Mechanism for the attenuation of neutrophil and complement hyperactivity by MSC exosomes.

Complements and neutrophils are two key players of the innate immune system that are widely implicated as drivers of severe COVID-19 pathogenesis, as evident by the direct correlation of respiratory failure and mortality with elevated levels of terminal complement complex C5b-9 and neutrophils. In this study, we identified a feed-forward loop between complements and neutrophils that could amplify and perpetuate the cytokine storm seen in severe SARS-CoV-2-infected patients. We observed for the first time that the terminal complement activation complex C5b-9 directly triggered neutrophil extracellular trap (NET) release and interleukin (IL)-17 production by neutrophils. This is also the first report that the production of NETs and IL-17 induced by C5b-9 assembly on neutrophils could be abrogated by mesenchymal stem cell (MSC) exosomes. Neutralizing anti-CD59 antibodies abolished this abrogation. Based on our findings, we hypothesize that MSC exosomes could alleviate the immune dysregulation in acute respiratory failure, such as that observed in severe COVID-19 patients, by inhibiting complement activation through exosomal CD59, thereby disrupting the feed-forward loop between complements and neutrophils to inhibit the amplification and perpetuation of inflammation during SARS-CoV-2 infection.

Critical considerations for the development of potency tests for therapeutic applications of mesenchymal stromal cell-derived small extracellular vesicles.

Mesenchymal stromal/stem cells (MSCs) have been widely tested against many diseases, with more than 1000 registered clinical trials worldwide. Despite many setbacks, MSCs have been approved for the treatment of graft-versus-host disease and Crohn disease. However, it is increasingly clear that MSCs exert their therapeutic functions in a paracrine manner through the secretion of small extracellular vesicles (sEVs) of 50-200 nm in diameter. Unlike living cells that can persist long-term, sEVs are non-living and non-replicative and have a transient presence in the body. Their small size also renders sEV preparations highly amenable to sterilization by filtration. Together, acellular MSC-sEV preparations are potentially safer and easier to translate into the clinic than cellular MSC products. Nevertheless, there are inherent challenges in the development of MSC-sEV drug products. MSC-sEVs are products of living cells, and living cells are sensitive to changes in the external microenvironment. Consequently, quality control metrics to measure key identity and potency features of MSC-sEV preparations have to be specified during development of MSC-sEV therapeutics. The authors have previously described quantifiable assays to define the identity of MSC-sEVs. Here the authors discuss requirements for prospective potency assays to predict the therapeutic effectiveness of the drug substance in accordance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. Although potency assays should ideally reflect the mechanism of action (MoA), this is challenging because the MoA for the reported efficacy of MSC-sEV preparations against multiple diseases of diverse underlying pathology is likely to be complex and different for each disease and difficult to fully elucidate. Nevertheless, robust potency assays could be developed by identifying the EV attribute most relevant to the intended biological activity in EV-mediated therapy and quantifying the EV attribute. Specifically, the authors highlight challenges and mitigation measures to enhance the manufacture of consistent and reproducibly potent sEV preparations, to identify and select the appropriate EV attribute for potency assays despite a complex "work-in-progress" MoA and to develop assays likely to be compliant with regulatory guidance for assay validation.

Topical Application of Mesenchymal Stem Cell Exosomes Alleviates the Imiquimod Induced Psoriasis-Like Inflammation.

Severe psoriasis, a chronic inflammatory skin disease is increasingly being effectively managed by targeted immunotherapy but long-term immunotherapy poses health risk and loss of response. Therefore, there is a need for alternative therapy strategies. Mesenchymal stem/stromal cell (MSC) exosomes are widely known for their potent immunomodulatory properties. Here we investigated if topically applied MSC exosomes could alleviate psoriasis-associated inflammation. Topically applied fluorescent exosomes on human skin explants were confined primarily to the stratum corneum with <1% input fluorescence exiting the explant over a 24-h period. Nevertheless, topically applied MSC exosomes in a mouse model of imiquimod (IMQ) psoriasis significantly reduced IL-17 and terminal complement activation complex C5b-9 in the mouse skin. MSC exosomes were previously shown to inhibit complement activation, specifically C5b-9 complex formation through CD59. Infiltration of neutrophils into the stratum corneum is characteristic of psoriasis and neutrophils are a major cellular source of IL-17 in psoriasis through the release of neutrophil extracellular traps (NETs). We propose that topically applied MSC exosomes inhibit complement activation in the stratum corneum and this alleviates IL-17 release by NETS from neutrophils that accumulate in and beneath the stratum corneum.

