Cell Surface Marker Profile Research Articles

Multiomic Single-Cell Analysis Identifies Von Willebrand Factor and TIM3-Expressing BCR-ABL1+ Chronic Myeloid Leukemia Stem Cells

Characterization of the leukemic stem cell (LSC) population in chronic myeloid leukemia (CML) may identify therapeutic targets for sustained elimination of leukemic cells. High-throughput single-cell RNAseq (scRNAseq) has paved the way for detailed transcriptional assessment of CML LSC. Building on this technique, multiomic CITE-seq approaches carry additional benefit in that the paired protein expression information may enable identification of associated cell surface marker profiles, subsequent FACS-based isolation and functional characterization. However, detection of BCR-ABL1 transcripts, which is arguably the only unequivocal marker to distinguish CML LSC from healthy hematopoietic stem cells (HSC), remains challenging using current high-throughput 3' end capture-based scRNAseq methods. For the present study, we performed single-cell CITE-seq analysis of the expression of 597 genes and 51 proteins in >70,000 stem and progenitor cells from 16 chronic phase CML patients and five healthy donors. Furthermore, we developed and employed a strategy allowing parallel detection of BCR-ABL1 transcripts at the single-cell level. In bone marrow samples from CML patients, we observed pronounced expansion of erythroid and myeloid progenitors within the CD14 -CD34 + compartment, and substantial heterogeneity within the traditionally defined CD34 +CD38 -/low LSC compartment. In-depth analysis of the latter identified a group of immature BCR-ABL1 +cells displaying a CD45RA -cKIT -CD26 + TKI resistance phenotype sitting atop a hierarchy of myeloid progenitors. Within this group of potential LSC, expression of both previously reported ( e.g. CD25 and CD26) and unreported gene and protein markers was found to distinguish these cells from their healthy counterparts. Unlike HSC, the immature LSC showed high surface expression of TIM3 and high transcription of the von Willebrand factor gene ( VWF). While overexpression of VWF within the stem cell population may be linked to the aberrant myeloid-biased hematopoiesis characterizing the disease, the cell surface upregulation of TIM3 suggests that its targeting may have merit in CML. In conclusion, we here performed detailed multiomic characterization of CML stem and progenitor cells, paired with BCR-ABL1 detection at the single-cell level. Our findings revealed LSC-specific expression patterns that may have implications for the phenotypic definition of CML LSC. Additionally, the results identify TIM3 as a conceivable target for sustained elimination of immature LSC in CML.

ChemR23 activation reprograms macrophages toward a less inflammatory phenotype and dampens carcinoma progression.

Tumor Associated Macrophages (TAM) are a major component of the tumor environment and their accumulation often correlates with poor prognosis by contributing to local inflammation, inhibition of anti-tumor immune response and resistance to anticancer treatments. In this study, we thus investigated the anti-cancer therapeutic interest to target ChemR23, a receptor of the resolution of inflammation expressed by macrophages, using an agonist monoclonal antibody, αChemR23. Human GM-CSF, M-CSF and Tumor Associated Macrophage (TAM)-like macrophages were obtained by incubation of monocytes from healthy donors with GM-CSF, M-CSF or tumor cell supernatants (Breast cancer (BC) or malignant pleural mesothelioma (MPM) cells). The effects of αChemR23 on macrophages were studied at the transcriptomic, protein and functional level. Datasets from The Cancer Genome Atlas (TCGA) were used to study CMKLR1 expression, coding for ChemR23, in BC and MPM tumors. In vivo, αChemR23 was evaluated on overall survival, metastasis development and transcriptomic modification of the metastatic niche using a model of resected triple negative breast cancer. We show that ChemR23 is expressed at higher levels in M-CSF and tumor cell supernatant differentiated macrophages (TAM-like) than in GM-CSF-differentiated macrophages. ChemR23 activation triggered by αChemR23 deeply modulates M-CSF and TAM-like macrophages including profile of cell surface markers, cytokine secretion, gene mRNA expression and immune functions. The expression of ChemR23 coding gene (CMKLR1) strongly correlates to TAM markers in human BC tumors and MPM and its histological detection in these tumors mainly corresponds to TAM expression. In vivo, treatment with αChemR23 agonist increased mouse survival and decreased metastasis occurrence in a model of triple-negative BC in correlation with modulation of TAM phenotype in the metastatic niche. These results open an attractive opportunity to target TAM and the resolution of inflammation pathways through ChemR23 to circumvent TAM pro-tumoral effects.

