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.

Assessment of Tumorigenic Potential in Mesenchymal-Stem/Stromal-Cell-Derived Small Extracellular Vesicles (MSC-sEV).

Mesenchymal-stem/stromal-cell-derived small extracellular vesicles (MSC-sEV) have been shown to ameliorate many diseases in preclinical studies. However, translating MSC-sEV into clinical use requires the development of scalable manufacturing processes for highly reproducible preparations of safe and potent MSC-sEVs. A major source of variability in MSC-sEV preparations is EV producer cells. To circumvent variability in producer cells, clonal immortalized MSC lines as EV producer lines are increasingly being used for sEV production. The use of sEVs from immortalized producer cells inevitably raises safety concerns regarding the tumorigenicity or tumor promoting potential of the EV products. In this study, cells from E1-MYC line, a MSC cell line immortalized with the MYC gene, were injected subcutaneously into athymic nude mice. At 84 days post-injection, no tumor formation was observed at the injection site, lungs, or lymph nodes. E1-MYC cells pre-and post-sEV production did not exhibit anchorage-independent growth in soft agar. Daily intraperitoneal injections of 1 or 5 µg sEVs from E1-MYC into athymic nude mice with FaDu human head and neck cancer xenografts for 28 days did not promote or inhibit tumor growth relative to the xenograft treated with vehicle control. Therefore, MYC-immortalized MSCs are not tumorigenic and sEVs from these MSCs do not promote tumor growth.

Conservation of oncofetal antigens on human embryonic stem cells enables discovery of monoclonal antibodies against cancer.

Monoclonal antibodies (mAbs) are used as targeted therapies against cancers. These mAbs kill cancer cells via various mechanisms of actions. In this study, human embryonic stem cells (hESCs) was used as the immunogen to generate a panel of antibodies. From this panel of mAbs, A19 was found to bind both hESC and various cancer cell lines. The antigen target of A19 was identified as Erbb-2 and glycan analysis showed that A19 binds to a N-glycan epitope on the antigen. A19 was elucidated to internalize into cancer cells following binding to Erbb-2 and hence developed as an antibody-drug conjugate (ADC). Using ADC as the mechanism of action, A19 was able to kill cancer cells in vitro and delayed the onset of tumour formation in mice xenograft model. When compared to Herceptin, A19 binds to different isoforms of Erbb-2 and does not compete with Herceptin for the same epitope. Hence, A19 has the potential to be developed as an alternative targeted therapeutic agent for cancers expressing Erbb-2.

Isolation and Characterization of Exosome from Human Embryonic Stem Cell-Derived C-Myc-Immortalized Mesenchymal Stem Cells.

Mesenchymal stem cells (MSC) are currently the cell type of choice in many cell therapy trials. The number of therapeutic applications for MSCs registered as product IND submissions with the FDA and initiation of registered clinical trials has increased substantially in recent years, in particular between 2006 and 2012. However, defined mechanisms of action underpinning the therapeutic efficacy of MSCs are lacking, but they are increasingly attributed to MSC trophic secretion rather than their differentiation potential. A promising secreted therapeutic candidate is an extracellular vesicle (EV) known as the exosome. The use of exosomes instead of cells as a therapeutic agent provides several advantages. A critical advantage is the prospect of a conventional pharmaceutical manufacturing process that is highly scalable and amenable to the stringent manufacturing process. For example, MSCs used as producers of therapeutics, and not as therapeutics per se, could be immortalized to generate infinitely expansible clonal lines to enhance the reproducible production of therapeutic exosomes. In this chapter, we will describe the immortalization of MSCs, and the production, isolation, and characterization of exosomes from immortalized MSC.

Calcium-Dependent Protein Kinase in Ginger Binds with Importin-α through Its Junction Domain for Nuclear Localization, and Further Interacts with NAC Transcription Factor.

