growth in xenografted immunodeficient NOD/SCID mice.Results The C1R-CD1d cell line was highly susceptible to specific CD1d-restricted T cell cytotoxicity in the presence α-GalCer in vitro. After adoptive transfer of CD1d-restricted T cells and α-GalCer to mice engrafted with both C1R-CD1c and C1R-CD1d, a reduction in tumor growth was observed only in CD1d+ masses. In addition, CD1d-restricted T-cell treatment plus α-GalCer eradicated small C1R-CD1d+ nodules. Immunohistochemical analysis revealed that infiltrating NKT cells were mainly observed in CD1d nodules.Conclusions Our results indicate that ex vivo expanded cytotoxic CD1d-restricted T cells and α-GalCer may represent a new immunotherapeutic tool for treatment of CD1d+ hematologic malignancies.

Mesenchymal stem cells (MSCs), the archetypal multipotent progenitor cells derived in cultures of developed organs, are of unknown identity and native distribution. We have prospectively identified perivascular cells, principally pericytes, in multiple human organs including skeletal muscle, pancreas, adipose tissue, and placenta, on CD146, NG2, and PDGF-R beta expression and absence of hematopoietic, endothelial, and myogenic cell markers. Perivascular cells purified from skeletal muscle or nonmuscle tissues were myogenic in culture and in vivo. Irrespective of their tissue origin, long-term cultured perivascular cells retained myogenicity; exhibited at the clonal level osteogenic, chondrogenic, and adipogenic potentials; expressed MSC markers; and migrated in a culture model of chemotaxis. Expression of MSC markers was also detected at the surface of native, noncultured perivascular cells. Thus, blood vessel walls harbor a reserve of progenitor cells that may be integral to the origin of the elusive MSCs and other related adult stem cells.

Pluripotent stem cells (PSCs) have been successfully differentiated to many cell types relevant to human neurodegenerative diseases. Moreover, induced pluripotent stem cell technology offers an unprecedented opportunity to study these diseases in a patient-specific manner. One impediment to this research is that neural differentiations from PSCs often yield heterogeneous cell cultures, which can hamper or preclude studies requiring pure cell populations. Researchers have partially overcome this hurdle by identifying cell surface signatures of neural stem cells, neurons, glia, and neural crest cells and utilized flow cytometry to isolate near-pure populations of these cells. These advancements have enabled groundbreaking research models of neurodegenerative diseases and have been instrumental in animal models of spinal cord injury. In addition, flow cytometry has been employed to facilitate quantification of neural differentiation cultures. The identification of novel cell surface signatures has also been instrumental in a more comprehensive characterization of pluripotent stem cell derivatives, as well as adult and cancer stem cells. All together, these studies have demonstrated that multiparameter flow cytometry is an indispensable tool for studying neurogenesis and other cell lineages utilizing human PSCs.