Issue highlights-May 2023.

In the era of personalized medicine, the list of targeted therapeutic drugs has been greatly expanded, many of which are employed to treat hematolymphoid neoplasms. These include drugs specifically targeting immune checkpoint signaling epitopes such as programmed cell death 1 (PD1) and its ligand PDL1 to inhibit T-cell activation. Alternatively, these drugs might target the surface antigens expressed by tumor cells such as CD19 and CD22 in B-cell neoplasms. Of the latter, targeted immunotherapies include monoclonal antibodies with or without drug conjugates, bispecific T-cell engagers, or chimeric antigen receptor (CAR) T-cell therapy. The binding sites of these therapeutic antibodies are often identical or in proximity with the binding sites of the diagnostic antibodies. Therefore, use of these therapies pose great challenges for clinical cytometry labs in the assessment of post-treatment samples, especially in the detection of measurable/minimal residual disease (MRD). In this issue, Chen, Gao, et al. (2023) provided an overview of MRD detection in B-lymphoblastic leukemia/lymphoma in the era of immunotherapy, and Gao et al. (2023) focused their review on the impact of targeted therapy on mature B- and plasma cell neoplasms utilizing flow cytometry assessments. In both reviews, the authors illustrated the challenges, identified the problems, and provided a list of available options and solutions. For each of the above-mentioned disease categories, optimal gating and analysis strategies were illustrated with literature review and inputs from the authors' experience and insights. The utility and interpretation of additional B-cell markers other than CD19 and CD20 for mature or immature B-cell neoplasms such as CD22, CD24, and cCD79a (Mikhailova et al., 2022) were studied as well as VS38, CD229, and CD319 (Pojero et al., 2016; Soh et al., 2021) for plasma cell neoplasms. The authors recommended that the flow cytometry assays in the era of targeted therapies must contain significant redundancy in the antibody panels allowing the detection of the cells of interest and additionally should be ready to utilize several gating strategies for accurate and consistent population identification. Neoplastic mature B-cells often show restricted kappa or lambda light chain expression; however, light chain expression may be absent in around 5%–10% of mature B-cell lymphoma (Li et al., 2002). It is suggested that mature B-cells lacking surface light chain expression can be used as a surrogate marker to diagnose mature B-cell lymphomas. Huang et al. (2023) reported a series of 89 cases of surface light chain negative B-cell lymphoma which consisted primarily of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Interestingly, the authors also reported no detectable light chain expression in normal/reactive mature B cells collected from body fluids and cystic fluids from 14 patients with no clinical and pathological evidence of lymphoma/leukemia. Body fluids are often proteaceous; thus additional washing prior to kappa/lambda staining is often required to obtain adequate light chain staining and caution is needed in the interpretation of light chain expression in B-cells collected from body fluids. It is known that out of the context of chronic myeloid leukemia (CML) (Soma et al., 2016), lymphoblastic crisis is extremely rare in other chronic myeloid neoplasms, such as myelodysplastic syndrome (MDS) (Xie et al., 2019) and Philadelphia negative myeloproliferative neoplasms (MPN). Chan et al. (2023) reported the detection of a low level of abnormal B-lymphoblasts ranging from 0.012% to 3.6% in 9 out of 1262 MDS or Ph-negative MPN. Cell sorting coupled with next-generational sequencing showed that the abnormal B- lymphoblasts in myeloid neoplasms were often, but not always, clonally related to the myeloid compartment, and the molecular genetic findings suggested a mutant multipotent progenitor cell with different lineage output that might be determined by the specific mutations and the particular cell stage where the mutations occurred. Importantly, unlike CML, the presence of abnormal B-lymphoblasts in MDS and Ph-neg MPNs did not necessarily indicate blast crisis, and therapy for the underlying myeloid neoplasms with close monitoring is suggested to be a reasonable approach. The last article in this issue (Chen, Zhang, et al., 2023) studied mucosal-associated invariant T cells (MAIT) (Godfrey et al., 2019) in aplastic anemia (AA), a type of bone marrow failure directly linked to abnormal immune responses. MAIT cells exhibit an effector-memory phenotype and express several NK receptors such as NKG2D and CD161 and play an important role in the development of autoimmune diseases. The authors showed an increased frequency of MAIT cells with an activation and effector function-related cell surface marker profile in patients with aplastic anemia. The specific property of NKG2D expression on MAIT cells suggests that these cells may have a distinct mechanism involved in AA immune pathogenesis.