Calcium-dependent protein kinases (CDPKs) are important sensors of Ca2+ elevations in plant cells regulating the gene expression linked with various cellular processes like stress response, growth and development, metabolism, and cytoskeleton dynamics. Ginger is an extensively used spice due to its unique flavor and immense medicinal value. The two major threats that interfere with the large scale production of ginger are the salinity and drought stress. ZoCDPK1 (Zingiber officinale Calcium-dependent protein kinase 1) is a salinity and drought-inducible CDPK gene isolated from ginger and undergoes dynamic subcellular localization during stress conditions. ZoCDPK1, with signature features of a typical Ca2+ regulated kinase, also possesses a bipartite nuclear localization sequence (NLS) in its junction domain (JD). A striking feature in ZoCDPK1 is the rare occurrence of a coupling between the NLS in JD and consensus sequences in regulatory domain. Here, we further identified its nature of nuclear localization and its interaction partners. In the homology model generated for ZoCDPK1, the regulatory domain mimics the crystal structure of the regulatory domain in Arabidopsis CDPK1. Molecular docking simulation of importin (ZoIMPα), an important protein involved in nuclear translocation, into the NLS of ZoCDPK1 was well-visualized. Furthermore, the direct interaction of ZoCDPK1 and ZoIMPα proteins was studied by the yeast 2-hybrid (Y2H) system, which confirmed that junction domain (JD) is an important interaction module required for ZoCDPK1 and ZoIMPα binding. The probable interacting partners of ZoCDPK1 were also identified using Y2H experiment. Of the 10 different stress-related interacting partners identified for ZoCDPK1, NAC transcription factor (TF) needs special mention, especially in the context of ZoCDPK1 function. The interaction between ZoCDPK1 and NAC TF, in fact, corroborate with the results of gene expression and over-expression studies of ZoCDPK1. Hence ZoCDPK1 is operating through NAC TF mediated ABA-independent, cold non-responsive stress signaling pathway in ginger.

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.

Over-expression of bael quinolone synthase in tobacco improves plant vigor under favorable conditions, drought, or salt stress.

Type III polyketide synthases (PKSs) catalyze the biosynthesis of various medicinally important secondary metabolites in plants, but their role in growth and stress response is unclear. Here, we overexpressed quinolone synthase (QNS) from bael in tobacco. QNS-overexpressing plants showed an overall increase in growth, photosynthetic efficiency and chlorophyll content compared to wild type plants. Second-generation (T2) transgenic plants grew to maturity, flowered early and set viable seeds under favorable conditions without yield penalty. An increased accumulation of flavonoids, phenols and alkaloids was associated with higher tolerance to drought and salinity stress in transgenic plants. Thus, bael QNS seems to function as a positive regulator of plant growth and stress response, and could be potentially used for engineering plants tolerant to abiotic stress.

CDPK1 from ginger promotes salinity and drought stress tolerance without yield penalty by improving growth and photosynthesis in Nicotiana tabacum.

In plants, transient changes in calcium concentrations of cytosol have been observed during stress conditions like high salt, drought, extreme temperature and mechanical disturbances. Calcium-dependent protein kinases (CDPKs) play important roles in relaying these calcium signatures into downstream effects. In this study, a stress-responsive CDPK gene, ZoCDPK1 was isolated from a stress cDNA generated from ginger using rapid amplification of cDNA ends (RLM-RACE) - PCR technique and characterized its role in stress tolerance. An important aspect seen during the analysis of the deduced protein is a rare coupling between the presence of a nuclear localization sequence in the junction domain and consensus sequence in the EF-hand loops of calmodulin-like domain. ZoCDPK1 is abundantly expressed in rhizome and is rapidly induced by high-salt stress, drought, and jasmonic acid treatment but not by low temperature stress or abscissic acid treatment. The sub-cellular localization of ZoCDPK1-GFP fusion protein was studied in transgenic tobacco epidermal cells using confocal laser scanning microscopy. Over-expression of ginger CDPK1 gene in tobacco conferred tolerance to salinity and drought stress as reflected by the high percentage of seed germination, higher relative water content, expression of stress responsive genes, higher leaf chlorophyll content, increased photosynthetic efficiency and other photosynthetic parameters. In addition, transgenic tobacco subjected to salinity/drought stress exhibited 50% more growth during stress conditions as compared to wild type plant during normal conditions. T3 transgenic plants are able to grow to maturity, flowers early and set viable seeds under continuous salinity or drought stress without yield penalty. The ZoCDPK1 up-regulated the expression levels of stress-related genes RD21A and ERD1 in tobacco plants. These results suggest that ZoCDPK1 functions in the positive regulation of the signaling pathways that are involved in the response to salinity and drought stress in ginger and it is likely operating in a DRE/CRT independent manner.

