Potential surrogate quantitative immunomodulatory potency assay for monitoring human umbilical cord-derived mesenchymal stem cells production

Mesenchymal stem cells (MSCs) play an important role as immune modulator through interaction with several immune cells, including macrophages. In this study, the immunomodulatory potency of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) was demonstrated in the in vivo middle cerebral artery occlusion (MCAo)-induced brain injury rat model and in vitro THP-1-derived macrophages model. At 24 h after induction of MCAo, hUC-MSCs was administered via tail vein as a single dose. Remarkably, hUC-MSCs could inhibit M1 polarization and promote M2 polarization of microglia in vivo after 14 days induction of MCAo. Compared with THP-1-derived macrophages which had been stimulated by lipopolysaccharide, the secretion of proinflammatory cytokines, tumor necrosis factor-α (TNF-α) and interferon-γ inducible protein (IP-10), were significantly reduced in the presence of hUC-MSCs. Moreover, the secretion of anti-inflammatory cytokine, interleukin-10 (IL-10), was significantly increased after cocultured with hUC-MSCs. Prostaglandins E2 (PGE2), secreted by hUC-MSCs, is one of the crucial immunomodulatory factors and could be inhibited in the presence of COX2 inhibitor, NS-398. PGE2 inhibition suppressed hUC-MSCs immunomodulatory capability, which was restored after addition of synthetic PGE2, establishing the minimum amount of PGE2 required for immunomodulation. In conclusion, our data suggested that PGE2 is a crucial potency marker involved in the therapeutic activity of hUC-MSCs through macrophages immune response modulation and cytokines regulation. This study provides the model for the development of a surrogate quantitative potency assay of immunomodulation in stem cells production.     

Development of a screening assay for surrogate markers of CHK1 inhibitor-induced cell cycle release

Chk1 is a key regulator of the S and G2/M checkpoints and is activated following DNA damage by agents such as the topoisomerase I inhibitor camptothecin (CPT). It has been proposed that Chk1 inhibitors used in combination with such a DNA damaging agent to treat tumors would potentiate cytotoxicity and increase the therapeutic index, particularly in tumors lacking functional p53. The aim of this study was to determine whether gene expression analysis could be used to inform lead optimization of a novel series of Chk1 inhibitors. The candidate small-molecule Chk1 inhibitors were used in combination with CPT to identify potential markers of functional Chk1 inhibition, as well as resulting cell cycle progression, using cDNA-based microarrays. Differential expression of several of these putative marker genes was further validated by RT-PCR for use as a medium-throughput assay. In the presence of DNA damage, Chk1 inhibitors altered CPT-dependent effects on the expression of cell cycle and DNA repair genes in a manner consistent with a Chk1-specific mechanism of action. Furthermore, differential expression of selected marker genes, cyclin E2, EGR1, and DDIT3, was dose dependent for Chk1 inhibition. RT-PCR results for these genes following treatment with a panel of Chk1 inhibitors showed a strong correlation between marker gene response and the ability of each compound to abrogate cell cycle arrest in situ following CPT-induced DNA damage. These results demonstrate the utility of global expression analysis to identify surrogate markers, providing an alternative method for rapid compound characterization to support advancement decisions in early drug discovery.     

The derivation of clinical-grade human embryonic stem cell lines

The pluripotent nature of human embryonic stem cells (hESC) has attracted great interest in using them as a source of cells or tissue in cell therapy. However, in order to be used in regenerative medicine, the pluripotent hESC lines should be established and propagated according to good manufacturing practice quality requirements. The cultures should be animal substance free in order to exclude the risk of infections and immunogenity. They should also be genetically and epigenetically normal. The detailed molecular mechanisms of their pluripotency are still not defined. Using human feeder cells, a medium containing only human proteins, the mechanical isolation of the inner cell mass and mechanical passaging of hESC, is a safe option until a functional defined medium containing physiological concentrations of regulatory factors is available.     