While flow cytometry continues to be a critical tool for immunologists, one of the more difficult aspects of this practice is panel design. Fully resolving dim populations of interest can be challenging. Low antigen density, fluorescence spillover, available fluorochromes and limited catalog offerings are just a few of the factors that make panel design difficult often leading to sub-optimal resolution. Previously, we have shown that following a basic set of principles can reduce the number of iterations needed to maximize the resolution for populations of interest. Here we show how recent developments, including the BD Horizon™ Guided Panel Solution (GPS) and BD OptiBuild™ reagents, help simplify panel design even further. The BD GPS was designed with the best practices of panel design at its core. By guiding the user through critical steps in this process and flagging potential problem areas in a proposed panel, the GPS minimizes the need for early panel iteration. BD OptiBuild reagents vastly improves the catalog offering of fluorochrome/specificity combinations, thus reducing the need for panel redesign due to limited reagent availability. To demonstrate the utility of these new tools, we walk through panel design for an 8 color assay on both 3 laser (8 parameter) and 5 laser (18 parameter) systems. The analysis of these panels shows that by using these tools, the chances of obtaining a successful panel increase. Additionally, panel design can be made easier and population resolution improved when utilizing a 5 laser system where impacts of spillover and spread can be minimized. Together, using these tools and approaches the goal is to minimize the number of iterations required to develop panels that meet the needs of the investigator.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Duchenne muscular dystrophy (DMD) is still an untreatable lethal X-linked disorder, which affects 1 in 3500 male births. It is caused by the absence of muscle dystrophin due to mutations in the dystrophin gene. The potential regenerative capacity as well as immune privileged properties of mesenchymal Stem Cells (MSC) has been under investigation for many years in an attempt to treat DMD. One of the questions to be addressed is whether stem cells from distinct sources have comparable clinical effects when injected in murine or canine muscular dystrophy animal models. Many studies comparing different stem cells from various sources were reported but these cells were obtained from different donors and thus with different genetic backgrounds. Here we investigated whether human pericytes obtained from 4 different tissues (muscle, adipose tissue, fallopian tube and endometrium) from the same donor have a similar clinical impact when injected in double mutant Utrn (tm1Ked) Dmd (mdx) /J mice, a clinically relevant model for DMD. After a weekly regimen of intraperitoneal injections of 10(6) cells per 8 weeks we evaluated the motor ability as well as the life span of the treated mice as compared to controls. Our experiment showed that only adipose tissue derived pericytes are able to increase significantly (39 days on average) the life span of affected mice. Microarray analysis showed an inhibition of the interferon pathway by adipose derived pericytes. Our results suggest that the clinical benefit associated with intraperitoneal injections of these adult stem cells is related to immune modulation rather than tissue regeneration.

An ideal mesenchymal stem cell (MSC) source for bone tissue engineering has yet to be identified. Such an MSC population would be easily harvested in abundance, with minimal morbidity and with high purity. Our laboratories have identified perivascular stem cells (PSCs) as a candidate cell source. PSCs are readily isolatable through fluorescent-activated cell sorting from adipose tissue and have been previously shown to be indistinguishable from MSCs in the phenotype and differentiation potential. PSCs consist of two distinct cell populations: (1) pericytes (CD146+, CD34-, and CD45-), which surround capillaries and microvessels, and (2) adventitial cells (CD146-, CD34+, and CD45-), found within the tunica adventitia of large arteries and veins. We previously demonstrated the osteogenic potential of pericytes by examining pericytes derived from the human fetal pancreas, and illustrated their in vivo trophic and angiogenic effects. In the present study, we used an intramuscular ectopic bone model to develop the translational potential of our original findings using PSCs (as a combination of pericytes and adventitial cells) from human white adipose tissue. We evaluated human PSC (hPSC)-mediated bone formation and vascularization in vivo. We also examined the effects of hPSCs when combined with the novel craniosynostosis-associated protein, Nel-like molecule I (NELL-1). Implants consisting of the demineralized bone matrix putty combined with NELL-1 (3 μg/μL), hPSC (2.5×10(5) cells), or hPSC+NELL-1, were inserted in the bicep femoris of SCID mice. Bone growth was evaluated using microcomputed tomography, histology, and immunohistochemistry over 4 weeks. Results demonstrated the osteogenic potential of hPSCs and the additive effect of hPSC+NELL-1 on bone formation and vasculogenesis. Comparable osteogenesis was observed with NELL-1 as compared to the more commonly used bone morphogenetic protein-2. Next, hPSCs induced greater implant vascularization than the unsorted stromal vascular fraction from patient-matched samples. Finally, we observed an additive effect on implant vascularization with hPSC+NELL-1 by histomorphometry and immunohistochemistry, accompanied by in vitro elaboration of vasculogenic growth factors. These findings hold significant implications for the cell/protein combination therapy hPSC+NELL-1 in the development of strategies for vascularized bone regeneration.

