Human Mesenchymal Stem Cell Processing for Clinical Applications Using a Closed Semi-Automated Workflow.

Human mesenchymal stem cells (hMSCs) are currently being explored as a promising cell-based therapeutic modality for various diseases, with more market approvals for clinical use expected over the next few years. To facilitate this transition, addressing the bottlenecks of scale, lot-to-lot reproducibility, cost, regulatory compliance, and quality control is critical. These challenges can be addressed by closing the process and adopting automated manufacturing platforms. In this study, we developed a closed and semi-automated process for passaging and harvesting Wharton's jelly (WJ)-derived hMSCs (WJ-hMSCs) from multi-layered flasks using counterflow centrifugation. The WJ-hMSCs were expanded using regulatory compliant serum-free xeno-free (SFM XF) medium, and they showed comparable cell proliferation (population doubling) and morphology to WJ-hMSCs expanded in classic serum-containing media. Our closed semi-automated harvesting protocol demonstrated high cell recovery (~98%) and viability (~99%). The cells washed and concentrated using counterflow centrifugation maintained WJ-hMSC surface marker expression, colony-forming units (CFU-F), trilineage differentiation potential, and cytokine secretion profiles. The semi-automated cell harvesting protocol developed in the study can be easily applied for the small- to medium-scale processing of various adherent and suspension cells by directly connecting to different cell expansion platforms to perform volume reduction, washing, and harvesting with a low output volume.

Acceleration of Translational Mesenchymal Stromal Cell Therapy Through Consistent Quality GMP Manufacturing.

Human mesenchymal stromal cell (hMSC) therapy has been gaining immense interest in regenerative medicine and quite recently for its immunomodulatory properties in COVID-19 treatment. Currently, the use of hMSCs for various diseases is being investigated in >900 clinical trials. Despite the huge effort, setting up consistent and robust scalable manufacturing to meet regulatory compliance across various global regions remains a nagging challenge. This is in part due to a lack of definitive consensus for quality control checkpoint assays starting from cell isolation to expansion and final release criterion of clinical grade hMSCs. In this review, we highlight the bottlenecks associated with hMSC-based therapies and propose solutions for consistent GMP manufacturing of hMSCs starting from raw materials selection, closed and modular systems of manufacturing, characterization, functional testing, quality control, and safety testing for release criteria. We also discuss the standard regulatory compliances adopted by current clinical trials to broaden our view on the expectations across different jurisdictions worldwide.

Multiomics analyses of cytokines, genes, miRNA, and regulatory networks in human mesenchymal stem cells expanded in stirred microcarrier-spinner cultures.

Mesenchymal stem cells (MSCs) are of great clinical interest as a form of allogenic therapy due to their excellent regenerative and immunomodulatory effects for various therapeutic indications. Stirred suspension bioreactors using microcarriers (MC) have been used for large-scale production of MSCs compared to planar cultivation systems. Previously, we have demonstrated that expansion of MSCs in MC-spinner cultures improved chondrogenic, osteogenic, and cell migration potentials as compared to monolayer-static cultures. In this study, we sought to address this by analyzing global gene expression patterns, miRNA profiles and secretome under both monolayer-static and MC-spinner cultures in serum-free medium at different growth phases. The datasets revealed differential expression patterns that correlated with potentially improved MSC properties in cells from MC-spinner cultures compared to those of monolayer-static cultures. Transcriptome analysis identified a unique expression signature for cells from MC-spinner cultures, which correlated well with miRNA expression, and cytokine secretion involved in key MSC functions. Importantly, MC-spinner cultures and conditioned medium showed increased expression of factors that possibly enhance pathways of extracellular matrix dynamics, cellular metabolism, differentiation potential, immunoregulatory function, and wound healing. This systematic analysis provides insights for the efficient optimization of stem cell bioprocessing and infers that MC-based bioprocess manufacturing could improve post-expansion cellular properties for stem cell therapies.

A model-driven approach towards rational microbial bioprocess optimization.