Expanded characterization of in vitro polarized M0, M1, and M2 human monocyte-derived macrophages: Bioenergetic and secreted mediator profiles.

Respiratory macrophage subpopulations exhibit unique phenotypes depending on their location within the respiratory tract, posing a challenge to in vitro macrophage model systems. Soluble mediator secretion, surface marker expression, gene signatures, and phagocytosis are among the characteristics that are typically independently measured to phenotype these cells. Bioenergetics is emerging as a key central regulator of macrophage function and phenotype but is often not included in the characterization of human monocyte-derived macrophage (hMDM) models. The objective of this study was to expand the phenotype characterization of naïve hMDMs, and their M1 and M2 subsets by measuring cellular bioenergetic outcomes and including an expanded cytokine profile. Known markers of M0, M1 and M2 phenotypes were also measured and integrated into the phenotype characterization. Peripheral blood monocytes from healthy volunteers were differentiated into hMDM and polarized with either IFN-γ + LPS (M1) or IL-4 (M2). As expected, our M0, M1, and M2 hMDMs exhibited cell surface marker, phagocytosis, and gene expression profiles indicative of their different phenotypes. M2 hMDMs however were uniquely characterized and different from M1 hMDMs by being preferentially dependent on oxidativte phosphorylation for their ATP generation and by secreting a distinct cluster of soluble mediators (MCP4, MDC, and TARC). In contrast, M1 hMDMs secreted prototypic pro-inflammatory cytokines (MCP1, eotaxin, eotaxin-3, IL12p70, IL-1α, IL15, TNF-β, IL-6, TNF-α, IL12p40, IL-13, and IL-2), but demonstrated a relatively constitutively heightened bioenergetic state, and relied on glycolysis for ATP generation. These data are similar to the bioenergetic profiles we previously observed in vivo in sputum (M1) and BAL (M2)-derived macrophages in healthy volunteers, supporting the notion that polarized hMDMs can provide an acceptable in vitro model to study specific human respiratory macrophage subtypes.

A comparison study of dental pulp stem cells derived from healthy and orthodontically intruded human permanent teeth for mesenchymal stem cell characterisation.

The objective of this study was to compare the characteristics of Dental Pulp Stem Cells (DPSCs) derived from healthy human permanent teeth with those that were orthodontically-intruded to serve as potential Mesenchymal Stem Cells (MSC). Recruited subjects were treated with orthodontic intrusion on one side of the maxillary first premolar while the opposite side served as the control for a period of six weeks before the dental pulp was extracted. Isolated DPSCs from both the control and intruded samples were analyzed, looking at the morphology, growth kinetics, cell surface marker profile, and multilineage differentiation for MSC characterisation. Our study showed that cells isolated from both groups were able to attach to the cell culture flask, exhibited fibroblast-like morphology under light microscopy, able to differentiate into osteogenic, adipogenic and chondrogenic lineages as well as tested positive for MSCs cell surface markers CD90 and CD105 but negative for haematopoietic cell surface markers CD34 and HLA-DR. Both groups displayed a trend of gradually increasing population doubling time from passage 1 to passage 5. Viable DPSCs from both groups were successfully recovered from their cryopreserved state. In conclusion, DPSCs in the dental pulp of upper premolar not only remained viable after 6 weeks of orthodontic intrusion using fixed appliances but also able to develop into MSCs.

Expansion of a CD8+ Temra Population and Activating T-Cell Interactions Characterize the Graft Versus Leukemia Response in Relapsed AML

