Surface Marker Profile Research Articles

Sequential Single Cell Transcriptional and Protein Marker Profiling Reveals Tigit As a Marker of CD19 CAR-T Cell Dysfunction in Patients with Non-Hodgkin's Lymphoma

Background: Chimeric antigen receptor T cell (CAR-T) therapy is known to produce durable remissions in the treatment of CD19 + relapsed/refractory B cell malignancies. Nonetheless, many patients receiving CAR-T cells will fail to respond for unknown reasons. Here, we employed single cell RNA sequencing and protein surface marker profiling of serial CAR-T cell samples from patients with non-Hodgkin's lymphoma (NHL) to reveal CAR-T cell evolution, identify biomarkers of response, and test for evidence of exhaustion in CAR-T cells of poor responders.Methods: To describe the evolution of CAR-T cells after infusion into NHL patients and to identify mechanisms and biomarkers of response, our study examined manufactured CAR-T cell products and FACS isolated CAR-T cells from post-infusion blood samples from patients treated for CD19 + relapsed/refractory NHL. Utilizing scRNA sequencing and flow cytometry, we investigated time points after infusion that are known from previous studies to be associated with peak expansion (day 14) and contraction (day 30) and represent key changes in CAR-T cell activity. Altogether, our datasets include 14 manufactured CAR-T cell products, 13 samples from day 14, and 12 samples from day 30. This sampling represents 10 patients with favorable response (complete or partial remission (CR; PR)) and 4 patients with poor response (stable or progressive disease (SD; PD)).To isolate CAR-T cells for scRNA sequencing, viable CD3 +CAR + cells were sorted from cryopreserved CAR-T cell products or PBMCs. Next, libraries were generated with the 10x Genomics Chromium single cell 3' platform with feature barcoding technology to allow simultaneous and paired quantification of transcriptional and cell surface protein expression in individual CAR-T cells. The libraries were sequenced and the data stringently filtered. Batch effect removal was applied to remove differences due to sample preparation or sequencing.Results: Post-filtering, our dataset contained 94,000 cells with an average of 3,917 cells per sample, 8,518 reads per cell, and 2263 unique detectable genes per cell. After dimension reduction and clustering, eleven high-frequency clusters were identified with cluster patterns corresponding with time point, cell cycle phase, cell type, or patient (Figure 1).At the transcriptional level, post-infusion CD8 CAR-T cells displayed significant upregulation of transcription factors (PRDM1, EOMES) and cytotoxic effector molecules (GZMB, PRF1, GZMK, CCL5) associated with differentiation into cytotoxic effector cells as well as transcription factors associated with exhaustion (TOX, TOX2, NR4A2, NR4A3) (p.adj < 0.05) (Figure 2). These data were corroborated by enrichment of effector and exhaustion gene set signatures after infusion as well as global increases in the protein expression of activation and exhaustion markers CD45RO, CD69, CD57, PD1 (CD279), and TIGIT.Contrasts of CAR-T cells between response groups displayed enrichment of an exhaustion profile in CD8 CAR-T cells of poor responders characterized by significant upregulation of the transcription factors FOS, JUNB, JUND, FOSB, JUN, NR4A2, NFKBIA, and PRDM1 and other markers of exhaustion at the RNA level (p.adj < 0.0001). At the protein level, the frequency of TIGIT expression was 20% greater on average in the CD8 CAR-T cells of poor responders compared to favorable responders (Figure 3). CD8 CAR-T cells expressing TIGIT compared to TIGIT negative CD8 CAR-T cells were enriched in an exhaustion transcriptional profile by gene set enrichment analysis and expressed consistently higher levels of exhaustion markers (PD1, CTLA4, LAG3, TIM3) by flow cytometry, regardless of response group or time point (Figure 4).Conclusions: At the transcriptional and protein levels, we note the evolution of CAR-T cells toward a non-proliferative, highly-differentiated, and exhausted state that is enriched in CAR-T cells of patients with poor response. Furthermore, we identified the checkpoint receptor TIGIT as a novel prognostic biomarker and potential driver of CAR-T cell exhaustion. [Display omitted] DisclosuresDropulic: Lentigen: Ended employment in the past 24 months, Patents & Royalties. Caimi: Kite Pharmaceuticals: Consultancy; XaTek: Patents & Royalties: Royalties from patents (wife); Seattle Genetics: Consultancy; Genentech: Research Funding; ADC Theraputics: Consultancy, Research Funding; Verastem: Consultancy; Amgen Therapeutics.: Consultancy; TG Therapeutics: Honoraria. de Lima: Miltenyi Biotec: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees.

Dexamethasone priming enhances stemness and immunomodulatory property of tissue-specific human mesenchymal stem cells