Due to sustainability concerns, bio-based production capitalizing on microbes as cell factories is in demand to synthesize valuable products. Nevertheless, the nonhomogenous variations of the extracellular environment in bioprocesses often challenge the biomass growth and the bioproduction yield. To enable a more rational bioprocess optimization, we have established a model-driven approach that systematically integrates experiments with modeling, executed from flask to bioreactor scale, and using ferulic acid to vanillin bioconversion as a case study. The impacts of mass transfer and aeration on the biomass growth and bioproduction performances were examined using minimal small-scale experiments. An integrated model coupling the cell factory kinetics with the three-dimensional computational hydrodynamics of bioreactor was developed to better capture the spatiotemporal distributions of bioproduction. Full-factorial predictions were then performed to identify the desired operating conditions. A bioconversion yield of 94% was achieved, which is one of the highest for recombinant Escherichia coli using ferulic acid as the precursor.

Self-Assembled Polymer Thin Films Towards Nanoarchitectonics for Respiration Monitoring.

Manipulation of ionic transport in the self-assembled polymer thin films using nanoarchitectonics approach can open the door for the development of novel electronic devices with ultrafast operation and low-power consumption. Here, we demonstrate a highly sensitive and ultrafast responsive flexible humidity sensor for human respiration monitoring. Humidity sensing behavior of the polymerbased planar devices, in which a polyethylene oxide-phosphotungstic acid (PEO-PWA) thin film is placed between an opposing inert electrodes, have been investigated by optimizing the device configuration and PWA salt concentration in the PEO matrix. The ultrafast response (~50 ms) and recovery (~52 ms) of the humidity sensor enabled us to study the real-time human respiration monitoring. Using morphological analysis, it is proposed that the ultrafast response-recovery time for this sensor is ascribed to their self-assembled lamellar-like structures of the PEO-PWA matrix polymer, which provides long-range continuous proton transport path in the polymer interface.

Rapid Detection of Senescent Mesenchymal Stromal Cells by a Fluorescent Probe.

Despite intense interest in human mesenchymal stromal cells (MSCs), monitoring of the progressive occurrence of senescence has been hindered by the lack of efficient detection tools. Here, the discovery of a novel MSC senescence-specific fluorescent probe (CyBC9) identified by a high-throughput screen is reported. Compared with the prototypical senescence-associated ß-galactosidase (SA-ß-gal) staining, the CyBC9 assay is rapid (2 h) and nontoxic and can thus be applied to live cells. It is shown that CyBC9 is able to stain early and late senescent populations both in monolayer- and in microcarrier-based cultures. Finally, to investigate the mechanism of CyBC9, colocalization assays are performed and it is found that CyBC9 is accumulated in the mitochondria of senescent MSCs presumably due to the loss of membrane potential. Taken together, it is expected that CyBC9 will be a useful tool to ameliorate cell therapy through rapid and early screening of senescent phenotypes in clinically relevant MSCs.

Programming the Dynamic Control of Bacterial Gene Expression with a Chimeric Ligand- and Light-Based Promoter System.

To program cells in a dynamic manner, synthetic biologists require precise control over the threshold levels and timing of gene expression. However, in practice, modulating gene expression is widely carried out using prototypical ligand-inducible promoters, which have limited tunability and spatiotemporal resolution. Here, we built two dual-input hybrid promoters, each retaining the function of the ligand-inducible promoter while being enhanced with a blue-light-switchable tuning knob. Using the new promoters, we show that both ligand and light inputs can be synchronously modulated to achieve desired amplitude or independently regulated to generate desired frequency at a specific amplitude. We exploit the versatile programmability and orthogonality of the two promoters to build the first reprogrammable logic gene circuit capable of reconfiguring into logic OR and N-IMPLY logic on the fly in both space and time without the need to modify the circuit. Overall, we demonstrate concentration- and time-based combinatorial regulation in live bacterial cells with potential applications in biotechnology and synthetic biology.

Cell-Free Optogenetic Gene Expression System.