Graft-versus-leukemia (GvL) activity underlies the curative potential of allogeneic hematopoietic stem cell transplantation. Its effectiveness is well-exemplified by donor lymphocyte infusion (DLI), an established adoptive cellular therapy for treatment of relapsed disease. Whereas ~80% of chronic myelogenous leukemia (CML) patients achieve durable remissions to DLI, responses for acute myelogenous leukemia (AML) are only 15-20%. We hypothesized that systematic characterization of immune cell composition and cellular interactions within the marrow tumor microenvironment in relation to DLI would illuminate the molecular basis of the disease-specific susceptibility to GvL. Given the heterogeneous nature of AML, we suspected AML response to comprise more complex leukemic-immune cell interactions than in CML. To these ends, we generated 5’ single-cell RNA- and TCR-sequencing (scTCRseq) and cell surface marker profiling (scCITE-seq) on 24 bone marrow biopsy samples collected before and after DLI from 9 patients with relapsed AML (5 responders [R], 4 nonresponders [NR]). We merged this dataset with our previously reported CML dataset (14 patients, 8 R, 6 NR [PMID: 34758319]) and batch corrected these data with scVI to obtain a total of 414,000 high-quality single-cell transcriptomes for analysis. Evaluation of the immune cell subset proportions over time revealed 10 populations with distinct dynamics in AML responders to DLI but not CML, while 3 other populations showed similar dynamics across diseases. Both CML and AML DLI response were associated with pre-treatment enrichment of an NK cell population highly expressing XCL1, XCL2, CCL3, CCL4, CCL4L2, and CCL5, consistent with a tumor-infiltrating expression profile. Unique to CML (i.e. not observed in AML), in addition to post-DLI enrichment of the TPEX population previously identified, we found a γδ-like T cell population and a classical dendritic cell population exhibiting post-DLI enrichment associated with response to DLI. In AML, response to DLI was most consistently associated with expansion of a CD8+ T cell population (6.03% of total pre-DLI cells vs 18.31% post-DLI for R [p=0.016] compared to 8.68% pre-DLI vs 9.55% post-DLI for NR [p=0.454]) and a naïve B cell population (4.02% pre-DLI vs 11.68% post-DLI for R [p=0.002] compared to 5.42% pre-DLI vs 5.17% post-DLI for NR [p=0.471]). The CD8+ T cell population was characterized by high expression of cytotoxicity genes (e.g. GZMA, GZMB, GZMH, GZMM, PRF1, KLRG1, TOX, EOMES, TBX21) and was most consistent with a T effector memory re-expressing RA (TEMRA) profile, supported by high CD45RA expression by scCITE-seq. This population also highly expressed ZNF683, which encodes the transcription factor Hobit, known to regulate cytotoxicity in CD8+ T cells. Differential gene expression between AML R and NR TEMRA cells revealed enriched expression of ZNF683, GZMH, NKG7, CCL5, and CXCR4 in R and the exhaustion marker TIGIT and the senescence marker KLRG1 in NR. scTCR-seq of the TEMRA subset demonstrated clonal expansion and differentiation into a more proliferative expression profile post-DLI. Among AML cases, 2-20% of the post-DLI repertoire of responders originated from the DLI product while no TCR from the DLI product was detected in the single NR patient available for analysis. We applied predictive interaction modeling among all 50 cell type pairs using CellPhoneDB (PMID: 32103204), which utilizes a publicly available curated database of receptor-ligand interactions. Predicted interactions unique to R between CD8+ TEMRA and AML leukemia cells included CD226-PVR, CD6-ALCAM, and CD46-JAG1, which have been associated with T cell activation. Interactions unique to NR between CD8+ TEMRA and AML leukemia included the inhibitory interactions TIGIT-NECTIN and PTPRC-MRC1. These interactions were not observed in CML patients in either R or NR between CD8+ TEMRA and CML cells, suggesting disease-specific response and resistance mechanisms. Here we show that response to DLI in AML is associated with largely distinct expansion of immune cell populations compared to CML, including not only T cells but an orchestration of B, NK, and myeloid cell subsets. AML responders were uniquely characterized by expansion of a TEMRA subset marked by high expression of the pro-cytotoxic gene ZNF683. Further investigation is needed to evaluate the role of this population in mediating GvL response.

Preoperative exercise induces endothelial progenitor cell mobilisation in patients undergoing major surgery – A prospective randomised controlled clinical proof-of-concept trial