BackgroundHuman Mesenchymal Stem Cells (hMSCs) represent a promising cell source for cell-based therapy in autoimmune diseases and other degenerative disorders due to their immunosuppressive, anti-inflammatory and regenerative potentials. Belonging to a glucocorticoid family, Dexamethasone (Dex) is a powerful anti-inflammatory compound that is widely used as therapy in autoimmune disease conditions or allogeneic transplantation. However, minimal immunomodulatory effect of hMSCs may limit their therapeutic uses. Moreover, the effect of glucocorticoids on the immunomodulatory molecules or other regenerative properties of tissue-specific hMSCs remains unknown.MethodHerein, we evaluated the in vitro effect of Dex at various dose concentrations and time intervals, 1000 ng/ml, 2000 ng/ml, 3000 ng/ml and 24 h, 48 h respectively, on the basic characteristics and immunomodulatory properties of Bone marrow derived MSC (BM-MSCs), Adipose tissue derived MSCs (AD-MSCs), Dental Pulp derived MSC (DP-MSCs) and Umbilical cord derived MSCs (UC-MSCs).ResultsThe present study indicated that the concentration of Dex did not ramify the cellular morphology nor showed cytotoxicity as well as conserved the basic characteristics of tissue specific hMSCs including cell proliferation and surface marker profiling. However, quite interestingly it was observed that the stemness markers (Oct-4, Sox-2, Nanog and Klf-4) showed a significant upregulation in DP-MSCs and AD-MSCs followed by UC-MSCs and BM-MSCs. Additionally, immunomodulatory molecules, Prostaglandin E-2 (PGE-2), Indoleamine- 2,3-dioxygenase (IDO) and Human Leukocyte Antigen-G (HLA-G) were seen to be upregulated in a dose-dependent manner. Moreover, there was a differential response of tissue specific hMSCs after pre-conditioning with Dex during mixed lymphocyte reaction, wherein UC-MSCs and DP-MSCs showed enhanced immunosuppression as compared to AD-MSCs and BM-MSCs, thereby proving to be a better candidate for therapeutic applications in immune-related diseases.ConclusionDex preconditioning improved the hMSCs immunomodulatory property and may have reduced the challenge associated with minimal potency and strengthen their therapeutic efficacy.Graphical Preconditioning of tissue specific hMSCs with dexamethasone biomanufacturers the enhanced potential hMSCs with better stemness and immunomodulatory properties for future therapeutics.

Characterization of Urine-Derived Stem Cells from Patients with End-Stage Liver Diseases and Application to Induced Acute and Chronic Liver Injury of Nude Mice Model.

Urine-derived stem cells (USCs) are adult stem cells isolated from urine with strong proliferative ability and differentiation potentials. Cell transplantation of USCs could partly repair liver injury. It has been reported that the proliferative ability of bone mesenchymal stem cells in patients with chronic liver failure is significantly lower than in patients without liver disease. The aim of this study was therefore to evaluate the biological characteristics of USCs from end-stage liver disease patients (LD-USCs, USCs from patients with liver disease) compared with those from normal healthy individuals (N-USCs, USCs from normal individuals), with a view to determining whether autologous USCs can be applied to the treatment of liver disease. In this study USCs were isolated from urine samples of male patients with end-stage liver disease. Adherent USCs exhibit a spindle- or rice grain-like morphology, and express CD24, CD29, CD73, CD90, and CD146 surface markers, but not CD31, CD34, CD45, and CD105. We observed no differences in cell morphology or cell surface marker profile between LD-USCs and N-USCs. LD-USCs exhibited similar proliferative, colony-forming, apoptotic, and migratory abilities to N-USCs. Both USCs demonstrated similar capacities for osteogenic, adipogenic, and chondrogenic differentiation. When USCs were transplanted into CCl4 treatment-induced acute and chronic liver fibrosis mouse models, we observed a decrease in liver index, recovery of alanine aminotransferase and aspartate aminotransferase levels, alleviation of liver tissue injury, and dramatic improvement of liver tissue structure. USC transplantation can effectively recover liver function and improve liver tissue damage in acute or chronic liver injury mouse models. According to the results, we concluded that the biological characteristics of LD-USCs are not affected by basic liver disease. This study provides further evidence of the stem cell characteristics and liver repair function of LD-USCs, which may serve as a theoretical and experimental foundation for autologous USC transplantation technology in the treatment of liver failure and end-stage liver diseases.

Selection of Highly Proliferative and Multipotent Meniscus Progenitors through Differential Adhesion to Fibronectin: A Novel Approach in Meniscus Tissue Engineering

Meniscus injuries can be highly debilitating and lead to knee osteoarthritis. Progenitor cells from the meniscus could be a superior cell type for meniscus repair and tissue-engineering. The purpose of this study is to characterize meniscus progenitor cells isolated by differential adhesion to fibronectin (FN-prog). Human osteoarthritic menisci were digested, and FN-prog were selected by differential adhesion to fibronectin. Multilineage differentiation, population doubling time, colony formation, and MSC surface markers were assessed in the FN-prog and the total meniscus population (Men). Colony formation was compared between outer and inner zone meniscus digest. Chondrogenic pellet cultures were performed for redifferentiation. FN-prog demonstrated multipotency. The outer zone FN-prog formed more colonies than the inner zone FN-prog. FN-prog displayed more colony formation and a higher proliferation rate than Men. FN-prog redifferentiated in pellet culture and mostly adhered to the MSC surface marker profile, except for HLA-DR receptor expression. This is the first study that demonstrates differential adhesion to fibronectin for the isolation of a progenitor-like population from the meniscus. The high proliferation rates and ability to form meniscus extracellular matrix upon redifferentiation, together with the broad availability of osteoarthritis meniscus tissue, make FN-prog a promising cell type for clinical translation in meniscus tissue-engineering.