Optogenetic tools provide a new and efficient way to dynamically program gene expression with unmatched spatiotemporal precision. To date, their vast potential remains untapped in the field of cell-free synthetic biology, largely due to the lack of simple and efficient light-switchable systems. Here, to bridge the gap between cell-free systems and optogenetics, we studied our previously engineered one component-based blue light-inducible Escherichia coli promoter in a cell-free environment through experimental characterization and mathematical modeling. We achieved >10-fold dynamic expression and demonstrated rapid and reversible activation of the target gene to generate oscillatory response. The deterministic model developed was able to recapitulate the system behavior and helped to provide quantitative insights to optimize dynamic response. This in vitro optogenetic approach could be a powerful new high-throughput screening technology for rapid prototyping of complex biological networks in both space and time without the need for chemical induction.

Chitosan-nickel film based interferometric optical fiber sensor for label-free detection of histidine tagged proteins.

An interferometric fiber sensor for detection of hexa-histidine tagged microcin (His-MccS) is reported and experimentally demonstrated. This intermodal fiber sensor is implemented by a no-core fiber (NCF) functionalized with chitosan (CS)-nickel (Ni) film for direct detection of small peptide: microcin. The fiber intermodal sensor relies on the refractive index modulations due to selective adsorption event at the chitosan (CS)-nickel (Ni) film. Owing to the strong affinity between Ni2+ ions and histidine, the immobilized Ni2+ ions in the chitosan film were utilized as binding agents for the direct detection of hexa-histidine tagged microcin. A comparative study in relation to different target size was conducted: full proteins trypsin, bovine serum albumin (BSA) and human serum albumin (HSA), with high histidine content on their surface and His-MccS (peptide, 11.6kDa), have been employed for sensor evaluation. Results have shown selectivity for His-MccS relative to trypsin, BSA and HSA. The most telling contribution of this study is the fast detection of small biomolecule His-MccS compared to standard detection procedures like SDS-PAGE and western blot. The proposed sensor exhibits His-MccS detection sensitivity of 0.0308nm/(ng/ml) in the range of (0-78) ng/ml with concentration detection limit of 0.8368ng/ml.

Repurposing a Two-Component System-Based Biosensor for the Killing of Vibrio cholerae.

New strategies to control cholera are urgently needed. This study develops an in vitro proof-of-concept sense-and-kill system in a wild-type Escherichia coli strain to target the causative pathogen Vibrio cholerae using a synthetic biology approach. Our engineered E. coli specifically detects V. cholerae via its quorum-sensing molecule CAI-1 and responds by expressing the lysis protein YebF-Art-085, thereby self-lysing to release the killing protein Art-085 to kill V. cholerae. For this report, we individually characterized YebF-Art-085 and Art-085 expression and their activities when coupled to our previously developed V. cholerae biosensing circuit. We show that, in the presence of V. cholerae supernatant, the final integrated sense-and-kill system in our engineered E. coli can effectively inhibit the growth of V. cholerae cells. This work represents the first step toward a novel probiotic treatment modality that could potentially prevent and treat cholera in the future.

Biosensing Vibrio cholerae with Genetically Engineered Escherichia coli.

Cholera is a potentially mortal, infectious disease caused by Vibrio cholerae bacterium. Current treatment methods of cholera still have limitations. Beneficial microbes that could sense and kill the V. cholerae could offer potential alternative to preventing and treating cholera. However, such V. cholerae targeting microbe is still not available. This microbe requires a sensing system to be able to detect the presence of V. cholera bacterium. To this end, we designed and created a synthetic genetic sensing system using nonpathogenic Escherichia coli as the host. To achieve the system, we have moved proteins used by V. cholerae for quorum sensing into E. coli. These sensor proteins have been further layered with a genetic inverter based on CRISPRi technology. Our design process was aided by computer models simulating in vivo behavior of the system. Our sensor shows high sensitivity to presence of V. cholerae supernatant with tight control of expression of output GFP protein.

Blue light-mediated transcriptional activation and repression of gene expression in bacteria.