IntroductionPrehabilitation is increasingly recognised as a therapeutic option to reduce postoperative complications. Investigating the beneficial effects of exercise on cellular mechanisms, we have previously shown that a single episode of exhaustive exercise effectively stimulates endothelial progenitor cells (a cell population associated with vascular maintenance, repair, angiogenesis, and neovascularization) in correlation with fewer postoperative complications, despite the ongoing debate about the appropriate cell surface marker profiles of these cells (common phenotypical definitions include CD45dim, CD133+, CD34+ and/or CD31+). In order to translate these findings into clinical application, a feasible prehabilitation programme achieving both functional and cellular benefits in a suitable timeframe to expedite surgery is necessary. ObjectiveThe objective of this study was to test the hypothesis that a four-week prehabilitation programme of vigorous-intensity interval exercise training is feasible, increases physical capacity (primary outcome) and the circulatory number of endothelial progenitor cells within peripheral blood. MethodsIn this unblinded, parallel-group, randomised controlled proof-of-concept clinical trial (German Clinical Trial Register number: DRKS00000527) conducted between 01st December 2014 and 30th November 2016, fifteen female adult patients scheduled for incontinence surgery with abdominal laparotomy at the University Hospital Cologne were allocated to either an exercise (n = 8, exclusion of 1 patient, analysed n = 7) or non-exercise group (n = 7, exclusion of 1 patient, analysed n = 6). The exercise group's intervention consisted of a vigorous-intensity interval training for four weeks preoperatively. Cardiopulmonary Exercise Testing accompanied by peripheral blood collection was performed before and after the (non-)training phase. Cellular investigations were conducted by flow cytometry and cluster-based analyses. ResultsVigorous-intensity interval training over four weeks was feasible in the exercise group (successful completion by 8 out of 8 patients without any harms), with significant improvements in patients' functional capacity (increased oxygen uptake at anaerobic threshold [intervention group mean + 1.71 ± 3.20 mL/min/kg vs. control group mean −1.83 ± 2.14 mL/min/kg; p = 0.042] and peak exercise [intervention group mean + 1.71 ± 1.60 mL/min/kg vs. control group mean −1.67 ± 1.37 mL/min/kg; p = 0.002]) and a significant increase in the circulatory number of endothelial progenitor cells (proportionate CD45dim/CD14dim/CD133+/CD309+/CD34+/CD31 + subpopulation within the circulating CD45-pool [p = 0.016]). ConclusionsWe introduce a novel prehabilitation concept that shows effective stimulation of an endothelial progenitor cell subpopulation within four weeks of preoperative exercise, serving as a clinical cell-mediated intervention with the aim to reduce surgical complications. FundingInstitutional funding. DFG (German Research Foundation, 491454339) support for the Article Processing Charge.

Dynamics of monocyte-derived macrophage diversity in experimental myocardial infarction.

Macrophages have a critical and dual role in post-ischaemic cardiac repair, as they can foster both tissue healing and damage. Multiple subsets of tissue resident and monocyte-derived macrophages coexist in the infarcted heart, but their precise identity, temporal dynamics, and the mechanisms regulating their acquisition of discrete states are not fully understood. To address this, we used multi-modal single-cell immune profiling, combined with targeted cell depletion and macrophage fate mapping, to precisely map monocyte/macrophage transitions after experimental myocardial infarction. We performed single-cell transcriptomic and cell-surface marker profiling of circulating and cardiac immune cells in mice challenged with acute myocardial infarction, and integrated single-cell transcriptomes obtained before and at 1, 3, 5, 7, and 11 days after infarction. Using complementary strategies of CCR2+ monocyte depletion and fate mapping of tissue resident macrophages, we determined the origin of cardiac macrophage populations. The macrophage landscape of the infarcted heart was dominated by monocyte-derived cells comprising two pro-inflammatory populations defined as Isg15hi and MHCII+Il1b+, alongside non-inflammatory Trem2hi cells. Trem2hi macrophages were observed in the ischaemic area, but not in the remote viable myocardium, and comprised two subpopulations sequentially populating the heart defined as Trem2hiSpp1hi monocyte-to-macrophage intermediates, and fully differentiated Trem2hiGdf15hi macrophages. Cardiac Trem2hi macrophages showed similarities to 'lipid-associated macrophages' found in mouse models of metabolic diseases and were observed in the human heart, indicating conserved features of this macrophage state across diseases and species. Ischaemic injury induced a shift of circulating Ly6Chi monocytes towards a Chil3hi state with granulocyte-like features, but the acquisition of the Trem2hi macrophage signature occurred in the ischaemic tissue. In vitro, macrophages acquired features of the Trem2hi signature following apoptotic-cell efferocytosis. Our work provides a comprehensive map of monocyte/macrophage transitions in the ischaemic heart, constituting a valuable resource for further investigating how these cells may be harnessed and modulated to promote post-ischaemic heart repair.