Bioreactor design for clinical-grade expansion of stem cells

The many clinical trials currently in progress will likely lead to the widespread use of stem cell-based therapies for an extensive variety of diseases, either in autologous or allogeneic settings. With the current pace of progress, in a few years' time, the field of stem cell-based therapy should be able to respond to the market demand for safe, robust and clinically efficient stem cell-based therapeutics. Due to the limited number of stem cells that can be obtained from a single donor, one of the major challenges on the roadmap for regulatory approval of such medicinal products is the expansion of stem cells using Good Manufacturing Practices (GMP)-compliant culture systems. In fact, manufacturing costs, which include production and quality control procedures, may be the main hurdle for developing cost-effective stem cell therapies. Bioreactors provide a viable alternative to the traditional static culture systems in that bioreactors provide the required scalability, incorporate monitoring and control tools, and possess the operational flexibility to be adapted to the differing requirements imposed by various clinical applications. Bioreactor systems face a number of issues when incorporated into stem cell expansion protocols, both during development at the research level and when bioreactors are used in on-going clinical trials. This review provides an overview of the issues that must be confronted during the development of GMP-compliant bioreactors systems used to support the various clinical applications employing stem cells.     

The generation of GLP-grade human embryonic stem cell banks from four clinical-grade cell lines for preclinical research

The Singapore Stem Cell Bank has generated human embryonic stem cell banks from clinical-grade cell lines ESI-017, ESI-035, ESI-049, and ESI-053. All banks were prepared and characterized according to principles of Good Laboratory Practice for quality assurance. Importantly, each cell line has clearly documented and approved ethical provenance and meets recognized standards for performance and safety. The banks are intended to facilitate the translation of stem cell research to clinical medicine by enabling early phase research and development with high-quality, low-cost cells that are also available as clinical-grade stocks.     

Improvement of in vitro potency assays by a resting step for clinical-grade chimeric antigen receptor engineered T cells

BackgroundChimeric antigen receptor engineered T (CAR-T) cell therapy is a promising approach currently revolutionizing the field of cancer immunotherapy. However, data concerning clinical-grade CAR-T cell stability and functionality after months of cryopreservation have not been released by companies so far. To investigate the effect of cryopreservation on CAR-T cells and to further optimize the potency assays, we performed this study.MethodsA third generation of CD19 CAR-T cells was manufactured according to Good Manufacturing Practice (GMP) requirements, which is applied to patients in an ongoing clinical phase 1 study. Quality control tests for sterility, endotoxin and mycoplasma were performed for each batch. Stability in terms of viability, recovery, transduction efficiency and functional capacity was determined using microscopy, multiparametric flow cytometry as well as chromium-51 release tests.ResultsUp to 90days of cryopreservation had no influence on viability, recovery and transduction efficiency of CAR-T cells. However, higher cell concentration for cryopreservation could alter the cell viability and recovery but not the transduction efficiency. Moreover, directly after thawing, both the quantity and quality of the functionality of CAR-T cells were transiently hampered by the negative effects of cryopreservation. Notably, the impaired functionality could be fully restored and even strengthened after an overnight resting process.DiscussionCryopreservation is a challenge for the functional activity of CAR-T cells. However, CAR-T cells regain their potency by overnight incubation at 37°C, which mimics the clinical application setting. Therefore, an overnight resting step should be included in in vitro potency assays.     

Development and validation of an egg-based potency assay for a trivalent live attenuated influenza vaccine

Live attenuated influenza vaccines (LAIVs) targeting seasonal influenza are produced in embryonated eggs and formulated as a trivalent preparation of three live attenuated vaccine strains, one A(H1N1) strain, one A(H3N2) strain and one type B strain. In this study, we describe an egg-based potency assay for estimating the 50% Egg infectious dose (EID50) of individual strains in the trivalent preparation by selective neutralisation of two strains and then estimating the infective titres of the non-neutralised strain. The test is highly specific, and no cross interference of heterologous antisera is observed in the estimation of individual titres. Individual strains with titres in the range of 6.5-7.0 log EID50 per 0.5 ml show intra-assay and inter-assay coefficients of variance ranging from 1.25% to 2.95%. This assay was developed to establish a simple, reliable and inexpensive egg-based assay for estimating the potency of individual strains in a trivalent preparation.     

Low angiogenic potency induced by the implantation of ex vivo expanded CD117(+) stem cells

Ex vivo expansion of stem cells might be a feasible method of resolving the problem of limited cell supply in cell-based therapy. The implantation of expanded CD34(+) endothelial progenitor cells has the capacity to induce angiogenesis. In this study, we tried to induce angiogenesis by implanting expanded CD117(+) stem cells derived from mouse bone marrow. After 2 wk of culture with the addition of several growth factors, the CD117(+) stem cells expanded approximately 20-fold and had an endothelial phenotype with high expression of CD34 and vascular endothelial-cadherin. However, >70% of these ex vivo expanded cells had a senescent phenotype by beta-galactosidase staining, and their survival and incorporation were poor after implantation into the ischemic limbs of mice. Compared with the PBS injection only, the microvessel density and the percentage of limb blood flow were significantly higher after the implantation of 2 x 10(5) freshly collected CD117(+) cells (P < 0.01) but not after the implantation of 2 x 10(5) expanded CD117(+) cells (P > 0.05). These data indicate that ex vivo expansion of CD117(+) stem cells has low potency for inducing therapeutic angiogenesis, which might be related to the cellular senescence during ex vivo expansion.     