Transcriptome Alterations of Prospectively Isolated Bone Marrow Stromal Cells Provide Potential Cues to MGUS and Multiple Myeloma Pathobiology

Multiple myeloma (MM) is the second most common hematopoietic malignancy characterized by the clonal population of plasma cells that invade and destroy adjacent bone tissue. Monoclonal gammopathy of undetermined significance (MGUS) is the premalignant precursor of MM with 1% of patients progressing to MM every year. Although both MGUS and MM plasma cells depend on interactions with bone marrow stromal cells for their survival and growth, little is known about the exact mechanisms of this crosstalk and if specific genetic events in the stromal compartment are correlated with disease state.Primary bone marrow mesenchymal stromal cells (BMSC) are essential components of the hematopoietic microenvironment and they give rise to the hematopoietic stroma in vivo. We have previously reported the phenotype of the bona-fide stroma stem cells in human bone marrow (Li et al., Stem Cell Reports, 3(6):965-74, 2014), and surface marker expression profiling studies on primary lin-CD45-CD71-CD235a-CD31-CD271+ BMSC from patients with MM (n=5), MGUS (n=8) and healthy age-matched controls (n=7) demonstrated clear differences in the expression of signature MSC surface markers, such as CD146, CD105, CD106, CD230, CD51, CD73, and W5C5.Motivated by these striking findings we then went on to transcriptionally interrogate prospectively isolated primary BMSC from MGUS and MM patients using RNAseq. Primary lin-CD45-CD71-CD235a-CD31-CD271+ stromal stem cells from patients undergoing diagnostic bone marrow examination (n=43) and age-matched healthy donors (n=7) were isolated from aspirated bone marrow samples by FACS. Fifteen and five of these patients were diagnosed with MGUS and MM, respectively, and RNA was extracted from bulk sorted populations of primary MM-BMSC, MGUS-BMSC and control-BMSC. The SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing was used for cDNA synthesis. Sequencing libraries were prepared using Nextera XT DNA Library Prep kit (Illumina) and run on a NextSeq500 system (Illumina) using the NextSeq 500/550 High Output Kit v2 (300 cycles).Unsupervised clustering of the data showed that MGUS-BMSC samples clustered partly with healthy controls, but partly also with MM-BMSC or as a distinct and completely separate cluster. Clearly, some MGUS-BMSC showed features of healthy donor BMSC and some exhibited already a transcriptome profile that was more similar to MM-BMSC. In the next step, we therefore restricted the analysis to those BMSC samples that clustered separately and thus represented the MGUS-, MM- and normal BMSC specific transcriptional profiles. Comparison of the transcription patterns of these three distinct groups (MGUS-BMSC, n=3; MM-BMSC, n=5; control-BMSC, n=5) showed that a total of 3,758 genes were differentially expressed between the three groups. Further clustering of the data into 16 non-overlapping subsets of differentially expressed gene groups showed intriguing expression patterns. In one cluster, 78 genes were downregulated only in the MGUS-BMSC group, whereas three clusters contained genes that were downregulated specifically in MM-BMSC, and two clusters showed genes that were upregulated in both, MGUS-BMSC and MM-BMSC compared to control-BMSC. GSEA analysis revealed enrichment of genes reported to be downregulated in MM plasma cells, genes involved in cell adhesion and angiogenesis as well as genes involved in osteoclast differentiation in MM-BMSC versus MGUS-BMSC. Furthermore, MM-BMSCs were enriched for genes involved in integrin-mediated signaling pathway, cell-adhesion, and growth factors and chemokines associated with MM support compared to both control-BMSC and MGUS-BMSC.Taken together, surface marker profiling of prospectively isolated BMSC identified marked differences between these important stromal elements in MGUS, MM, and normal bone marrow. RNAseq of the stroma stem cells identified genes that were specifically upregulated/suppressed in MM-BMSC compared to MGUS-BMSC and control-BMSC. These genes thus represent potential candidates that might be implicated in the disruption of normal stroma function, thus leading to disease development and progression. Accordingly, studies are underway to investigate the effects of selected genes on stroma-myeloma crosstalk (dys)function. DisclosuresNo relevant conflicts of interest to declare.

Human Acquired Aplastic Anemia Patients’ Bone-Marrow-Derived Mesenchymal Stem Cells Are Not Influenced by Hematopoietic Compartment and Maintain Stemness and Immune Properties

Methods In the current study, we investigated the morphological differences, proliferation capacity, population doubling time (PDT), surface marker profiling, trilineage differentiation potential, and immunosuppressive ability of BM Mesenchymal Stem Cells (BM-MSCs) from untreated aAA patients and in the same number of age- and gender-matched controls. Results We observed similar morphology, proliferation capacity, phenotype, trilineage differentiation potential, and immunomodulatory properties of BM-MSCs in aAA patients and control subjects. Conclusion Our results confirm that the basic and immunosuppressive properties of BM-MSCs from aAA patients do not differ from normal BM-MSCs. Our data suggest that BM-MSCs from aAA patients might not be involved in disease pathogenesis. However, owing to a smaller number of samples, it is not conclusive, and future studies with more exhaustive investigation at transcriptome level are warranted.