Mesenchymal stem/stromal cells (MSCs) are adult multipotent progenitors of great promise for cell therapy. MSCs can mediate tissue regeneration, immunomodulation, and hematopoiesis support. Despite the unique properties of MSCs and their broad range of potential clinical applications, the very nature of these cells has been uncertain. Furthermore, MSCs are heterogeneous and only defined subpopulations of these are endowed with the particular abilities to sustain hematopoietic stem cells, regulate immune responses, or differentiate into mesodermal cell lineages. It is becoming evident that current criteria used to define cultured polyclonal MSCs (expression of nonspecific markers and in vitro mesodermal differentiation) are not sufficient to fully understand and exploit the potential of these cells. Here, we describe how flow cytometry has been used to reveal a perivascular origin of MSCs. As a result, the prospective purification of MSCs and specialized subsets thereof is now possible, and the clinical use of purified autologous MSCs is now within reach.

Human microvascular pericytes (CD146+/34−/45−/56−) contain multipotent precursors and repair/regenerate defective tissues, notably skeletal muscle. However, their ability to repair the ischemic heart remains unknown. We investigated the therapeutic potential of human pericytes, purified from skeletal muscle, for treating ischemic heart disease and mediating associated repair mechanisms in mice. Echocardiography revealed that pericyte transplantation attenuated left ventricular dilatation and significantly improved cardiac contractility, superior to CD56+ myogenic progenitor transplantation, in acutely infarcted mouse hearts. Pericyte treatment substantially reduced myocardial fibrosis and significantly diminished infiltration of host inflammatory cells at the infarct site. Hypoxic pericyte-conditioned medium suppressed murine fibroblast proliferation and inhibited macrophage proliferation in vitro. High expression by pericytes of immunoregulatory molecules, including interleukin-6, leukemia inhibitory factor, cyclooxygenase-2, and heme oxygenase-1, was sustained under hypoxia, except for monocyte chemotactic protein-1. Host angiogenesis was significantly increased. Pericytes supported microvascular structures in vivo and formed capillary-like networks with/without endothelial cells in three-dimensional cocultures. Under hypoxia, pericytes dramatically increased expression of vascular endothelial growth factor-A, platelet-derived growth factor-β, transforming growth factor-β1 and corresponding receptors while expression of basic fibroblast growth factor, hepatocyte growth factor, epidermal growth factor, and angiopoietin-1 was repressed. The capacity of pericytes to differentiate into and/or fuse with cardiac cells was revealed by green fluorescence protein labeling, although to a minor extent. In conclusion, intramyocardial transplantation of purified human pericytes promotes functional and structural recovery, attributable to multiple mechanisms involving paracrine effects and cellular interactions.

Lysophosphatidic acid (LPA) is a pleiotropic phospholipid present in the blood and certain tissues at high concentrations; its diverse effects are mediated through differential, tissue specific expression of LPA receptors. Our goal was to determine if LPA exerts lineage-specific effects during normal human hematopoiesis. In vitro stimulation of CD34+ human hematopoietic progenitors by LPA induced myeloid differentiation but had no effect on lymphoid differentiation. LPA receptors were expressed at significantly higher levels on Common Myeloid Progenitors (CMP) than either multipotent Hematopoietic Stem/Progenitor Cells (HSPC) or Common Lymphoid Progenitors (CLP) suggesting that LPA acts on committed myeloid progenitors. Functional studies demonstrated that LPA enhanced migration, induced cell proliferation and reduced apoptosis of isolated CMP, but had no effect on either HSPC or CLP. Analysis of adult and fetal human bone marrow sections showed that PPAP2A, (the enzyme which degrades LPA) was highly expressed in the osteoblastic niche but not in the perivascular regions, whereas Autotaxin (the enzyme that synthesizes LPA) was expressed in perivascular regions of the marrow. We propose that a gradient of LPA with the highest levels in peri-sinusoidal regions and lowest near the endosteal zone, regulates the localization, proliferation and differentiation of myeloid progenitors within the bone marrow marrow.