Characterization of a topologically aberrant plasmid population from pilot-scale production of clinical-grade DNA

As part of a program to develop DNA vaccines for pharmaceutical applications, we recently established a manufacturing process for the production of clinical grade plasmid DNA. In an evaluation of two cell separation methods, the cell culture experienced a temperature spike in a new tangential flow filtration rig, resulting in an aberrant plasmid HPLC peak. Analysis by agarose gel electrophoresis and HPLC demonstrated that the aberrant plasmid material's overall primary structure, methylation pattern and topological integrity was indistinguishable from that of reference material. Transmission electron microscopy and high-resolution agarose gel electrophoresis revealed that the unknown plasmid form exhibited a very low level of supercoiling, whereas the normal supercoiled fraction contained highly twisted DNA. We hypothesized that an enzymatic process, induced by stress during the temperature spike, caused the distinct plasmid topology. This idea was supported by a lab-scale fermentation experiment, where plasmid topology was shown to be similarly altered by conditions designed to induce metabolic stress.     

Characterization of a single reporter-gene potency assay for T-cell-dependent bispecific molecules

ABSTRACT

T-cell-dependent bispecific antibodies (TDBs) are promising cancer immunotherapies that recruit patients’ T cells to kill cancer cells. There are many TDBs in clinical trials, demonstrating their widely recognized therapeutic potential. However, their complex, multi-step mechanism of action (MoA), which includes bispecific antigen binding, T-cell activation, and target-cell killing, presents unique challenges for biological characterization and potency assay selection. Here, we describe the development of a single reporter-gene potency assay for a TDB (TDB1) that is MoA reflective and sensitive to binding of both antigens. Our reporter-gene assay measures T-cell activation using Jurkat cells engineered to express luciferase under the control of an NFkB response element. The potencies of select samples were measured both by this assay and by a flow-cytometry-based cell-killing assay using human lymphocytes as effector cells. Correlating the two sets of potency results clearly establishes our reporter-gene assay as MoA reflective. Furthermore, correlating potencies for the same panel of samples against binding data measured by binding assays for each individual arm demonstrates that the reporter-gene potency assay reflects dual-antigen binding and can detect changes in affinity for either arm. This work demonstrates that one reporter-gene assay can be used to measure the potency of TDB1 while capturing key aspects of its MoA, thus serving as a useful case study of selection and justification of reporter-gene potency assays for TDBs. Furthermore, our strategy of correlating reporter-gene potency, target-cell killing, and antigen binding for each individual arm serves as a useful example of a thorough, holistic approach to biological characterization for TDBs that can be applied to other bispecific molecules.     

Standardization and validation of Vero cell assay for potency estimation of diphtheria antitoxin serum

Diphtheria toxin has the capacity to block protein synthesis in cultured mammalian cells, and thus causing cell death. This capacity of diphtheria toxin was utilized for in-vitro neutralization test to determine antibody titer, using Vero cells, which have been found to be susceptible to diphtheria toxin. In the present study, a Vero cell assay was standardized and validated for potency estimation of diphtheria antitoxin serum (DATS). The results obtained by Vero cell assay were compared with in-vivo biological assay. High degree of correlation (+0.98) was found between in-vivo biological assay and in-vitro Vero cell assay. The assay has also been found to be effective in determining the rising antibody titer in the equines inducted in DATS production. The present study indicated that although biological assays hold the key for final potency estimations till date but in the future scenario in-vitro Vero cell assay may be a good alternative to in-vivo biological assay.     

Development of a one-step embryonic stem cell-based assay for the screening of sprouting angiogenesis

Background  

Angiogenesis assays are important tools for the identification of regulatory molecules and the potential development of therapeutic strategies to modulate neovascularization. Although numerous in vitro angiogenesis models have been developed in the past, they exhibit limitations since they do not recapitulate the entire angiogenic process or correspond to multi-step procedures that are not easy to use. Convenient, reliable, easily quantifiable and physiologically relevant assays are still needed for pharmacological screenings of angiogenesis.     

