Mesenchymal and induced pluripotent stem cell-based therapeutics: a comparison.

Stem cell-based cell therapeutics and especially those based on human mesenchymal stem cells (hMSCs) and induced pluripotent stem cells (hiPSCs) are said to have enormous developmental potential in the coming years. Their applications range from the treatment of orthopedic disorders and cardiovascular diseases to autoimmune diseases and even cancer. However, while more than 27 hMSC-derived therapeutics are currently commercially available, hiPSC-based therapeutics have yet to complete the regulatory approval process. Based on a review of the current commercially available hMSC-derived therapeutic products and upcoming hiPSC-derived products in phase 2 and 3, this paper compares the cell therapy manufacturing process between these two cell types. Moreover, the similarities as well as differences are highlighted and the resulting impact on the production process discussed. Here, emphasis is placed on (i) hMSC and hiPSC characteristics, safety, and ethical aspects, (ii) their morphology and process requirements, as well as (iii) their 2- and 3-dimensional cultivations in dependence of the applied culture medium and process mode. In doing so, also downstream processing aspects are covered and the role of single-use technology is discussed. KEY POINTS: • Mesenchymal and induced pluripotent stem cells exhibit distinct behaviors during cultivation • Single-use stirred bioreactor systems are preferred for the cultivation of both cell types • Future research should adapt and modify downstream processes to available single-use devices.

Slowed Diffusion and Excluded Volume Both Contribute to the Effects of Macromolecular Crowding on Alcohol Dehydrogenase Steady-State Kinetics.

To understand the consequences of macromolecular crowding, studies have largely employed in vitro experiments with synthetic polymers assumed to be both pure and "inert". These polymers alter enzyme kinetics by excluding volume that would otherwise be available to the enzymes, substrates, and products. Presented here is evidence that other factors, in addition to excluded volume, must be considered in the interpretation of crowding studies with synthetic polymers. Dextran has a weaker effect on the Michaelis-Menten kinetic parameters of yeast alcohol dehydrogenase (YADH) than its small molecule counterpart, glucose. For glucose, the decreased Vmax values directly correlate with slower translational diffusion and the decreased Km values likely result from enhanced substrate binding due to YADH stabilization. Because dextran is unable to stabilize YADH to the same extent as glucose, this polymer's ability to decrease Km is potentially due to the nonideality of the solution, a crowding-induced conformational change, or both. Chronoamperometry reveals that glucose and dextran have surprisingly similar ferricyanide diffusion coefficients. Thus, the reduction in Vmax values for glucose is partially offset by an additional macromolecular crowding effect with dextran. Finally, this is the first report that supplier-dependent impurities in dextran affect the kinetic parameters of YADH. Taken together, our results reveal that caution should be used when interpreting results obtained with inert synthetic polymeric agents, as additional effects from the underlying monomer need to be considered.

A novel approach for enumeration of extracellular vesicles from crude and purified cell culture samples.

The interest in extracellular vesicles (EVs) has been increased in recent years due to their potential application in diagnosis and therapy of severe diseases. The versatile fields of application due to the numerous possible cargos and the targeted delivery system make them a promising biopharmaceutical product. However, their broad size range as well as varied surface protein content result in challenges for the purification, characterization, and quantification. In this study a novel method, based on high-resolution flow cytometry, was examined for the enumeration of EVs in purified as well as crude process samples. In addition to quantification, samples were characterized by dynamic light scattering, zeta potential measurement, and analytical size exclusion chromatography. It has been demonstrated that EVs were successfully enumerated with the novel method, offering great benefits for development and monitoring of EV processes.

The Interplay of Electrostatics and Chemical Positioning in the Evolution of Antibiotic Resistance in TEM ß-Lactamases.

The interplay of enzyme active site electrostatics and chemical positioning is important for understanding the origin(s) of enzyme catalysis and the design of novel catalysts. We reconstruct the evolutionary trajectory of TEM-1 ß-lactamase to TEM-52 toward extended-spectrum activity to better understand the emergence of antibiotic resistance and to provide insights into the structure-function paradigm and noncovalent interactions involved in catalysis. Utilizing a detailed kinetic analysis and the vibrational Stark effect, we quantify the changes in rates and electric fields in the Michaelis and acyl-enzyme complexes for penicillin G and cefotaxime to ascertain the evolutionary role of electric fields to modulate function. These data are combined with MD simulations to interpret and quantify the substrate-dependent structural changes during evolution. We observe that this evolutionary trajectory utilizes a large preorganized electric field and substrate-dependent chemical positioning to facilitate catalysis. This governs the evolvability, substrate promiscuity, and protein fitness landscape in TEM ß-lactamase antibiotic resistance.

Testing the Limitations of MD-Based Local Electric Fields Using the Vibrational Stark Effect in Solution: Penicillin G as a Test Case.

Noncovalent interactions underlie nearly all molecular processes in the condensed phase from solvation to catalysis. Their quantification within a physically consistent framework remains challenging. Experimental vibrational Stark effect (VSE)-based solvatochromism can be combined with molecular dynamics (MD) simulations to quantify the electrostatic forces in solute-solvent interactions for small rigid molecules and, by extension, when these solutes bind in enzyme active sites. While generalizing this approach toward more complex (bio)molecules, such as the conformationally flexible and charged penicillin G (PenG), we were surprised to observe inconsistencies in MD-based electric fields. Combining synthesis, VSE spectroscopy, and computational methods, we provide an intimate view on the origins of these discrepancies. We observe that the electric fields are correlated to conformation-dependent effects of the flexible PenG side chain, including both the local solvation structure and solute conformational sampling in MD. Additionally, we identified that MD-based electric fields are consistently overestimated in three-point water models in the vicinity of charged groups; this cannot be entirely ameliorated using polarizable force fields (AMOEBA) or advanced water models. This work demonstrates the value of the VSE as a direct method for experiment-guided refinements of MD force fields and establishes a general reductionist approach to calibrating vibrational probes for complex (bio)molecules.