Preclinical Activity of Embryonic Annexin A2-Specific Chimeric Antigen Receptor T Cells Against Ovarian Cancer.

Chimeric antigen receptors (CARs) have found clinical success in B cell malignancies, but a dearth of potential targets limits their wider clinical application, especially in solid tumours. Here, we describe the development of an anti-annexin A2 CAR, CAR(2448), derived from an antibody found to have activity against epithelial ovarian cancer cell lines. The spacer length of CAR(2448) was optimised based on in vitro cytotoxic activity against ovarian cancer (OC) cell lines via a real-time cytotoxicity assay. The longer spacer CAR(2448)L T cells exhibit significant effector activity, inducing inflammatory cytokine release and cytotoxicity against OC cell lines. Furthermore, CAR(2448)L-BBz T cells induced enhanced survival in an in vivo OC xenograft model and reduced tumour volume by 76.6%. Our preclinical studies of CAR(2448) suggest its potential for the unmet need of novel strategies for the treatment of ovarian cancer.

Clustering and curation of electropherograms: an efficient method for analyzing large cohorts of capillary electrophoresis glycomic profiles for bioprocessing operations.

The accurate assessment of antibody glycosylation during bioprocessing requires the high-throughput generation of large amounts of glycomics data. This allows bioprocess engineers to identify critical process parameters that control the glycosylation critical quality attributes. The advances made in protocols for capillary electrophoresis-laser-induced fluorescence (CE-LIF) measurements of antibody N-glycans have increased the potential for generating large datasets of N-glycosylation values for assessment. With large cohorts of CE-LIF data, peak picking and peak area calculations still remain a problem for fast and accurate quantitation, despite the presence of internal and external standards to reduce misalignment for the qualitative analysis. The peak picking and area calculation problems are often due to fluctuations introduced by varying process conditions resulting in heterogeneous peak shapes. Additionally, peaks with co-eluting glycans can produce peaks of a non-Gaussian nature in some process conditions and not in others. Here, we describe an approach to quantitatively and qualitatively curate large cohort CE-LIF glycomics data. For glycan identification, a previously reported method based on internal triple standards is used. For determining the glycan relative quantities our method uses a clustering algorithm to 'divide and conquer' highly heterogeneous electropherograms into similar groups, making it easier to define peaks manually. Open-source software is then used to determine peak areas of the manually defined peaks. We successfully applied this semi-automated method to a dataset (containing 391 glycoprofiles) of monoclonal antibody biosimilars from a bioreactor optimization study. The key advantage of this computational approach is that all runs can be analyzed simultaneously with high accuracy in glycan identification and quantitation and there is no theoretical limit to the scale of this method.

Combining Glucose Units, m/z, and Collision Cross Section Values: Multiattribute Data for Increased Accuracy in Automated Glycosphingolipid Glycan Identifications and Its Application in Triple Negative Breast Cancer.

Glycan head-groups attached to glycosphingolipids (GSLs) found in the cell membrane bilayer can alter in response to external stimuli and disease, making them potential markers and/or targets for cellular disease states. To identify such markers, comprehensive analyses of glycan structures must be undertaken. Conventional analyses of fluorescently labeled glycans using hydrophilic interaction high-performance liquid chromatography (HILIC) coupled with mass spectrometry (MS) provides relative quantitation and has the ability to perform automated glycan assignments using glucose unit (GU) and mass matching. The use of ion mobility (IM) as an additional level of separation can aid the characterization of closely related or isomeric structures through the generation of glycan collision cross section (CCS) identifiers. Here, we present a workflow for the analysis of procainamide-labeled GSL glycans using HILIC-IM-MS and a new, automated glycan identification strategy whereby multiple glycan attributes are combined to increase accuracy in automated structural assignments. For glycan matching and identification, an experimental reference database of GSL glycans containing GU, mass, and CCS values for each glycan was created. To assess the accuracy of glycan assignments, a distance-based confidence metric was used. The assignment accuracy was significantly better compared to conventional HILIC-MS approaches (using mass and GU only). This workflow was applied to the study of two Triple Negative Breast Cancer (TNBC) cell lines and revealed potential GSL glycosylation signatures characteristic of different TNBC subtypes.