Mesenchymal stem/stromal cells (MSC) are currently the best candidate therapeutic cells for regenerative medicine related to osteoarticular, muscular, vascular and inflammatory diseases, although these cells remain heterogeneous and necessitate a better biological characterization. We and others recently described that MSC originate from two types of perivascular cells, namely pericytes and adventitial cells and contain the in situ counterpart of MSC in developing and adult human organs, which can be prospectively purified using well defined cell surface markers. Pericytes encircle endothelial cells of capillaries and microvessels and express the adhesion molecule CD146 and the PDGFRβ, but lack endothelial and haematopoietic markers such as CD34, CD31, vWF (von Willebrand factor), the ligand for Ulex europaeus 1 (UEA1) and CD45 respectively. The proteoglycan NG2 is a pericyte marker exclusively associated with the arterial system. Besides its expression in smooth muscle cells, smooth muscle actin (αSMA) is also detected in subsets of pericytes. Adventitial cells surround the largest vessels and, opposite to pericytes, are not closely associated to endothelial cells. Adventitial cells express CD34 and lack αSMA and all endothelial and haematopoietic cell markers, as for pericytes. Altogether, pericytes and adventitial perivascular cells express in situ and in culture markers of MSC and display capacities to differentiate towards osteogenic, adipogenic and chondrogenic cell lineages. Importantly, adventitial cells can differentiate into pericyte-like cells under inductive conditions in vitro. Altogether, using purified perivascular cells instead of MSC may bring higher benefits to regenerative medicine, including the possibility, for the first time, to use these cells uncultured.

The interaction of tumor cells with the tumor vasculature is mainly studied for its role in tumor angiogenesis and intravascular metastasis of circulating tumor cells. In addition, a specific interaction of tumor cells with the abluminal surfaces of vessels, or angiotropism, may promote the migration of angiotropic tumor cells along the abluminal vascular surfaces in a pericytic location. This process has been termed extravascular migratory metastasis. The abluminal vascular surface may also provide a vascular niche inducing or sustaining stemness to angiotropic tumor cells. This pilot study investigated if angiotropic melanoma cells might represent a subset population with pericytic and embryonic or stem cell properties. Through microarray analysis, we showed that the interaction between melanoma cells and the abluminal surface of endothelial cells triggers significant differential expression of several genes. The most significantly differentially expressed genes have demonstrated properties linked to cancer cell migration (CCL2, ICAM1 and IL6), cancer progression (CCL2, ICAM1, SELE, TRAF1, IL6, SERPINB2 and CXCL6), epithelial to mesenchymal transition (CCL2 and IL6), embryonic/stem cell properties (CCL2, PDGFB, EVX1 and CFDP1) and pericytic recruitment (PDGFB). In addition, bioinformatics-based analysis of the differentially expressed genes has shown that the most significantly enriched functional groups included development, cell movement, cancer, and embryonic development. Finally, the investigation of pericyte/mesenchymal stem cells markers via immunostaining of human melanoma samples revealed expression of PDGFRB, NG2 and CD146 by angiotropic melanoma cells. Taken together, these preliminary data are supportive of the “pericytic mimicry” by angiotropic melanoma cells, and suggest that the interaction between melanoma cells and the abluminal vascular surface induce differential expression of genes linked to cancer migration and embryonic/stem cell properties.