Enabling a robust scalable manufacturing process for therapeutic exosomes through oncogenic immortalization of human ESC-derived MSCs.

BACKGROUND: Exosomes or secreted bi-lipid vesicles from human ESC-derived mesenchymal stem cells (hESC-MSCs) have been shown to reduce myocardial ischemia/reperfusion injury in animal models. However, as hESC-MSCs are not infinitely expansible, large scale production of these exosomes would require replenishment of hESC-MSC through derivation from hESCs and incur recurring costs for testing and validation of each new batch. Our aim was therefore to investigate if MYC immortalization of hESC-MSC would circumvent this constraint without compromising the production of therapeutically efficacious exosomes. METHODS: The hESC-MSCs were transfected by lentivirus carrying a MYC gene. The transformed cells were analyzed for MYC transgene integration, transcript and protein levels, and surface markers, rate of cell cycling, telomerase activity, karyotype, genome-wide gene expression and differentiation potential. The exosomes were isolated by HPLC fractionation and tested in a mouse model of myocardial ischemia/reperfusion injury, and infarct sizes were further assessed by using Evans' blue dye injection and TTC staining. RESULTS: MYC-transformed MSCs largely resembled the parental hESC-MSCs with major differences being reduced plastic adherence, faster growth, failure to senesce, increased MYC protein expression, and loss of in vitro adipogenic potential that technically rendered the transformed cells as non-MSCs. Unexpectedly, exosomes from MYC-transformed MSCs were able to reduce relative infarct size in a mouse model of myocardial ischemia/reperfusion injury indicating that the capacity for producing therapeutic exosomes was preserved. CONCLUSION: Our results demonstrated that MYC transformation is a practical strategy in ensuring an infinite supply of cells for the production of exosomes in the milligram range as either therapeutic agents or delivery vehicles. In addition, the increased proliferative rate by MYC transformation reduces the time for cell production and thereby reduces production costs.

Defined and serum-free media support undifferentiated human embryonic stem cell growth.

Four commercially available serum-free and defined culture media tested on 2 human embryonic stem cell (hESC) lines were all found to support undifferentiated growth for >10 continuous passages. For hESC cultured with defined StemPro and mTeSR1 media, the cells were maintained feeder-free on culture dishes coated with extracellular matrices (ECMs) with no requirement of feeder-conditioned media (CM). For xeno-free serum replacer (XSR), HEScGRO, and KnockOut media, mitotically inactivated human foreskin feeders (hFFs) were required for hESC growth. Under the different media conditions, cells continued to exhibit alkaline phosphatase activity and expressed undifferentiated hESC markers Oct-4, stage-specific embryonic antigens 4 (SSEA-4), and Tra-1-60. In addition, hESC maintained the expression of podocalyxin-like protein-1 (PODXL), an antigen recently reported in another study to be present in undifferentiated hESC. The cytotoxic antibody mAb 84 binds via PODXL expressed on hESC surface and kills >90% of hESC within 45 min of incubation. When these cells were spontaneously differentiated to form embryoid bodies, derivatives representing the 3 germ layers were obtained. Injection of hESC into animal models resulted in teratomas and the formation of tissue types indicative of ectodermal, endodermal, and mesodermal lineages were observed. Our data also suggested that StemPro and mTeSR1 media were more optimal for hESC proliferation compared to cells grown on CM because the growth rate of hESC increased by 30%-40%, higher split ratio was thus required for weekly passaging. This is advantageous for the large-scale cultivation of hESC required in clinical applications.