In vivo surveillance and elimination of teratoma-forming human embryonic stem cells with monoclonal antibody 2448 targeting annexin A2.

This study describes the use of a previously reported chimerised monoclonal antibody (mAb), ch2448, to kill human embryonic stem cells (hESCs) in vivo and prevent or delay the formation of teratomas. ch2448 was raised against hESCs and was previously shown to effectively kill ovarian and breast cancer cells in vitro and in vivo. The antigen target was subsequently found to be Annexin A2, an oncofetal antigen expressed on both embryonic cells and cancer cells. Against cancer cells, ch2448 binds and kills via antibody-dependent cell-mediated cytotoxicity (ADCC) and/or antibody-drug conjugate (ADC) routes. Here, we investigate if the use of ch2448 can be extended to hESC. ch2448 was found to bind specifically to undifferentiated hESC but not differentiated progenitors. Similar to previous study using cancer cells, ch2448 kills hESC in vivo either indirectly by eliciting ADCC or directly as an ADC. The treatment with ch2448 post-transplantation eliminated the in vivo circulating undifferentiated cells and prevented or delayed the formation of teratomas. This surveillance role of ch2448 adds an additional layer of safeguard to enhance the safety and efficacious use of pluripotent stem cell-derived products in regenerative medicine. Thereby, translating the use of ch2448 in the treatment of cancers to a proof of concept study in hESC (or pluripotent stem cell [PSC]), we show that mAbs can also be used to eliminate teratoma forming cells in vivo during PSC-derived cell therapies. We propose to use this strategy to complement existing methods to eliminate teratoma-forming cells in vitro. Residual undifferentiated cells may escape in vitro removal methods and be introduced into patients together with the differentiated cells.

Opportunities for Antibody Discovery Using Human Pluripotent Stem Cells: Conservation of Oncofetal Targets.

Pluripotent stem cells (PSCs) comprise both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). The application of pluripotent stem cells is divided into four main areas, namely: (i) regenerative therapy, (ii) the study and understanding of developmental biology, (iii) drug screening and toxicology and (iv) disease modeling. In this review, we describe a new opportunity for PSCs, the discovery of new biomarkers and generating antibodies against these biomarkers. PSCs are good sources of immunogen for raising monoclonal antibodies (mAbs) because of the conservation of oncofetal antigens between PSCs and cancer cells. Hence mAbs generated using PSCs can potentially be applied in two different fields. First, these mAbs can be used in regenerative cell therapy to characterize the PSCs. In addition, the mAbs can be used to separate or eliminate contaminating or residual undifferentiated PSCs from the differentiated cell product. This step is critical as undifferentiated PSCs can form teratomas in vivo. The mAbs generated against PSCs can also be used in the field of oncology. Here, novel targets can be identified and the mAbs developed as targeted therapy to kill the cancer cells. Conversely, as new and novel oncofetal biomarkers are discovered on PSCs, cancer mAbs that are already approved by the FDA can be repurposed for regenerative medicine, thus expediting the route to the clinics.

Characterization of H type 1 and type 1 N-acetyllactosamine glycan epitopes on ovarian cancer specifically recognized by the anti-glycan monoclonal antibody mAb-A4.