Light-regulated modules offer unprecedented new ways to control cellular behavior in precise spatial and temporal resolution. The availability of such tools may dramatically accelerate the progression of synthetic biology applications. Nonetheless, current optogenetic toolbox of prokaryotes has potential issues such as lack of rapid and switchable control, less portable, low dynamic expression and limited parts. To address these shortcomings, we have engineered a novel bidirectional promoter system for Escherichia coli that can be induced or repressed rapidly and reversibly using the blue light dependent DNA-binding protein EL222. We demonstrated that by modulating the dosage of light pulses or intensity we could control the level of gene expression precisely. We show that both light-inducible and repressible system can function in parallel with high spatial precision in a single cell and can be switched stably between ON- and OFF-states by repetitive pulses of blue light. In addition, the light-inducible and repressible expression kinetics were quantitatively analysed using a mathematical model. We further apply the system, for the first time, to optogenetically synchronize two receiver cells performing different logic behaviors over time using blue light as a molecular clock signal. Overall, our modular approach layers a transformative platform for next-generation light-controllable synthetic biology systems in prokaryotes.

Novel phytochemical-antibiotic conjugates as multitarget inhibitors of Pseudomononas aeruginosa GyrB/ParE and DHFR.

BACKGROUND: There is a dearth of treatment options for community-acquired and nosocomial Pseudomonas infections due to several rapidly emerging multidrug resistant phenotypes, which show resistance even to combination therapy. As an alternative, developing selective promiscuous hybrid compounds for simultaneous modulation of multiple targets is highly appreciated because it is difficult for the pathogen to develop resistance when an inhibitor has activity against multiple targets. METHODS: In line with our previous work on phytochemical-antibiotic combination assays and knowledge-based methods, using a fragment combination approach we here report a novel drug design strategy of conjugating synergistic phytochemical-antibiotic combinations into a single hybrid entity for multi-inhibition of P. aeruginosa DNA gyrase subunit B (GyrB)/topoisomerase IV subunit B (ParE) and dihydrofolate reductase (DHFR) enzymes. The designed conjugates were evaluated for their multitarget specificity using various computational methods including docking and dynamic simulations, drug-likeness using molecular properties calculations, and pharmacophoric features by stereoelectronic property predictions. RESULTS: Evaluation of the designed hybrid compounds based on their physicochemical properties has indicated that they are promising drug candidates with drug-like pharmacotherapeutic profiles. In addition, the stereoelectronic properties such as HOMO (highest occupied molecular orbital), LUMO (lowest unoccupied molecular orbital), and MEP (molecular electrostatic potential) maps calculated by quantum chemical methods gave a good correlation with the common pharmacophoric features required for multitarget inhibition. Furthermore, docking and dynamics simulations revealed that the designed compounds have favorable binding affinity and stability in both the ATP-binding sites of GyrB/ParE and the folate-binding site of DHFR, by forming strong hydrogen bonds and hydrophobic interactions with key active site residues. CONCLUSION: This new design concept of hybrid "phyto-drug" scaffolds, and their simultaneous perturbation of well-established antibacterial targets from two unrelated pathways, appears to be very promising and could serve as a prospective lead in multitarget drug discovery.

Hybrid-drug design targeting Pseudomonas aeruginosa dihydropteroate synthase and dihydrofolate reductase

In this study, we successfully present the dual-target design hypothesis to inhibit both dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) enzymes using a novel scheme that integrates our previous antibiotic-phytochemical interaction data, fragment combination and knowledge-based methods. Both the enzymes are well established antibacterial targets from folate biosynthesis pathway and their synergistic modulation by a single hybrid entity may have profound therapeutic benefits. Evaluation of the designed hybrid compounds based on their physico-chemical properties has indicated them as promising drug candidates with drug-like pharmacotherapeutic profiles. In addition, the stereo-electronic properties such as HOMO, LUMO and MEP maps calculated by quantum chemical methods gave a good correlation with the common pharmacophoric features required for dual-site interactions. Furthermore, docking and dynamics simulation studies reveal that the designed hybrid compounds have favorable binding affinity and stability in both pterin-binding site of DHPS and folate-binding site of DHFR by forming strong hydrogen bonds and hydrophobic interactions with key active-site residues. Looking forward this study could serve as a prospective lead in the process of new natural-product based hybrid-drugs development.