Different phenotypes and chondrogenic responses of human menstrual blood and bone marrow mesenchymal stem cells to activin A and TGF-\u03b23

BackgroundDue to its low capacity for self-repair, articular cartilage is highly susceptible to damage and deterioration, which leads to the development of degenerative joint diseases such as osteoarthritis (OA). Menstrual blood-derived mesenchymal stem/stromal cells (MenSCs) are much less characterized, as compared to bone marrow mesenchymal stem/stromal cells (BMMSCs). However, MenSCs seem an attractive alternative to classical BMMSCs due to ease of access and broader differentiation capacity. The aim of this study was to evaluate chondrogenic differentiation potential of MenSCs and BMMSCs stimulated with transforming growth factor β (TGF-β3) and activin A.MethodsMenSCs (n = 6) and BMMSCs (n = 5) were isolated from different healthy donors. Expression of cell surface markers CD90, CD73, CD105, CD44, CD45, CD14, CD36, CD55, CD54, CD63, CD106, CD34, CD10, and Notch1 was analyzed by flow cytometry. Cell proliferation capacity was determined using CCK-8 proliferation kit and cell migration ability was evaluated by scratch assay. Adipogenic differentiation capacity was evaluated according to Oil-Red staining and osteogenic differentiation according to Alizarin Red staining. Chondrogenic differentiation (activin A and TGF-β3 stimulation) was investigated in vitro and in vivo (subcutaneous scaffolds in nude BALB/c mice) by expression of chondrogenic genes (collagen type II, aggrecan), GAG assay and histologically. Activin A protein production was evaluated by ELISA during chondrogenic differentiation in monolayer culture.ResultsMenSCs exhibited a higher proliferation rate, as compared to BMMSCs, and a different expression profile of several cell surface markers. Activin A stimulated collagen type II gene expression and glycosaminoglycan synthesis in TGF-β3 treated MenSCs but not in BMMSCs, both in vitro and in vivo, although the effects of TGF-β3 alone were more pronounced in BMMSCs in vitro.ConclusionThese data suggest that activin A exerts differential effects on the induction of chondrogenic differentiation in MenSCs vs. BMMSCs, which implies that different mechanisms of chondrogenic regulation are activated in these cells. Following further optimization of differentiation protocols and the choice of growth factors, potentially including activin A, MenSCs may turn out to be a promising population of stem cells for the development of cell-based therapies with the capacity to stimulate cartilage repair and regeneration in OA and related osteoarticular disorders.

Phenotypic characterization of leukemia-initiating stem cells in chronic myelomonocytic leukemia.

Chronic myelomonocytic leukemia (CMML) is a stem cell-derived neoplasm characterized by dysplasia, uncontrolled expansion of monocytes and a substantial risk to transform to secondary acute myeloid leukemia (sAML). So far, little is known about CMML-initiating cells. We found that leukemic stem cells (LSC) in CMML reside in a CD34+/CD38– fraction of the malignant clone. Whereas CD34+/CD38– cells engrafted NSGS mice with overt CMML, no CMML was produced by CD34+/CD38+ progenitors or the bulk of CD34– monocytes. CMML LSC invariably expressed CD33, CD117, CD123 and CD133. In a subset of patients, CMML LSC also displayed CD52, IL-1RAP and/or CLL-1. CMML LSC did not express CD25 or CD26. However, in sAML following CMML, the LSC also expressed CD25 and high levels of CD114, CD123 and IL-1RAP. No correlations between LSC phenotypes, CMML-variant, mutation-profiles, or clinical course were identified. Pre-incubation of CMML LSC with gemtuzumab-ozogamicin or venetoclax resulted in decreased growth and impaired engraftment in NSGS mice. Together, CMML LSC are CD34+/CD38– cells that express a distinct profile of surface markers and target-antigens. During progression to sAML, LSC acquire or upregulate certain cytokine receptors, including CD25, CD114 and CD123. Characterization of CMML LSC should facilitate their enrichment and the development of LSC-eradicating therapies.

Analysis of Key Distinct Biological Characteristics of Human Placenta-Derived Mesenchymal Stromal Cells and Individual Heterogeneity Attributing to Donors

For potential clinical applications in the future, we investigated the distinct biological features of mesenchymal stromal cells (MSCs) derived from different origin areas of human placenta and individual heterogeneity among different donors. Chorionic plate MSCs (CP-MSCs), amniotic membrane MSCs (AM-MSCs), and decidual plate MSCs (DP-MSCs) were isolated from 5 human placentae and were analyzed in terms of main features of MSCs including surface marker profile, growth, differentiation potential, immune regulation capability, and tubulin acetylation (Ac-tubulin). The expression profile of surface markers in the 3 types of MSCs derived from the 5 donors was relatively stable. Heterogeneity was found in growth, differentiation potential, and immune regulation among MSCs according to the different areas of isolation and different donors. CP-MSCs and AM-MSCs derived from the placentae of donors 1–3 had a higher osteogenic differentiation potential than the corresponding DP-MSCs, but those derived from the placentae of donors 4 and 5 had a markedly lower osteogenic differentiation potential than DP-MSCs. All CP-MSCs derived from donors 1–3 had the highest adipogenic differentiation potential, but CP-MSCs derived from donors 4 and 5 did not show strong capability of adipogenic differentiation. CP-MSCs markedly inhibited the proliferation of peripheral blood mononuclear cells (PBMCs) induced by phytohemagglutinin, whereas AM- and DP-MSCs did not. All MSCs decreased the proportion of CD3+/CD8–/IFN-γ+ Th1 and CD3+/CD8–/IL17+ Th17 cells, but increased the proportion of Treg cells in PBMCs, with individual differences among the 5 donors. DP-MSCs from donors 1 and 2 had higher levels of Ac-tubulin compared with CP- and AM-MSCs. However, the levels of Ac-tubulin in AM-MSCs from donors 3 and 5 were higher than those of the other 2 types of MSCs. Our results revealed that there was tissue-specific heterogeneity among the 3 types of MSCs from different origin tissues of placenta and individual heterogeneity among donors. In future, the pre-selected placenta-derived MSCs with specific biological advantages may improve the curative effect of cell therapy in different situations.