Cancer-specific glycans of ovarian cancer are promising epitopes for targeting with monoclonal antibodies (mAb). Despite their potential, structural characterization of these glycan epitopes remains a significant challenge in mAb preclinical development. Our group generated the monoclonal antibody mAb-A4 against human embryonic stem cells (hESC), which also bound specifically to N-glycans present on 11 of 19 ovarian cancer (OC) and 8 of 14 breast cancer cell lines tested. Normal cell lines and tissue were unstained by mAb-A4. To characterize the N-linked glycan epitopes on OC cell lines targeted by mAb-A4, we used glycosidases, glycan microarray, siRNA, and advanced high sensitivity matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The mAb-A4 epitopes were found to be Fucα1-2Galß1-3GlcNAcß (H type 1) and Galß1-3GlcNAcß (type 1 LacNAc). These structures were found to be present on multiple proteins from hESC and OC. Importantly, endo-ß-galactosidase coupled with MALDI-MS allowed these two epitopes, for the first time, to be directly identified on the polylactosamines of N-glycans of SKOV3, IGROV1, OV90, and OVCA433. Furthermore, siRNA knockdown of B3GALT5 expression in SKOV3 demonstrated that mAb-A4 binding was dependent on B3GALT5, providing orthogonal evidence of the epitopes' structures. The recognition of oncofetal H type 1 and type 1 LacNAc on OC by mAb-A4 is a novel and promising way to target OC and supports the theory that cancer can acquire stem-like phenotypes. We propose that the orthogonal framework used in this work could be the basis for advancing anti-glycan mAb characterization.

mAb C19 targets a novel surface marker for the isolation of human cardiac progenitor cells from human heart tissue and differentiated hESCs.

AIMS: Cardiac progenitor cells (CPCs) have been isolated from adult and developing hearts using an anti-mouse Sca-1 antibody. However, the absence of a human Sca-1 homologue has hampered the clinical application of the CPCs. Therefore, we generated novel monoclonal antibodies (mAbs) specifically raised against surface markers expressed by resident human CPCs. Here, we explored the suitability of one of these mAbs, mAb C19, for the identification, isolation and characterization of CPCs from fetal heart tissue and differentiating cultures of human embryonic stem cells (hESCs). METHODS & RESULTS: Using whole-cell immunization, mAbs were raised against Sca-1+ CPCs and screened for reactivity to various CPC lines by flow cytometry. mAb C19 was found to be specific for Sca-1+ CPCs, with high cell surface binding capabilities. mAb C19 stained small stem-like cells in cardiac tissue sections. Moreover, during differentiation of hESCs towards cardiomyocytes, a transient population of cells with mAb C19 reactivity was identified and isolated using magnetic-activated cell sorting. Their cell fate was tracked and found to improve cardiomyocyte purity from hESC-derived cultures. mAb C19+ CPCs, from both hESC differentiation and fetal heart tissues, were maintained and expanded in culture, while retaining their CPC-like characteristics and their ability to further differentiate into cardiomyocytes by stimulation with TGFß1. Finally, gene expression profiling of these mAb C19+ CPCs suggested a highly angiogenic nature, which was further validated by cell-based angiogenesis assays. CONCLUSION: mAb C19 is a new surface marker for the isolation of multipotent CPCs from both human heart tissues and differentiating hESCs.

Multiple reaction monitoring mass spectrometry for the discovery and quantification of O-GlcNAc-modified proteins.

O-linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification regulating proteins involved in a variety of cellular processes and diseases. Unfortunately, O-GlcNAc remains challenging to detect and quantify by shotgun mass spectrometry (MS) where it is time-consuming and tedious. Here, we investigate the potential of Multiple Reaction Monitoring Mass Spectrometry (MRM-MS), a targeted MS method, to detect and quantify native O-GlcNAc modified peptides without extensive labeling and enrichment. We report the ability of MRM-MS to detect a standard O-GlcNAcylated peptide and show that the method is robust to quantify the amount of O-GlcNAcylated peptide with a method detection limit of 3 fmol. In addition, when diluted by 100-fold in a trypsin-digested whole cell lysate, the O-GlcNAcylated peptide remains detectable. Next, we apply this strategy to study glycogen synthase kinase-3 beta (GSK-3ß), a kinase able to compete with O-GlcNAc transferase and modify identical site on proteins. We demonstrate that GSK-3ß is itself modified by O-GlcNAc in human embryonic stem cells (hESC). Indeed, by only using gel electrophoresis to grossly enrich GSK-3ß from whole cell lysate, we discover by MRM-MS a novel O-GlcNAcylated GSK-3ß peptide, bearing 3 potential O-GlcNAcylation sites. We confirm our finding by quantifying the increase of O-GlcNAcylation, following hESC treatment with an O-GlcNAc hydrolase inhibitor. This novel O-GlcNAcylation could potentially be involved in an autoinhibition mechanism. To the best of our knowledge, this is the first report utilizing MRM-MS to detect native O-GlcNAc modified peptides. This could potentially facilitate rapid discovery and quantification of new O-GlcNAcylated peptides/proteins.