78-OR: LRH-1/NR5A2 Agonism Triggers an Anti-Inflammatory Phenotypic Switch of Immune Cells from Patients with Type 1 Diabetes and Improves Islet Engraftment/Function

Administration of the LRH-1 agonist BL0refrains hyperglycemia in 3 mouse models of type 1 diabetes (T1DM) . This antidiabetes action correlated with beta cell mass preservation and the presence of anti, rather than pro, inflammatory immune cells, indicating tolerization. Herein, we sought to translate these data to human immune cells of T1DM patients to demonstrate BL0benefits to re-educate the immune system and to assess its therapeutic value on human islet engraftment/function. Blood mononuclear cells purified from blood samples (healthy and T1DM) were derived in culture into 1) pro-inflammatory macrophages (MDM1) , 2) mature dendritic cells (mDCs) , 3) T-cells and 4) B-cells. Cells were then treated with BL0 and processed for 1) cell surface marker profiling, 2) cytokine secretome profiling, 3) T-cell proliferation and 4) RNAseq. In T1DM samples, BL0reduced the expression of pro-inflammatory markers on MDM1 (CD80) and mDCs (CD54 and CXCR4) and the secretion of the pro-inflammatory cytokines, CXCL6 and CCL24 (MDM1) as well as IL7 (mDCs) . Moreover, in these samples, BL0also increased Tregs, but not B-cells. T-cell proliferation was blunted by BL001-treated mDCs and Tregs. RNAseq analysis of BL001-treated MDM1 revealed increased expression of genes encoding components of complex I, III, IV, and V of the respiratory chain consistent with a transition from a glycolytic driven MDM1 to an oxidative phosphorylation driven MDM2 anti-inflammatory phenotype. BL0administration to STZ-treated mice transplanted with human islet improved survival correlating with reduced hyperglycemia, increased circulating human C-peptide, preserved beta cell mass, and reduced CD4+ T-cell infiltration. Our results establish that BL0can induce a pro-to-anti-inflammatory phenotypic switch to human immune cells, and improve human islet engraftment/function, supporting the therapeutic benefits of BL0in human. Disclosure N. Cobo-Vuilleumier: None. S. Rodriguez-Fernandez: Employee; Ahead Therapeutics SL. P.I. Lorenzo: None. A. Dorronsoro: None. C.C. Lachaud: None. L. Almenara-Fuentes: Employee; Ahead Therapeutics SL. M. Ramos-Rodríguez: None. I. Merida: None. M. Aguilar-Diosdado: None. L. Piemonti: None. L. Pasquali: None. F. Martin: None. I. Garcia-Sanchez: None. M. Martinez-Brocca: None. M. Vives-Pi: Research Support; Ahead Therapeutics S.L. B.R. Gauthier: None. Funding JDRF (2-SRA-2019-837-S-B) Consejeria de Salud y Familia, junta de Andalucia (PI-0001-2020)

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.

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.

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.

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.

An update on human periapical cyst-mesenchymal stem cells and their potential applications in regenerative medicine.

The broad clinical applications of Mesenchymal Stem Cells (MSCs) in the regenerative medicine field is attributed to their ability to self-renew and differentiate into multiple cellular lineages. Nowadays, MSCs can be derived from a variety of adult and fetal tissues including bone marrow, adipose tissue, umbilical cord and placenta. The difficulties associated with the isolation of MSCs from certain tissues such as bone marrow promoted the search for alternative tissues which are easily accessible. Oral derived MSCs include dental pulp stem cells (DPSCs), dental follicle progenitor cells (DFPC), and periodontal ligament stem cells (PDLSC). Being abundant and easily accessible, oral derived MSCs represent an interesting alternative MSC type to be employed in regenerative medicine. Human periapical cyst-mesenchymal stem cells (hPCy-MSCs) correspond to a newly discovered and characterized MSC subtype. Interestingly, hPCy-MSCs are collected from periapical cysts, which are a biological waste, without any influence on the other healthy tissues in oral cavity. hPCy-MSCs exhibit cell surface marker profile similar to that of other oral derived MSCs, show high proliferative potency, and possess the potential to differentiate into different cell types such as osteoblasts, adipocytes and neurons-like cells. hPCy-MSCs, therefore, represent a novel promising MSCs type to be applied in regenerative medicine domain. In this review, we will compare the different types of dental derived MSCs, we will highlight the isolation technique, the characteristics, and the therapeutic potential of hPCy-MSCs.