Cell migration occurs during physiological and pathological processes, which include wound healing and tumor metastasis. In vitro migration assays are necessary to understand the mechanism underlying cell migration and also to identify inhibitory or stimulatory molecules. The Boyden chamber assay is commonly used to measure cell motility in vitro where the number of cells that migrate through the transwell membrane is quantitated manually using an inverted microscope. Alternatively, cells can be detached from the membrane and stained with a fluorescent probe, prior to counting cells using a plate reader. These conventional assays are limited by an inability to simultaneously characterize individual cells while monitoring migration. To address this, we have developed a multi-parameter flow cytometry-based migration assay for the quantitative measurement of cell migration and simultaneous immunophenotypic analysis of the migrated cells. Invasive HT-1080 and non-invasive MCF-7 cancer cell lines were plated in the upper layer of a cell permeable membrane, as per the standard Boyden chamber assay. BD Horizon™ Fixable Viability Stain (FVS) was used to determine the optimal detachment conditions providing maximum yield with minimal impact on cell viability. Migrated cells were also co-stained with multiple conjugated antibodies to assess cell surface marker expression. Cells were analyzed on BD Accuri™ flow cytometer for rapid quantitation of migrated cells and analysis of up to 4 parameters. Alternatively, BD FACSCelesta™ flow cytometer was used for higher parameter analysis. Migrated cells were identified based on light scatter properties and viability staining with FVS, while immunophenotype was assessed in live cells. Our results demonstrate that this flow cytometric assay can be used to rapidly quantify changes in the migratory ability of different cell lines in the presence of inhibitors or stimulators. Additionally, we were able to assess the expression of hallmark cancer cell markers (CD44, CD24, CD326 (EpCAM)) before and after migration. The ability to quantify the number of migrating cells in response to specific stimuli and to simultaneously define cell immunophenotype represents a significant advancement, as compared to conventional cell motility assays. Our results demonstrate that this novel approach allows for a deeper analysis of cell migration that could enable complex drug discovery studies and the discovery of new cell signatures associated with metastatic progression. Citation Format: Mirko Corselli, Xiao Wang, Lissette Wilensky, Aaron Middlebrook, Smita Ghanekar, Jacob Rabenstein, Nil Emre. A quantitative and multi-parameter flow cytometry assay to simultaneously assess cell migration and immunophenotype. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5082.

Adipose tissue is an ideal source of mesenchymal stem cells for bone tissue engineering: it is largely dispensable and readily accessible with minimal morbidity. However, the stromal vascular fraction (SVF) of adipose tissue is a heterogeneous cell population, which leads to unreliable bone formation. In the present study, we prospectively purified human perivascular stem cells (PSCs) from adipose tissue and compared their bone-forming capacity with that of traditionally derived SVF. PSCs are a population (sorted by fluorescence-activated cell sorting) of pericytes (CD146+CD34−CD45−) and adventitial cells (CD146−CD34+CD45−), each of which we have previously reported to have properties of mesenchymal stem cells. Here, we found that PSCs underwent osteogenic differentiation in vitro and formed bone after intramuscular implantation without the need for predifferentiation. We next sought to optimize PSCs for in vivo bone formation, adopting a demineralized bone matrix for osteoinduction and tricalcium phosphate particle formulation for protein release. Patient-matched, purified PSCs formed significantly more bone in comparison with traditionally derived SVF by all parameters. Recombinant bone morphogenetic protein 2 increased in vivo bone formation but with a massive adipogenic response. In contrast, recombinant Nel-like molecule 1 (NELL-1; a novel osteoinductive growth factor) selectively enhanced bone formation. These studies suggest that adipose-derived human PSCs are a new cell source for future efforts in skeletal regenerative medicine. Moreover, PSCs are a stem cell-based therapeutic that is readily approvable by the U.S. Food and Drug Administration, with potentially increased safety, purity, identity, potency, and efficacy. Finally, NELL-1 is a candidate growth factor able to induce human PSC osteogenesis.