A simple and highly sensitive LC-MS workflow for characterization and quantification of ADC cleavable payloads.

Antibody-drug conjugates (ADC) payloads are cleavable drugs that act as the warhead to exert an ADC's cytotoxic effects on cancer cells intracellularly. A simple and highly sensitive workflow is developed and validated for the simultaneous quantification of six ADC payloads, namely SN-38, MTX, DXd, MMAE, MMAF and Calicheamicin (CM). The workflow consists of a short and simple sample extraction using a methanol-ethanol mixture, followed by a fast liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. The results showed that well-validated linear response ranges of 0.4-100 nM for SN38, MTX and DXd, 0.04-100 nM for MMAE and MMAF, 0.4-1000 nM for CM were achieved in mouse serum. Recoveries for all six payloads at three different concentrations (low, medium and high) were more than 85%. An ultra-low sample volume of only 5 µL of serum is required due to the high sensitivity of the method. This validated method was successfully applied to a pharmacokinetic study to quantify MMAE in mouse serum samples.

Antibody glycan quality predicted from CHO cell culture media markers and machine learning.

N-glycosylation can have a profound effect on the quality of mAb therapeutics. In biomanufacturing, one of the ways to influence N-glycosylation patterns is by altering the media used to grow mAb cell expression systems. Here, we explore the potential of machine learning (ML) to forecast the abundances of N-glycan types based on variables related to the growth media. The ML models exploit a dataset consisting of detailed glycomic characterisation of Anti-HER fed-batch bioreactor cell cultures measured daily under 12 different culture conditions, such as changes in levels of dissolved oxygen, pH, temperature, and the use of two different commercially available media. By performing spent media quantitation and subsequent calculation of pseudo cell consumption rates (termed media markers) as inputs to the ML model, we were able to demonstrate a small subset of media markers (18 selected out of 167 mass spectrometry peaks) in a Chinese Hamster Ovary (CHO) cell cultures are important to model N-glycan relative abundances (Regression - correlations between 0.80-0.92; Classification - AUC between 75.0-97.2). The performances suggest the ML models can infer N-glycan critical quality attributes from extracellular media as a proxy. Given its accuracy, we envisage its potential applications in biomaufactucuring, especially in areas of process development, downstream and upstream bioprocessing.

Inter-laboratory multiplex bead-based surface protein profiling of MSC-derived EV preparations identifies MSC-EV surface marker signatures.

Mesenchymal stromal cells (MSCs) are promising regenerative therapeutics that primarily exert their effects through secreted extracellular vesicles (EVs). These EVs - being small and non-living - are easier to handle and possess advantages over cellular products. Consequently, the therapeutic potential of MSC-EVs is increasingly investigated. However, due to variations in MSC-EV manufacturing strategies, MSC-EV products should be considered as highly diverse. Moreover, the diverse array of EV characterisation technologies used for MSC-EV characterisation further complicates reliable interlaboratory comparisons of published data. Consequently, this study aimed to establish a common method that can easily be used by various MSC-EV researchers to characterise MSC-EV preparations to facilitate interlaboratory comparisons. To this end, we conducted a comprehensive inter-laboratory assessment using a novel multiplex bead-based EV flow cytometry assay panel. This assessment involved 11 different MSC-EV products from five laboratories with varying MSC sources, culture conditions, and EV preparation methods. Through this assay panel covering a range of mostly MSC-related markers, we identified a set of cell surface markers consistently positive (CD44, CD73 and CD105) or negative (CD11b, CD45 and CD197) on EVs of all explored MSC-EV preparations. Hierarchical clustering analysis revealed distinct surface marker profiles associated with specific preparation processes and laboratory conditions. We propose CD73, CD105 and CD44 as robust positive markers for minimally identifying MSC-derived EVs and CD11b, CD14, CD19, CD45 and CD79 as reliable negative markers. Additionally, we highlight the influence of culture medium components, particularly human platelet lysate, on EV surface marker profiles, underscoring the influence of culture conditions on resulting EV products. This standardisable approach for MSC-EV surface marker profiling offers a tool for routine characterisation of manufactured EV products in pre-clinical and clinical research, enhances the quality control of MSC-EV preparations, and hopefully paves the way for higher consistency and reproducibility in the emerging therapeutic MSC-EV field.