Mesenchymal stem cells derived from iPSCs expressing interleukin-24 inhibit the growth of melanoma in the tumor-bearing mouse model

BackgroundInterleukin-24 (IL-24) is a therapeutic gene for melanoma, which can induce melanoma cell apoptosis. Mesenchymal stem cells (MSCs) show promise as a carrier to delivery anti-cancer factors to tumor tissues. Induced pluripotent stem cells (iPSCs) are an alternative source of mesenchymal stem cells (MSCs). We previously developed a novel non-viral gene targeting vector to target IL-24 to human iPSCs. This study aims to investigate whether MSCs derived from the iPSCs with the site-specific integration of IL-24 can inhibit the growth of melanoma in a tumor-bearing mouse model via retro-orbital injection.MethodsIL-24-iPSCs were differentiated into IL-24-iMSCs in vitro, of which cellular properties and potential of differentiation were characterized. The expression of IL-24 in the IL-24-iMSCs was measured by qRT-PCR, Western Blotting, and ELISA analysis. IL-24-iMSCs were transplanted into the melanoma-bearing mice by retro-orbital intravenous injection. The inhibitory effect of IL-24-iMSCs on the melanoma cells was investigated in a co-culture system and tumor-bearing mice. The molecular mechanisms underlying IL-24-iMSCs in exerting anti-tumor effect were also explored.ResultsiPSCs-derived iMSCs have the typical profile of cell surface markers of MSCs and have the ability to differentiate into osteoblasts, adipocytes, and chondroblasts. The expression level of IL-24 in IL-24-iMSCs reached 95.39 ng/106 cells/24 h, which is significantly higher than that in iMSCs, inducing melanoma cells apoptosis more effectively in vitro compared with iMSCs. IL-24-iMSCs exerted a significant inhibitory effect on the growth of melanoma in subcutaneous mouse models, in which the migration of IL-24-iMSCs to tumor tissue was confirmed. Additionally, increased expression of Bax and Cleaved caspase-3 and down-regulation of Bcl-2 were observed in the mice treated with IL-24-iMSCs.ConclusionMSCs derived from iPSCs with the integration of IL-24 at rDNA locus can inhibit the growth of melanoma in tumor-bearing mouse models when administrated via retro-orbital injection.

Regulation of somatostatin expression by vitamin D3 and valproic acid in human adipose-derived mesenchymal stem cells

BackgroundAdipose-derived mesenchymal stem cells (ADMSC) are non-haematopoietic, fibroblast-like multipotent progenitor cells. They have the potential for trilineage (adipocyte, chondrocyte and osteocyte) differentiation as well as differentiation into endocrine pancreatic progenitors. In diabetic or cancer therapy, somatostatin (SST) expression plays a vital role. Small molecules such as valproic acid (VPA) and micronutrients like vitamin D3 have differentiation potential in ADMSC. Therefore, the aim of this study was to investigate the role of vitamin D3 machinery and its metabolic enzymes in ADMSC. Furthermore, the reprogramming effect of vitamin D3 and VPA was evaluated on somatostatin expression in pancreatic lineage differentiation.MethodsADMSC were characterised based on their cell surface marker profile using flow cytometry. Specific adipogenic and osteogenic differentiation protocols were used in this study. Gene expression of several pluripotent, endodermal, pancreatic progenitor and pancreatic endocrine lineage markers were investigated in native ADMSC and after stimulation with different concentration of vitamin D3 for five consecutive days (0, 50, 100, 150 nM) and VPA (0.5, 1, 1.5, 2 mM) by real-time PCR. Furthermore, somatostatin expression was confirmed with ELISA and immunocytochemistry.ResultsIn ADMSC, the expression of somatostatin mRNA, the vitamin D receptor (VDR) and its metabolising enzymes 1 α-Hydroxylase, 24-Hydroxylase and 25-Hydroxylase were detected. Upon stimulation with vitamin D3, nuclear translocation of vitamin D receptor (VDR) was observed. Interestingly, the presence of vitamin D3 reduced the transcription of the somatostatin gene. By contrast, VPA treatment of cultivated ADMSC showed enhancing effect on somatostatin gene expression. No other pluripotent, endodermal, pancreatic progenitor or pancreatic endocrine lineage mRNA expression was modulated under the influence of vitamin D3 and VPA.ConclusionHuman ADMSC carry the VDR. The vitamin D metabolising enzyme 25-Hydroxylase responded to the addition of vitamin D3. Moreover, our results demonstrate that somatostatin expression in ADMSC is constitutive, partially secreted and regulated by vitamin D3 and VPA.

CD49b, CD87, and CD95 Are Markers for Activated Cancer-Associated Fibroblasts Whereas CD39 Marks Quiescent Normal Fibroblasts in Murine Tumor Models.