Therapies with CCL25 require controlled release via microparticles to avoid strong inflammatory reactions

BackgroundChemokine therapy with C–C motif chemokine ligand 25 (CCL25) is currently under investigation as a promising approach to treat articular cartilage degeneration. We developed a delayed release mechanism based on Poly (lactic-co-glycolic acid) (PLGA) microparticle encapsulation for intraarticular injections to ensure prolonged release of therapeutic dosages. However, CCL25 plays an important role in immune cell regulation and inflammatory processes like T-cell homing and chronic tissue inflammation. Therefore, the potential of CCL25 to activate immune cells must be assessed more thoroughly before further translation into clinical practice. The aim of this study was to evaluate the reaction of different immune cell subsets upon stimulation with different dosages of CCL25 in comparison to CCL25 released from PLGA particles.ResultsImmune cell subsets were treated for up to 5 days with CCL25 and subsequently analyzed regarding their cytokine secretion, surface marker expression, polarization, and migratory behavior. The CCL25 receptor C–C chemokine receptor type 9 (CCR9) was expressed to a different extent on all immune cell subsets. Direct stimulation of peripheral blood mononuclear cells (PBMCs) with high dosages of CCL25 resulted in strong increases in the secretion of monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), interleukin-1β (IL-1β), tumor-necrosis-factor-α (TNF-α) and interferon-γ (IFN-γ), upregulation of human leukocyte antigen-DR (HLA-DR) on monocytes and CD4+ T-cells, as well as immune cell migration along a CCL25 gradient. Immune cell stimulation with the supernatants from CCL25 loaded PLGA microparticles caused moderate increases in MCP-1, IL-8, and IL-1β levels, but no changes in surface marker expression or migration. Both CCL25-loaded and unloaded PLGA microparticles induced an increase in IL-8 and MCP-1 release in PBMCs and macrophages, and a slight shift of the surface marker profile towards the direction of M2-macrophage polarization.ConclusionsWhile supernatants of CCL25 loaded PLGA microparticles did not provoke strong inflammatory reactions, direct stimulation with CCL25 shows the critical potential to induce global inflammatory activation of human leukocytes at certain concentrations. These findings underline the importance of a safe and reliable release system in a therapeutic setup. Failure of the delivery system could result in strong local and systemic inflammatory reactions that could potentially negate the benefits of chemokine therapy.

Human PD-1hiCD8+ T Cells Are a Cellular Source of IL-21 in Rheumatoid Arthritis.

BackgroundRheumatoid arthritis (RA) is a prototypical autoantibody-driven autoimmune disease in which T-B interactions play a critical role. Recent comprehensive analysis suggests that PD-1+CD8+ T cells as well as two distinct IL-21-producing PD-1+CD4+ T cell subsets, follicular helper T (Tfh) and peripheral helper T (Tph) cells, are involved in the pathogenesis of RA. Herein, we aimed to clarify a generation mechanism of IL-21-producing CD8+ T cells in humans, and to characterize this novel subset in patients with RA.MethodsCD8+ T cells in the peripheral blood (PB) and synovial fluid (SF) of healthy control (HC) and patients with RA were subject to the analysis of IL-21 mRNA and protein. We evaluated the surface marker, cytokine and transcription profiles of IL-21-producing CD8+ T cells in HCPB, RAPB and RASF.ResultsIL-21-producing CD8+ T cells were enriched in the CD45RA-(memory) PD-1+, especially PD-1hi subpopulation, and IL-12 and IL-21 synergistically induced IL-21 production by naïve CD8+ T cells. Memory PD-1hiCD8+ T cells in HCPB facilitated plasmablast differentiation and IgG production in an IL-21-dependent manner. In addition, PD-1hiCD8+ T cells in RASF and RAPB produced large amounts of IL-21 and were characterized by high levels of CD28, ICOS, CD69, HLA-DR, and CCR2 but not CXCR5. Furthermore, PD-1hiCD8+ T cells expressed high levels of transcripts of MAF and PRDM1, a feature observed in Tph cells.ConclusionsIdentification of IL-21-producing PD-1hiCD8+ T cells expands our knowledge of T cell subsets with B helper functions in RA. Selective targeting of these subsets could pave an avenue for the development of novel treatment strategies for this disease.

Proinflammatory T cell polarization is already present in patients with early knee osteoarthritis