A theoretical inverse relationship exists between osteogenic (bone forming) and adipogenic (fat forming) mesenchymal stem cell (MSC) differentiation. This inverse relationship in theory partially underlies the clinical entity of osteoporosis, in which marrow MSCs have a preference for adipose differentiation that increases with age. Two pro-osteogenic cytokines have been recently studied that each also possesses antiadipogenic properties: Sonic Hedgehog (SHH) and NELL-1 proteins. In the present study, we assayed the potential additive effects of the biologically active N-terminus of SHH (SHH-N) and NELL-1 protein on osteogenic and adipogenic differentiation of human primary adipose-derived stromal cell (hASCs). We observed that both recombinant SHH-N and NELL-1 protein significantly enhanced osteogenic differentiation and reduced adipose differentiation across all markers examined (alkaline phosphatase, Alizarin red and Oil red O staining, and osteogenic gene expression). Moreover, SHH-N and NELL-1 directed signaling produced additive effects on the pro-osteogenic and antiadipogenic differentiation of hASCs. NELL-1 treatment increased Hedgehog signaling pathway expression; coapplication of the Smoothened antagonist Cyclopamine reversed the pro-osteogenic effect of NELL-1. In summary, Hedgehog and Nell-1 signaling exert additive effects on the pro-osteogenic and antiadipogenic differentiation of ASCs. These studies suggest that the combination cytokines SHH-N+NELL-1 may represent a viable future technique for inducing the osteogenic differentiation of MSCs.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Multipotent stem/progenitor cells with similar developmental potentials have been independently identified from diverse human tissue/organ cultures. The increasing recognition of the vascular/perivascular origin of mesenchymal precursors suggested blood vessels being a systemic source of adult stem/progenitor cells. Our group and other laboratories recently isolated multiple stem/progenitor cell subsets from blood vessels of adult human tissues. Each of the three structural layers of blood vessels: intima, media, and adventitia has been found to include at least one precursor population, that is, myogenic endothelial cells (MECs), pericytes, and adventitial cells (ACs), respectively. MECs and pericytes efficiently regenerate myofibers in injured and dystrophic skeletal muscles as well as improve cardiac function after myocardial infarction. The applications of ACs in vascular remodeling and angiogenesis/vasculogenesis have been examined. Our recent finding that MECs and pericytes can be purified from cryogenically banked human primary muscle cell culture further indicates their potential applications in personalized regenerative medicine.

The search for novel sources of stem cells other than bone marrow mesenchymal stem cells (MSCs) for bone regeneration and repair has been a critical endeavor. We previously established an effective protocol to homogeneously purify human pericytes from multiple fetal and adult tissues, including adipose, bone marrow, skeletal muscle, and pancreas, and identified pericytes as a primitive origin of human MSCs. In the present study, we further characterized the osteogenic potential of purified human pericytes combined with a novel osteoinductive growth factor, Nell-1. Purified pericytes grown on either standard culture ware or human cancellous bone chip (hCBC) scaffolds exhibited robust osteogenic differentiation in vitro. Using a nude mouse muscle pouch model, pericytes formed significant new bone in vivo as compared to scaffold alone (hCBC). Moreover, Nell-1 significantly increased pericyte osteogenic differentiation, both in vitro and in vivo. Interestingly, Nell-1 significantly induced pericyte proliferation and was observed to have pro-angiogenic effects, both in vitro and in vivo. These studies suggest that pericytes are a potential new cell source for future efforts in skeletal regenerative medicine, and that Nell-1 is a candidate growth factor able to induce pericyte osteogenic differentiation.

Mesenchymal stem cells proliferate extensively in cultures of unselected, total cell isolates from multiple fetal and adult organs. Perivascular cells, principally pericytes surrounding capillaries and microvessels, but also adventitial cells located around larger arteries and veins, have been recently identified as possible originators of mesenchymal stem cells, first by phenotypic analogies and eventually following stringent cell sorting. While it is clear that purified perivascular cells exhibit multiple mesodermal developmental potentials and become indistinguishable from conventionally derived mesenchymal stem cells after in vitro culture, the possible roles played by these blood vessel-bound cells in organogenesis and adult tissue repair remain elusive. Unsolved questions regarding the identity of mesenchymal stem cells have not compromised the consideration of these cells as outstanding candidates for cell therapies. Better knowledge of the lineage affiliation, tissue distribution and molecular identity of mesenchymal stem cells will contribute to the development of more efficient, safer therapeutic cells.