Optimizing effector functions of monoclonal antibodies via tailored N-glycan engineering using a dual landing pad CHO targeted integration platform.

Monoclonal antibodies (mAbs) eliminate cancer cells via various effector mechanisms including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which are influenced by the N-glycan structures on the Fc region of mAbs. Manipulating these glycan structures on mAbs allows for optimization of therapeutic benefits associated with effector functions. Traditional approaches such as gene deletion or overexpression often lead to only all-or-nothing changes in gene expression and fail to modulate the expression of multiple genes at defined ratios and levels. In this work, we have developed a CHO cell engineering platform enabling modulation of multiple gene expression to tailor the N-glycan profiles of mAbs for enhanced effector functions. Our platform involves a CHO targeted integration platform with two independent landing pads, allowing expression of multiple genes at two pre-determined genomic sites. By combining with internal ribosome entry site (IRES)-based polycistronic vectors, we simultaneously modulated the expression of α-mannosidase II (MANII) and chimeric ß-1,4-N-acetylglucosaminyl-transferase III (cGNTIII) genes in CHO cells. This strategy enabled the production of mAbs carrying N-glycans with various levels of bisecting and non-fucosylated structures. Importantly, these engineered mAbs exhibited different degrees of effector cell activation and CDC, facilitating the identification of mAbs with optimal effector functions. This platform was demonstrated as a powerful tool for producing antibody therapeutics with tailored effector functions via precise engineering of N-glycan profiles. It holds promise for advancing the field of metabolic engineering in mammalian cells.

The quantitative proteomes of human-induced pluripotent stem cells and embryonic stem cells.

Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important, given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here, we present an in-depth quantitative mass spectrometry-based analysis of hESCs, two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless, a small group of 58 proteins, mainly related to metabolism, antigen processing and cell adhesion, was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap, highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature.

Activated T cells modulate immunosuppression by embryonic-and bone marrow-derived mesenchymal stromal cells through a feedback mechanism.

BACKGROUND AIMS: Human embryonic stem cell (hESC)-derived mesenchymal stromal cells (MSC) (hESC-MSC) are an alternative source of MSC to bone marrow (BM)-derived MSC (BM-MSC), which are being investigated in clinical trials for their immunomodulatory potential. hESC-MSC have the advantage of being consistent because each batch can be generated from hESC under defined conditions. In contrast, BM-MSC have a limited proliferative capacity. METHODS: The ability to suppress the proliferation of anti-CD3/CD28-stimulated CD4 (+) T cells by hESC-MSC was compared with adult BM-MSC and neonatal foreskin fibroblast (Fb). RESULTS: hESC-MSC suppress the proliferation of CD4 (+) T cells in both contact and transwell systems, although inhibition is less in the transwell system. hESC-MSC are approximately 2-fold less potent (67 cells/100 T cells) than BM-MSC and Fb (37 and 34 cells/100 T cells, respectively) at suppressing T-cell proliferation by 50% in a transwell [inhibitory concentration(IC)(50)]. The anti-proliferative effect is not contact-dependent but requires the presence of factors such as interferon (IFN)-γ produced by activated T cells. IFN-γ induces the expression of indoleamine-2,3-dioxygenase (IDO) in hESC-MSC, BM-MSC and Fb, contributing to their immunosuppressive property. CONCLUSIONS: The feedback loop between MSC or Fb and activated T cells may limit the immunosuppressive effects of MSC and Fb to sites containing ongoing immunologic or inflammatory responses where activated T cells induce the up-regulation of IDO and immunomodulatory properties of MSC and Fb. These data demonstrate that hESC-MSC may be evaluated further as an allogeneic cell source for therapeutic applications requiring immunosuppression.