Insights into antifolate activity of phytochemicals against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic drug resistant pathogen. Drug interaction studies for phytochemicals (protocatechuic acid (PA), gallic acid (GA), quercetin (QUER), and myricetin (MYR)) in combination with antifolates (sulfamethoxazole (SMX) and trimethoprim (TMP)) are presented. Our results show that the combinations of SMX and phytochemicals are synergistic, whereas TMP in combination with phytochemicals results in additive mode of interaction. Molecular docking of phytochemicals in the active site of modeled P. aeruginosa dihydrofolate reductase (DHFR), an important enzyme in the folic acid biosynthesis pathway, shows that the phytochemicals QUER and MYR dock in the active site of P. aeruginosa DHFR with promoted binding at the NADP site, PA, and GA dock in the active site of P. aeruginosa DHFR with promoted binding at the folate binding site. Possible mode of action of these phytochemicals as anti-DHFR compounds in this bacterium is suggested. Taken together, the above findings provide novel insights to mode of interactions of these phytochemicals with antibiotics and may have significance as prospective leads in the development of antipseudomonal drug developments.

Activity and interactions of antibiotic and phytochemical combinations against Pseudomonas aeruginosa in vitro.

In this study the in vitro activities of seven antibiotics (ciprofloxacin, ceftazidime, tetracycline, trimethoprim, sulfamethoxazole, polymyxin B and piperacillin) and six phytochemicals (protocatechuic acid, gallic acid, ellagic acid, rutin, berberine and myricetin) against five P. aeruginosa isolates, alone and in combination are evaluated. All the phytochemicals under investigation demonstrate potential inhibitory activity against P. aeruginosa. The combinations of sulfamethoxazole plus protocatechuic acid, sulfamethoxazole plus ellagic acid, sulfamethoxazole plus gallic acid and tetracycline plus gallic acid show synergistic mode of interaction. However, the combinations of sulfamethoxazole plus myricetin shows synergism for three strains (PA01, DB5218 and DR3062). The synergistic combinations are further evaluated for their bactericidal activity against P. aeruginosa ATCC strain using time-kill method. Sub-inhibitory dose responses of antibiotics and phytochemicals individually and in combination are presented along with their interaction network to suggest on the mechanism of action and potential targets for the phytochemicals under investigation. The identified synergistic combinations can be of potent therapeutic value against P. aeruginosa infections. These findings have potential implications in delaying the development of resistance as the antibacterial effect is achieved with lower concentrations of both drugs (antibiotics and phytochemicals).

In vitro combinations of antibiotics and phytochemicals against Pseudomonas aeruginosa.

BACKGROUND AND PURPOSE: Antibiotic combinations are used to enhance antibacterial efficacy and to prevent the development of resistance. In this study, the in vitro activities of antibiotic and phytochemical combinations against Pseudomonas aeruginosa were tested by the fractional inhibitory concentration method, derived from the minimal inhibitory concentrations (MICs) of the agents in combination. METHODS: The antimicrobial activity of phytochemicals, alone and in combination with antibiotics, was evaluated using the checkerboard assay and time-kill curve methods. RESULTS: There was synergism between gentamicin and caffeic acid, and sulfadiazine and the 3 phytochemicals under investigation (protocatechuic acid, quercetin, caffeic acid). The MIC of sulfadiazine was 256 microg/mL, and of gentamicin was 2 microg/mL. When gentamicin was combined with one-quarter the MIC of caffeic acid, the MIC of gentamicin was reduced 4-fold. When sulfadiazine was tested with one-quarter the MIC of protocatechuic acid, quercetin, and caffeic acid, the MIC was reduced 4-fold in combination with each of the drugs. CONCLUSIONS: These results indicate the potential efficacy of phytochemicals in combination with antibiotics for enhancing total biological activity.