BackgroundInvestigating the pathophysiological mechanisms of early osteoarthritis (OA) is of utmost interest since this stage holds the strongest promise for therapeutic interventions. The aims of this study were to analyze if synovial inflammation is already present in early OA and to characterize the involved cell populations, by investigating synovial fluid (SF) and synovial membrane (SM) of early OA patients for the presence and polarization status of CD4 T cells.MethodsA quantitative analysis of CD4+ T cell infiltration in SF and SM compared to peripheral blood (PB) was performed in patients with early stages of OA. We further investigated intracellular staining (ICS), surface marker, and chemokine receptor expression profiles of CD4+ T cells in SF, SM, and PB, as well as cytokine expression in native SF and PB. Matched samples of SF, SM, and PB were harvested from 40 patients with early OA at the time of surgery. Early OA was confirmed by independent surgeons intraoperatively. Samples were analyzed by flow cytometry for surface markers and cytokines, which are preferentially expressed by distinct T cell subsets (Th1, Th2, Th17, regulatory T cells). Furthermore, we analyzed native SF and PB supernatants using MACSPlex for multiple cytokine expression profiles.ResultsSF and SM showed a distinct infiltration of CD4+ T lymphocytes, with significantly increased expression of chemokine receptors CXCR3/CCR5, cytokine IFN-γ (preferentially expressed by Th1 cells), and CD161 (preferentially expressed by IL-17 producing Th17 cells) compared to PB. Furthermore, the percentage of CD4+ T cells polarized to Treg was significantly increased in SM compared to SF and PB. No significant differences were observed for CCR3 and CCR4 (preferentially expressed by Th2 cells), although IL-4 values were significantly higher in SM and SF compared to PB. Cytokine analysis showed comparable results between PB and SF, with only IL-6 being significantly increased in SF.ConclusionsEarly OA joints show already significant inflammation through CD4+ T cell infiltration, with predominant Th1 cell polarization. Inflammation seems to be driven by direct proinflammatory cell interaction. Cytokine signaling seems to be negligible at the site of inflammation in early OA, with only IL-6 being significantly increased in SF compared to PB.

Human Dental Pulp-Derived Mesenchymal Stem Cell Potential to Differentiate into Smooth Muscle-Like Cells In Vitro.

Previous studies have shown that mesenchymal stem cells (MSCs) derived from various tissue sources can be differentiated into smooth muscle-like cells (SMLCs) in vitro. In this paper, dental pulp-derived mesenchymal stem cells (DPSCs) were evaluated for their differentiation ability towards smooth muscle-like cells (SMLCs) under the effect of widely used cytokines (TGF-β1 and PDGF-BB) with special focus on different culturing environments. For this purpose, both the commercially used culturing plates (Norm-c) and 0.1% gelatin-precoated (Gel-c) plates were used. Isolated cells displayed plastic adherence, pluripotency and cell surface marker profiling, and adipogenic and osteogenic differentiation potential with lineage specific marker expression. Differentiated cells induced under different culturing plates showed successful differentiation into SMLCs by positively expressing smooth muscle cell (SMC) specific markers both at the mRNA and protein levels. Gelatin coating could substantially enhance DPSC differentiation potential than Norm-c-induced cells. However, the absence of mature marker MHY-11 by immunostaining results from all treatment groups further indicated the development of immature and synthetic SMLCs. Finally, it was concluded that DPSC differentiation ability into SMLCs can be enhanced under cytokine treatment as well as by altering the cellular niche by precoating the culturing plates with suitable substrates. However, to get fully functional, contractile, and mature SMLCs, still many different cytokine cocktail combinations and more suitable coating substrates will be needed.

Delineation of target expression profiles in CD34+/CD38− and CD34+/CD38+ stem and progenitor cells in AML and CML

AbstractIn an attempt to identify novel markers and immunological targets in leukemic stem cells (LSCs) in acute myeloid leukemia (AML) and chronic myeloid leukemia (CML), we screened bone marrow (BM) samples from patients with AML (n = 274) or CML (n = 97) and controls (n = 288) for expression of cell membrane antigens on CD34+/CD38− and CD34+/CD38+ cells by multicolor flow cytometry. In addition, we established messenger RNA expression profiles in purified sorted CD34+/CD38− and CD34+/CD38+ cells using gene array and quantitative polymerase chain reaction. Aberrantly expressed markers were identified in all cohorts. In CML, CD34+/CD38− LSCs exhibited an almost invariable aberration profile, defined as CD25+/CD26+/CD56+/CD93+/IL-1RAP+. By contrast, in patients with AML, CD34+/CD38− cells variably expressed “aberrant” membrane antigens, including CD25 (48%), CD96 (40%), CD371 (CLL-1; 68%), and IL-1RAP (65%). With the exception of a subgroup of FLT3 internal tandem duplication–mutated patients, AML LSCs did not exhibit CD26. All other surface markers and target antigens detected on AML and/or CML LSCs, including CD33, CD44, CD47, CD52, CD105, CD114, CD117, CD133, CD135, CD184, and roundabout-4, were also found on normal BM stem cells. However, several of these surface targets, including CD25, CD33, and CD123, were expressed at higher levels on CD34+/CD38− LSCs compared with normal BM stem cells. Moreover, antibody-mediated immunological targeting through CD33 or CD52 resulted in LSC depletion in vitro and a substantially reduced LSC engraftment in NOD.Cg-PrkdcscidIl2rgtm1Wjl /SzJ (NSG) mice. Together, we have established surface marker and target expression profiles of AML LSCs and CML LSCs, which should facilitate LSC enrichment, diagnostic LSC phenotyping, and development of LSC-eradicating immunotherapies.