Fibroblasts are thought to be key players in the tumor microenvironment. Means to identify and isolate fibroblasts as well as an understanding of their cancer-specific features are essential to dissect their role in tumor biology. To date, the identification of cancer-associated fibroblasts is widely based on generic markers for activated fibroblasts in combination with their origin in tumor tissue. This study was focused on a deep characterization of the cell surface marker profile of cancer-associated fibroblasts in widely used mouse tumor models and defining aberrant expression profiles by comparing them to their healthy counterparts. We established a generic workflow to isolate healthy and cancer-associated fibroblasts from solid tissues, thereby reducing bias, and background noise introduced by non-target cells. We identified CD87, CD44, CD49b, CD95, and Ly-6C as cancer-associated fibroblast cell surface markers, while CD39 was identified to mark normal fibroblasts from healthy tissues. In addition, we found a functional association of most cancer-related fibroblast markers to proliferation and a systemic upregulation of CD87, and CD49b in tumor-bearing mice, even in non-affected tissues. These novel markers will facilitate the characterization of fibroblasts and shed further light in their functions and implication in cancer progression.

Phenotypic Characterization of Bone Marrow Mononuclear Cells and Derived Stromal Cell Populations from Human Iliac Crest, Vertebral Body and Femoral Head.

(1) In vitro, bone marrow-derived stromal cells (BMSCs) demonstrate inter-donor phenotypic variability, which presents challenges for the development of regenerative therapies. Here, we investigated whether the frequency of putative BMSC sub-populations within the freshly isolated mononuclear cell fraction of bone marrow is phenotypically predictive for the in vitro derived stromal cell culture. (2) Vertebral body, iliac crest, and femoral head bone marrow were acquired from 33 patients (10 female and 23 male, age range 14–91). BMSC sub-populations were identified within freshly isolated mononuclear cell fractions based on cell-surface marker profiles. Stromal cells were expanded in monolayer on tissue culture plastic. Phenotypic assessment of in vitro derived cell cultures was performed by examining growth kinetics, chondrogenic, osteogenic, and adipogenic differentiation. (3) Gender, donor age, and anatomical site were neither predictive for the total yield nor the population doubling time of in vitro derived BMSC cultures. The abundance of freshly isolated progenitor sub-populations (CD45−CD34−CD73+, CD45−CD34−CD146+, NG2+CD146+) was not phenotypically predictive of derived stromal cell cultures in terms of growth kinetics nor plasticity. BMSCs derived from iliac crest and vertebral body bone marrow were more responsive to chondrogenic induction, forming superior cartilaginous tissue in vitro, compared to those isolated from femoral head. (4) The identification of discrete progenitor populations in bone marrow by current cell-surface marker profiling is not predictive for subsequently derived in vitro BMSC cultures. Overall, the iliac crest and the vertebral body offer a more reliable tissue source of stromal progenitor cells for cartilage repair strategies compared to femoral head.

Liver-Derived TGF-β Maintains the EomeshiTbetlo Phenotype of Liver Resident Natural Killer Cells

The adult human liver hosts a complex repertoire of liver resident and transient natural killer (NK) cell populations with diverse phenotypes and functions. Liver resident NK cells are CD56bright NK cells defined by a unique expression profile of transcription factors and cell surface markers (EomeshiTbetloTIGIT+CD69+CXCR6+CD49e−). Despite extensive characterization of the phenotype of liver resident NK cells, it remains unclear how factors within the liver microenvironment induce and maintain this unique phenotype. In this study, we have explored the factors regulating the phenotype of liver resident NK cells. Isolation of healthy liver resident NK cells from donor liver perfusate and in vitro culture results in the gradual loss of the characteristic Tbetlo phenotype, with the cells increasing Tbet expression significantly at day 7. This phenotypic loss could be halted through the dose-dependent addition of liver conditioned media (LCM), generated from the ex vivo culture of liver biopsies from healthy organ donors. TGF-β, but not IL-10, replicated the Tbet suppressive effects of LCM in both liver resident and peripheral blood NK cells. Furthermore, blocking TGF-β receptor signaling using the inhibitor SB431542, reversed the effect of LCM treatment on liver resident NK cells, causing the loss of tissue resident Eomeshi Tbetlo phenotype. Our findings identify liver-derived TGF-β as an important component of the liver microenvironment, which acts to regulate and maintain the phenotype of liver resident NK cells.