Biosynthetic Incorporation of Site-Specific Isotopes in ß-Lactam Antibiotics Enables Biophysical Studies.

A biophysical understanding of the mechanistic, chemical, and physical origins underlying antibiotic action and resistance is vital to the discovery of novel therapeutics and the development of strategies to combat the growing emergence of antibiotic resistance. The site-specific introduction of stable-isotope labels into chemically complex natural products is particularly important for techniques such as NMR, IR, mass spectrometry, imaging, and kinetic isotope effects. Toward this goal, we developed a biosynthetic strategy for the site-specific incorporation of 13C labels into the canonical ß-lactam carbonyl of penicillin G and cefotaxime, the latter via cephalosporin C. This was achieved through sulfur-replacement with 1-13C-l-cysteine, resulting in high isotope incorporations and milligram-scale yields. Using 13C NMR and isotope-edited IR difference spectroscopy, we illustrate how these molecules can be used to interrogate interactions with their protein targets, e.g., TEM-1 ß-lactamase. This method provides a feasible route to isotopically labeled penicillin and cephalosporin precursors for future biophysical studies.

Vibrational Stark Spectroscopy of Fluorobenzene Using Quantum Cascade Laser Dual Frequency Combs.

We demonstrate the performance of a dual frequency comb quantum cascade laser (QCL) spectrometer for the application of vibrational Stark spectroscopy. Measurements performed on fluorobenzene with the dual-comb spectrometer (DCS) were compared to results obtained using a conventional Fourier transform infrared (FT-IR) instrument in terms of spectral response, parameter estimation, and signal-to-noise ratio (S/N). The dual-comb spectrometer provided similar qualitative and quantitative data as the FT-IR setup in 250 times shorter acquisition time. For fluorobenzene, the DCS measurement resulted in a more precise estimation of the fluorobenzene Stark tuning rate ((0.81 ± 0.09) cm-1/(MV/cm)) than with the FT-IR system ((0.89 ± 0.15) cm-1/(MV/cm)). Both values are in accordance with the previously reported value of 0.84 cm-1/(MV/cm). We also point to an improvement of signal-to-noise ratio in the DCS configuration. Additional characteristics of the dual-comb spectrometer applicable to vibrational Stark spectroscopy and their scaling properties for future applications are discussed.

Solvent-Independent Anharmonicity for Carbonyl Oscillators.

The physical origins of vibrational frequency shifts have been extensively studied in order to understand noncovalent intermolecular interactions in the condensed phase. In the case of carbonyls, vibrational solvatochromism, MD simulations, and vibrational Stark spectroscopy suggest that the frequency shifts observed in simple solvents arise predominately from the environment's electric field due to the vibrational Stark effect. This is contrary to many previously invoked descriptions of vibrational frequency shifts, such as bond polarization, whereby the bond's force constant and/or partial nuclear charges are altered due to the environment, often illustrated in terms of favored resonance structures. Here we test these hypotheses using vibrational solvatochromism as measured using 2D IR to assess the solvent dependence of the bond anharmonicity. These results indicate that the carbonyl bond's anharmonicity is independent of solvent as tested using hexanes, DMSO, and D2O and is supported by simulated 2D spectra. In support of the linear vibrational Stark effect, these 2D IR measurements are consistent with the assertion that the Stark tuning rate is unperturbed by the electric field generated by both hydrogen and non-hydrogen bonding environments and further extends the general applicability of carbonyl probes for studying intermolecular interactions.

Vibrational Stark Effects of Carbonyl Probes Applied to Reinterpret IR and Raman Data for Enzyme Inhibitors in Terms of Electric Fields at the Active Site.

IR and Raman frequency shifts have been reported for numerous probes of enzyme transition states, leading to diverse interpretations. In the case of the model enzyme ketosteroid isomerase (KSI), we have argued that IR spectral shifts for a carbonyl probe at the active site can provide a connection between the active site electric field and the activation free energy (Fried et al. Science 2014, 346, 1510-1514). Here we generalize this approach to a much broader set of carbonyl probes (e.g., oxoesters, thioesters, and amides), first establishing the sensitivity of each probe to an electric field using vibrational Stark spectroscopy, vibrational solvatochromism, and MD simulations, and then applying these results to reinterpret data already in the literature for enzymes such as 4-chlorobenzoyl-CoA dehalogenase and serine proteases. These results demonstrate that the vibrational Stark effect provides a general framework for estimating the electrostatic contribution to the catalytic rate and may provide a metric for the design or modification of enzymes. Opportunities and limitations of the approach are also described.

A ring fragmentation approach to medium-sized cyclic 2-alkynones.

Bicyclic γ-silyloxy-ß-hydroxy-α-diazoketones, in which the Cß-Cγ bond is the ring fusion bond, productively fragment when treated with tin(IV) chloride to provide medium-sized cyclic 2-alkynones. This method provides good to excellent yields of 10-, 11-, and 12-membered alkynone products.