Closing the system: production of viral antigen-presenting dendritic cells eliciting specific CD8+ T cell activation in fluorinated ethylene propylene cell culture bags

BackgroundA major obstacle to anti-viral and -tumor cell vaccination and T cell immunotherapy is the ability to produce dendritic cells (DCs) in a suitable clinical setting. It is imperative to develop closed cell culture systems to accelerate the translation of promising DC-based cell therapy products to the clinic. The objective of this study was to investigate whether viral antigen-loaded monocyte-derived DCs (Mo-DCs) capable of eliciting specific T cell activation can be manufactured in fluorinated ethylene propylene (FEP) bags.MethodsMo-DCs were generated through a protocol applying cytokine cocktails combined with lipopolysaccharide or with a CMV viral peptide antigen in conventional tissue culture polystyrene (TCPS) or FEP culture vessels. Research-scale (< 10 mL) FEP bags were implemented to increase R&D throughput. DC surface marker profiles, cytokine production, and ability to activate antigen-specific cytotoxic T cells were characterized.ResultsMonocyte differentiation into Mo-DCs led to the loss of CD14 expression with concomitant upregulation of CD80, CD83 and CD86. Significantly increased levels of IL-10 and IL-12 were observed after maturation on day 9. Antigen-pulsed Mo-DCs activated antigen-responsive CD8+ cytotoxic T cells. No significant differences in surface marker expression or tetramer-specific T cell activating potency of Mo-DCs were observed between TCPS and FEP culture vessels.ConclusionsOur findings demonstrate that viral antigen-loaded Mo-DCs produced in downscaled FEP bags can elicit specific T cell responses. In view of the dire clinical need for closed system DC manufacturing, FEP bags represent an attractive option to accelerate the translation of promising emerging DC-based immunotherapies.

Transcriptome profiles acquired during cell expansion and licensing validate mesenchymal stromal cell lineage genes

BackgroundMesenchymal stromal cells (MSCs) are rapidly advancing as commercial therapeutics. However, there are still no adequate tools to validate the identity of MSCs and support standardization of MSC-based products. Currently accepted metrics include cell surface marker profiling and tri-lineage differentiation assays, neither of which is definitive. Transcript profiling represents a cost- and time-effective approach amenable to MSC manufacturing processes. Two independent labs recently reported non-overlapping MSC-specific transcriptomic signatures of 489 and 16 genes.MethodsHere, we interrogated our repository of transcriptome data to determine whether routine culture manipulations including cell expansion and immune activation affect expression of the reported MSC lineage genes. These data sets comprise 4 donor populations of human umbilical cord (UC) MSCs serially cultured from cryopreservation thaw through pre-senescence, and 3 donor populations each of naïve UC and bone marrow (BM) MSCs and licensed by 3 different cytokines.ResultsOverall, 437 of 456 proposed signature genes assessed in these data sets were reliably expressed, representing an enduring lineage profile in 96% agreement with the previous studies. Serial passaging resulted in the downregulation of 3 signature genes, and one was silenced. Cytokine stimulation downregulated expression of 16 signature genes, and 3 were uniformly silenced in one or the other MSC type. Fifteen additional genes were unreliably detected, independent of culture manipulation.ConclusionThese results validate and refine the proposed transcriptomic tools for reliable identification of MSCs after isolation through cell expansion and after inflammatory activation. We propose a 24-gene signature to support standardized and accessible MSC characterization.

Tissue-specific angiogenic and invasive properties of human neonatal thymus and bone MSCs: Role of SLIT3-ROBO1.

Although mesenchymal stem/stromal cells (MSCs) are being explored in numerous clinical trials as proangiogenic and proregenerative agents, the influence of tissue origin on the therapeutic qualities of these cells is poorly understood. Complicating the functional comparison of different types of MSCs are the confounding effects of donor age, genetic background, and health status of the donor. Leveraging a clinical setting where MSCs can be simultaneously isolated from discarded but healthy bone and thymus tissues from the same neonatal patients, thereby controlling for these confounding factors, we performed an in vitro and in vivo paired comparison of these cells. We found that both neonatal thymus (nt)MSCs and neonatal bone (nb)MSCs expressed different pericytic surface marker profiles. Further, ntMSCs were more potent in promoting angiogenesis in vitro and in vivo and they were also more motile and efficient at invading ECM in vitro. These functional differences were in part mediated by an increased ntMSC expression of SLIT3, a factor known to activate endothelial cells. Further, we discovered that SLIT3 stimulated MSC motility and fibrin gel invasion via ROBO1 in an autocrine fashion. Consistent with our findings in human MSCs, we found that SLIT3 and ROBO1 were expressed in the perivascular cells of the neonatal murine thymus gland and that global SLIT3 or ROBO1 deficiency resulted in decreased neonatal murine thymus gland vascular density. In conclusion, ntMSCs possess increased proangiogenic and invasive behaviors, which are in part mediated by the paracrine and autocrine effects of SLIT3.

Isolation and characterization of mesenchymal stem cells derived from amniotic fluid: A prospective study

Objectives: The study aims to isolate, expand, and check the feasibility and differentiation potential of amniotic fluid mesenchymal stem cell (AF-MSC) from the minimum amount of amniotic fluid. Materials and Methods: Amniotic fluid samples were collected from women undergoing 2nd trimester amniocentesis between 16 and 24 weeks of period of gestation. MSCs were isolated and characterized by MSCs surface marker profiling and were expanded in specific growth media to assess their differentiation capability into osteocytes, chondrocytes, and adipocytes. The differentiation was confirmed using specific staining. Results: The isolated AF-MSCs showed successful stem cell population for 18 samples out of 23. All the isolated AF-MSCs showed positivity for MSCs surface markers. For osteocyte differentiation, cells were cultured in osteogenic induction media for 4 weeks, and the differentiation was confirmed by staining with Alizarin Red S stain, which showed extracellular matrix mineralization. For adipocytes differentiation, the induction media exhibited lipid droplets and positive staining with Oil Red O stain. Similarly, cells cultured in chondrocytes differentiation media, showed positive staining with Alcian Blue. Conclusion: AF-MSCs have the capacity to differentiate into common mesodermal cell types. Considering their easy accessibility, amniotic fluid could be a good source for MSCs with a greater potential for cellular therapy in various chronic disabling diseases, for example, spinal cord injuries, massive bone and cartilage damage, and demyelinating diseases.