Immortalized feeders for the scale-up of human embryonic stem cells in feeder and feeder-free conditions.

Human embryonic stem cells (hESC) are pluripotent cells that proliferate indefinitely in culture, whilst retaining their capacity for differentiation into different cell types. However, hESC cultures require culture in direct contact with feeder cells or conditioned medium (CM) from feeder cells. The most common source of feeders has been primary mouse embryonic fibroblast (MEF). In this study, we immortalized a primary MEF line with the E6 and E7 genes from HPV16. The immortal line, DeltaE-MEF, was able to proliferate beyond 7-9 passages and has an extended lifespan beyond 70 passages. When tested for its ability to support hESC growth, it was found that hESC continue to maintain the undifferentiated morphology for >40 passages both in co-culture with DeltaE-MEF and in feeder-free cultures supplemented with CM from DeltaE-MEF. The cultures also continue to express the pluripotent markers, Oct-4, SSEA-4, Tra-1-60, Tra-1-81, alkaline phosphatase and maintain a normal karyotype. In addition, these hESC formed teratomas when injected into SCID mice. Lastly, we demonstrated the feasibility of scaling-up significant quantities of undifferentiated hESC (>10(8) cells) using DeltaE-MEF in cell factories. The results from this study suggest that immortalized feeders can provide a consistent and reproducible source of feeders for hESC expansion and research.

High density cultures of embryonic stem cells.

Embryonic stem cells (ESC) have the unique ability to differentiate into a variety of tissue types. However, the realization of regenerative medicine will require the production of large quantities of ESC which subsequently have to be differentiated into the final phenotype. Thus, we sought to develop a simple and scaleable bioprocess to increase densities of ESC to achieve this goal. Using mouse embryonic stem cells (mESC) as a model, by combining automated feeding and culture of mESC on petriperm dishes, cell densities were enhanced up to 6.4 x 10(6) cells/cm2 compared to conventional petri dish culture which only reached 0.2 to 1.4 x 10(6) cells/cm2. It was found that mESC from all experiments maintained excellent viability, pluripotency, and genetic stability after growing for 6 days in petriperm cultures with automated feeding. The expression of Oct-4 transcription factor was observed in all cultures, mESC formed embryoid bodies in differentiated cultures and teratomas in SCID mice, confirming their pluripotency, and karyotype of the cultures was normal. This culture method was stable for routine passaging and a second mESC cell line was shown to perform in a similar manner on petriperm with automated feeding. This work represents an important step towards achieving high density cultures of ESC.

Expansion of pluripotent human embryonic stem cells on human feeders.

Human embryonic stem cells (HES) hold great potential for regenerative medicine because of their ability to differentiate to any cell type. However, a limitation is that HES cells require a feeder layer to stay undifferentiated. Routinely, mouse embryonic fibroblast is used. However, for therapeutic applications, contamination with mouse cells may be considered unacceptable. In this study, we evaluated three commercially available human foreskin feeder (HF) lines for their ability to support HES cell growth in media supplemented with serum or serum replacer. HES cells on HF in serum replacer-supplemented media were cultured for >30 passages. They remained undifferentiated, maintained a normal karyotype, and continued to be positive for the pluripotent markers Oct-4, SOX-2, SSEA-4, GCTM-2, Tra-1-60, Tra-1-81, and alkaline phosphatase. In vivo, HES cells formed teratomas in SCID mouse models that represent the three embryonic germ layers. In contrast, HES cells cultured on HF in serum-supplemented media differentiated after three passages. Morphologically, the cells became cystic with a loss of intracellular Oct-4. We have successfully adapted and cultured undifferentiated HES cells on three human feeder lines for >30 passages. No difficulties were observed with the exception of serum in the media. This study reveals a safe and accessible source for feeders for HES cell research and potential therapeutic applications.