Estrogens regulate osteoblast differentiation and mineralization. We identified GATA4 as a transcription factor expressed in osteoblasts and directly regulated by 17β-estradiol in this cell type but not in breast cancer cells, another estrogen-responsive tissue. Chromatin immunoprecipitation sequencing (chromatin immunoprecipitation sequencing) reveals that estrogen receptor α (ERα) binds to chromatin near GATA4 at five different enhancers. GATA4 and ERα are both recruited to ERα binding sites near genes that are specifically expressed in osteoblasts and control osteoblast differentiation. Maximal binding of GATA4 precedes ERα binding, and GATA4 is necessary for histone 3 lysine 4 dimethylation at ERα binding sites, suggesting that GATA4 is a pioneer factor for ERα. As such, knockdown of GATA4 reduced recruitment of ERα to DNA. Our study illustrates that GATA4 is a pioneer factor for ERα recruitment to osteoblast-specific enhancers.

Tissue and organ injury results in alterations of the local microenvironment, including the reduction in oxygen concentration and degradation of the extracellular matrix (ECM). The response of perivascular stem cells to these microenvironment changes are of particular interest because of their wide distribution throughout the body and their potential involvement in tissue and organ response to injury. The chemotactic, mitogenic, and phenotypic responses of this stem cell population were evaluated in response to a combination of decreased oxygen concentration and the presence of ECM degradation products. Culture in low-oxygen conditions resulted in increased proliferation and migration of the cells and increased activation of the ERK signaling pathway and associated integrins without a change in cell surface marker phenotype. The addition of ECM degradation products were additive to these processes. Reactive oxygen species within the cells were increased in association with the mitogenic and chemotactic responses. The increased proliferation and chemotactic properties of this stem cell population without any changes in phenotype and differentiation potential has important implications for both in vitro cell expansion and for in vivo behavior of these cells at the site of injury.

Independent studies by numerous investigators have shown that it is possible to harvest multipotent progenitor cells from diverse dissociated and cultured fetal, perinatal, and principally adult developed tissues. Despite the increasingly recognized medical value of these progenitor cells, the archetype of which remains the mesenchymal stem cell, this indirect extraction method has precluded the understanding of their native identity, tissue distribution, and frequency. Consistent with other researchers, we have hypothesized that blood vessels in virtually all organs harbor ubiquitous stem cells. We have identified, marked, and sorted to homogeneity by flow cytometry endothelial and perivascular cells in a large selection of human fetal, perinatal, and adult organs. Perivascular cells, including pericytes in the smallest blood vessels and adventitial cells around larger ones, natively express mesenchymal stem cell markers and produce in culture a long-lasting progeny of multilineage mesodermal progenitor cells. Herein, we review results from our and other laboratories that suggest a perivascular origin for mesenchymal stem cells and other adult progenitor cells. Recent experiments illustrate the therapeutic potential of human pericytes to regenerate skeletal muscle and promote functional recovery in the diseased heart and kidney.

Two putative types of circulating endothelial progenitor cells have been recently identified in vitro: (1) endothelial colony-forming cell (ECFC) and (2) colony-forming unit–endothelial cell (CFU-EC). Only the former is now recognized to belong to endothelial lineage. We have used the ECFC and CFU-EC assays to readdress the issue of the clonal relation between endothelial progenitor cells and hematopoietic stem cells in patients with Philadelphia-positive and Philadelphia-negative chronic myeloproliferative disorders. Both ECFCs and CFU-ECs were cultured from peripheral blood mononuclear cells, and either BCR-ABL rearrangement or JAK2-V617F mutation were assessed in both types of endothelial colonies. We found that ECFCs lack the disease-specific markers, which are otherwise present in CFU-ECs, thus reinforcing the concept that the latter belongs to the hematopoietic lineage, and showing that in chronic myeloproliferative disorders the cell that gives rise to circulating ECFC has a distinct origin from the cell of the hematopoietic malignant clone.