Feasibility of defined, animal component free, protein free, salt base freezing solution composed of 5% DMSO for cryopreservation of HMSC toward clinical applications

Background & Aim hMSC are a critical raw material for cell-based therapies and usually a large number of cells are required for clinical applications. Cryopreservation is currently the only method to preserve cells with maintained functional properties and genetic stability for long term storage. To date, a common practice is use of homebrew cryo-medium that is composed of animal-derived raw materials and low Mw Cryoprotective agents (CPAs), usually 10-20% DMSO. Alternatively, animal component free (ACF) freezing solutions were developed for clinical applications, but they are still based on culture medium and are composed of 10% DMSO (e.g. NutriFreez™ D10). In addition, a few products were developed with a reduced concentration of DMSO or even without DMSO. However, these products appear not to be optimal for hMSC. Moreover, most of the commercially available DMSO-free products are actually composed of other potentially toxic permeable CPAs (e.g. Ethylene Glycol, EG). Exposure of cells to these materials can impact the quality, safety and efficacy of cell-based therapeutic product. Facing strict regulatory requirements, the development of defined, ACF, salt base freezing solution with reduced concentration of DMSO is essential. Methods, Results & Conclusion The current study presents the feasibility of a novel defined, ACF, protein free, salt base freezing solution composed of 5% DMSO for cryopreservation of hMSC from various sources. Results show that the novel cryopreservation solution efficiently maintains high cell yield and viability, and supports the recovery of hMSC while maintaining the normal hMSC features: typical fibroblast-like cell morphology, phenotypic surface marker profile, differentiation capability as well as self-renewal potential. Clinically accepted, cryopreservation solution for hMSC holds a unique opportunity to facilitate the translation of these cells to cellular therapy applications.

Phenotypic and Functional Characterization of human Umbilical Vein Endothelial Cells (hUVEC) Cultures Established under Physiologic vs. Atmospheric Oxygen Tension

Primary cultures of human umbilical vein endothelial cells (hUVEC) have historically been established at atmospheric oxygen (O2) tension (21%) and their surface marker profiles and angiogenic potential in are understood. However, physiologic O2 tension ranges from ~1% in cartilage to ~13% in major arteries, thus 21% O2 represents a supraphysiologic O2 exposure. We conducted experiments to establish and characterize hUVEC at 21% O2 and physiologically relevant oxygen tension (5% O2) from three independent donors.hUVEC were isolated by standard methods with dissociation and culture steps conducted at 21% or 5% O2. Cells were maintained in standard endothelial cell growth media and were cryopreserved at the end of passage 1. Cells were thawed, used to establish live passage 2 cultures, and confirmed to be sterile and free of mycoplasma and endotoxin. hUVEC established at 5% O2 (hUVEC‐LO) were compared to hUVEC established at 21% O2 (hUVEC‐HO) with respect to population doubling time, surface marker expression by flow cytometry, and angiogenic potential by tube formation assay.Both hUVEC‐LO and hUVEC‐HO were immunopositive for CD31, VE‐cadherin, CD146, CD202b, and vEGFR1, and were negative for CD45 and CD90. hUVEC‐LO uniquely expressed a greater percentage of markers associated with an endothelial progenitor phenotype, including CD117, Slug, CD133, and vEGFR2. The calculated population doubling time of hUVEC‐LO was on average 10% faster than the donor‐matched hUVEC‐HO. Angiogenic potential was confirmed by tube formation assay for each lot at the oxygen tension at which they were established. Crossover experiments were conducted to assess tube formation in hUVEC‐LO and hUVEC‐HO under assay conditions of 21% or 5% O2. No statistical differences were noted after 6 hours of assay duration with respect to: number of junctions, segments, meshes, and total length of segments, branches, or extremities. Significant differences were observed beyond 6 hours between hUVEC‐LO and hUVEC‐HO in mean mesh size and number of branches, but the magnitude and pattern of differences varied from donor‐to‐donor. In contrast, the early phase of angiogenesis (0–6 hours) revealed notable differences between hUVEC‐LO and hUVEC‐HO, with hUVEC‐LO generally exhibiting greater variance with respect to number of junctions, mesh area, number of meshes, and number of segments.Taken together, these results suggest that hUVEC‐LO may have a more stem‐like phenotype characterized by the retention of progenitor‐associated surface markers and a faster doubling time compared to hUVEC‐HO from the same donor. While the ultimate capacity for tube formation did not differ between hUVEC‐LO and hUVEC‐HO assayed at 21% or 5% O2, the preliminary data suggest that hUVEC‐LO respond more actively to adapt to changes in oxygen tension, undergoing significant fluctuations in multiple measures at early time points. Further experiments are needed to elucidate the processes and mechanism(s) that lead to differential phenotype and/or performance of hUVEC‐LO and hUVEC‐HO, in particular during the early initiating events of angiogenesis.