The human tumor stem cell assay revisited

The human tumor stem cell assay (HTSCA) is a bilayer soft agar system for growing fresh human tumor specimens in vitro to determine drug sensitivity and improve our understanding of tumor biology. Recent clinical correlations of 60% accuracy for predicting a positive clinical response and a 90% accuracy for predicting a lack of response to therapeutic agents suggest promising clinical usefulness. However, the clinician should be aware of the assay's inherent pitfalls, such as heterogeneity of the tumor specimen, inability to obtain pure single-cell suspensions, low cloning efficiency, unusual drug dose-dependent survival curves, uncertain validity of in vitro pharmacology, non-standardized criteria for in vitro sensitivity, and the variability of in vitro results. A brief summary of the concepts, potential, and limitations of this assay are discussed.     

Development of assay system for immunoglobulin production regulatory factors using whole cell cultures of mouse splenocytes

We tried to establish an assay system for screening and assessment of immunoregulatory factors using whole cell cultures of mouse splenocytes and found that splenic adhesive cells markedly increased immunogobulin (Ig) production of splenocytes. In the absence of adhesive cells, lipopolysaccharides, pokeweed mitogen, and phytohemagglutinin stimulated the production of IgA, IgG, and IgM in a class-dependent manner. Adhesive cells increased more markedly Ig production of splenocytes stimulated with these mitogens. When mouse splenocytes were cultured with milk proteins in the absence of adhesive cells, lactoferrin, beta-lactoglobulin, alpha-casein, and beta-casein stimulated IgA and IgG production. Adhesive cells increased IgA production of splenocytes stimulated with milk proteins, especially. These results suggest that the assay system is useful for assessment of Ig production-regulating factors.     

Type 2 diabetes alters mesenchymal stem cell secretome composition and angiogenic properties

This study aimed at characterizing the impact of type 2 diabetes mellitus (T2DM) on the bone marrow mesenchymal stem cell (BMMSC) secretome and angiogenic properties. BMMSCs from Zucker diabetic fatty rats (ZDF) (a T2DM model) and Zucker LEAN littermates (control) were cultured. The supernatant conditioned media (CM) from BMMSCs of diabetic and control rats were collected and analysed. Compared to results obtained using CM from LEAN‐BMMSCs, the bioactive content of ZDF‐BMMSC CM (i) differently affects endothelial cell (HUVEC) functions in vitro by inducing increased (3.5‐fold; P < 0.01) formation of tubule‐like structures and migration of these cells (3‐fold; P < 0.001), as well as promotes improved vascular formation in vivo, and (ii) contains different levels of angiogenic factors (e.g. IGF1) and mediators, such as OSTP, CATD, FMOD LTBP1 and LTBP2, which are involved in angiogenesis and/or extracellular matrix composition. Addition of neutralizing antibodies against IGF‐1, LTBP1 or LTBP2 in the CM of BMMSCs from diabetic rats decreased its stimulatory effect on HUVEC migration by approximately 60%, 40% or 40%, respectively. These results demonstrate that BMMSCs from T2DM rats have a unique secretome with distinct angiogenic properties and provide new insights into the role of BMMSCs in aberrant angiogenesis in the diabetic milieu.     

Critical appraisal of the side population assay in stem cell and cancer stem cell research

The "Side Population" (SP) discrimination assay is a flow cytometry method used to detect stem cells based on the dye efflux properties of ABC transporters. We discuss the SP assay and its applications in stem cell?biology, with an emphasis on the technical challenges related to sample preparation, data acquisition, analysis, and interpretation. We highlight the value of multicolor phenotyping, the impact of DNA ploidy, and the importance of distinguishing graft versus host cells for an appropriate SP discrimination. To improve the consistency and reliability of data between laboratories, we propose a set of recommendations for SP assay data reporting.     

Application of methods for viral clearance in stem cell production

Regenerative medicine therapies will allow in the future the transplant of cells of human origin in some diseases that until now have been incurable. The assurance of the safety and quality, especially from a microbiological point of view, is very important for these therapeutic products. Depending on the starting material, there are several sources of pathogen presence, mainly human viruses. Also, the use of feeders of animal origin as layers in which the stem cells can grow may permit the transmission of animal pathogens to these cells. However, cell sources are limited due to the low availability of spare in vitro fecundation human embryos and the low rate of success in the derivation of human stem cell lines. Thus, in several cases, it will be necessary to evaluate the possibility of removing or inactivating these microorganisms. In this paper, we summarize the main methods of viral clearance and we have provided an overview of the main features taking into account in the viral clearance techniques.