In-depth qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immunoaffinity purification and stable isotope dimethyl labeling.

Several mass spectrometry-based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative, and not be cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope-labeled) starting protein material are required to enable the detection of low abundance phosphotyrosine peptides. Here, we adopted and refined a peptidecentric immunoaffinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immunoaffinity purification, even for the large quantities (mg) of peptide material used, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or epidermal growth factor stimulation. Analysis of the epidermal growth factor-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data were found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immunoaffinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach, a rather complete qualitative and quantitative picture of tyrosine phosphorylation signaling events can be generated.

Mesenchymal stem cell secretes microparticles enriched in pre-microRNAs.

Intercellular exchange of protein and RNA-containing microparticles is an increasingly important mode of cell-cell communication. Here we investigate if mesenchymal stem cells (MSCs) known for secreting therapeutic paracrine factors also secrete RNA-containing microparticles. We observed that human embryonic stem cell (hESC)-derived MSC conditioned medium contained small RNAs (less than 300 nt) encapsulated in cholesterol-rich phospholipid vesicles as evidenced by their RNase sensitivity only in the presence of a sodium dodecyl sulfate-based cell lysis buffer, phospholipase A2 and a chelator of cholesterol, cyclodextrin and the restoration of their lower than expected density by detergent or phospholipase A2 treatment. MicroRNAs (miRNAs) such as hsa-let-7b and hsa-let-7g were present in a high precursor (pre)- to mature miRNA ratio by microarray analysis and quantitative reverse transcription-polymerase chain reaction. The pre-miRNAs were cleaved to mature miRNA by RNase III in vitro. High performance liquid chromatography-purified RNA-containing vesicles have a hydrodynamic radius of 55-65 nm and were readily taken up by H9C2 cardiomyocytes. This study suggests that MSCs could facilitate miRNA-mediated intercellular communication by secreting microparticles enriched for pre-miRNA.

Mesenchymal Stem Cell Exosomes Promote Functional Osteochondral Repair in a Clinically Relevant Porcine Model.

BACKGROUND: Previous studies have reported the efficacy of human mesenchymal stem cell (MSC) exosomes for the repair of osteochondral defects in rats and rabbits. However, the safety and efficacy of MSC exosomes remain to be validated in a clinically relevant large animal model. PURPOSE: To validate the safety and efficacy of human MSC exosomes for osteochondral repair in a clinically relevant micropig model. STUDY DESIGN: Controlled laboratory study. METHODS: Bilateral osteochondral defects (6-mm diameter and 1-mm depth) were surgically created in the medial femoral condyles in knees of 12 micropigs. The pigs then received 2-mL intra-articular injections of MSC exosomes and hyaluronic acid (HA) (Exosome+HA) or HA alone after surgery and thereafter at 8 and 15 days. Osteochondral repair was assessed by magnetic resonance imaging (MRI) at 15 days and at 2 and 4 months after surgery as well as by macroscopic, histological, biomechanical, and micro-computed tomography (micro-CT) analyses at 4 months after surgery. RESULTS: Exosome+HA-treated defects demonstrated significantly better MRI scores than HA-treated defects at 15 days and at 2 and 4 months. Additionally, Exosome+HA-treated defects demonstrated functional cartilage and subchondral bone repair, with significantly better macroscopic and histological scores and biomechanical properties (Young modulus and stiffness) than HA-treated defects at 4 months. Micro-CT further showed significantly higher bone volume and trabecular thickness in the subchondral bone of Exosome+HA-treated defects than that of HA-treated defects. Importantly, no adverse response or major systemic alteration was observed in any of the animals. CONCLUSION: This study shows that the combination of MSC exosomes and HA administered at a clinically acceptable frequency of 3 weekly intra-articular injections can promote functional cartilage and subchondral bone repair, with significantly improved morphological, histological, and biomechanical outcomes in a clinically relevant porcine model. CLINICAL RELEVANCE: Our findings provide a robust scientific rationale to support a phase 1/2 clinical trial to test MSC exosomes in patients with osteochondral lesions.