Quantitatively characterizing drug-induced arrhythmic contractile motions of human stem cell-derived cardiomyocytes.

Quantification of abnormal contractile motions of cardiac tissue has been a noteworthy challenge and significant limitation in assessing and classifying the drug-induced arrhythmias (i.e., Torsades de pointes). To overcome these challenges, researchers have taken advantage of computational image processing tools to measure contractile motion from cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs). However, the amplitude and frequency analysis of contractile motion waveforms does not produce sufficient information to objectively classify the degree of variations between two or more sets of cardiac contractile motions. In this paper, we generated contractile motion data from beating hiPSC-CMs using motion tracking software based on optical flow analysis, and then implemented a computational algorithm, phase space reconstruction (PSR), to derive parameters (embedding, regularity, and fractal dimensions) to further characterize the dynamic nature of the cardiac contractile motions. Application of drugs known to cause cardiac arrhythmia induced significant changes to these resultant dimensional parameters calculated from PSR analysis. Integrating this new computational algorithm with the existing analytical toolbox of cardiac contractile motions will allow us to expand current assessments of cardiac tissue physiology into an automated, high-throughput, and quantifiable manner which will allow more objective assessments of drug-induced proarrhythmias.

Navigating the Future of Cardiovascular Drug Development-Leveraging Novel Approaches to Drive Innovation and Drug Discovery: Summary of Findings from the Novel Cardiovascular Therapeutics Conference.

PURPOSE: The need for novel approaches to cardiovascular drug development served as the impetus to convene an open meeting of experts from the pharmaceutical industry and academia to assess the challenges and develop solutions for drug discovery in cardiovascular disease. METHODS: The Novel Cardiovascular Therapeutics Summit first reviewed recent examples of ongoing or recently completed programs translating basic science observations to targeted drug development, highlighting successes (protein convertase sutilisin/kexin type 9 [PCSK9] and neprilysin inhibition) and targets still under evaluation (cholesteryl ester transfer protein [CETP] inhibition), with the hope of gleaning key lessons to successful drug development in the current era. Participants then reviewed the use of innovative approaches being explored to facilitate rapid and more cost-efficient evaluations of drug candidates in a short timeframe. RESULTS: We summarize observations gleaned from this summit and offer insight into future cardiovascular drug development. CONCLUSIONS: The rapid development in genetic and high-throughput drug evaluation technologies, coupled with new approaches to rapidly evaluate potential cardiovascular therapies with in vitro techniques, offer opportunities to identify new drug targets for cardiovascular disease, study new therapies with better efficiency and higher throughput in the preclinical setting, and more rapidly bring the most promising therapies to human testing. However, there must be a critical interface between industry and academia to guide the future of cardiovascular drug development. The shared interest among academic institutions and pharmaceutical companies in developing promising therapies to address unmet clinical needs for patients with cardiovascular disease underlies and guides innovation and discovery platforms that are significantly altering the landscape of cardiovascular drug development.

Directing cell migration and organization via nanocrater-patterned cell-repellent interfaces.

Although adhesive interactions between cells and nanostructured interfaces have been studied extensively, there is a paucity of data on how nanostructured interfaces repel cells by directing cell migration and cell-colony organization. Here, by using multiphoton ablation lithography to pattern surfaces with nanoscale craters of various aspect ratios and pitches, we show that the surfaces altered the cells' focal-adhesion size and distribution, thus affecting cell morphology, migration and ultimately localization. We also show that nanocrater pitch can disrupt the formation of mature focal adhesions to favour the migration of cells towards higher-pitched regions, which present increased planar area for the formation of stable focal adhesions. Moreover, by designing surfaces with variable pitch but constant nanocrater dimensions, we were able to create circular and striped cellular patterns. Our surface-patterning approach, which does not involve chemical treatments and can be applied to various materials, represents a simple method to control cell behaviour on surfaces.

Branching Analysis of Multivalent Conjugates Using Size Exclusion Chromatography-Multiangle Light Scattering.

Multivalent conjugates (MVCs) (conjugation of multiple proteins to a linear polymer chain) are powerful for improving the bioactivity and pharmacokinetics of a bioactive molecule. Since this effect is highly dependent upon the valency of the conjugated proteins, it is imperative to have a technique for analysis of the conjugation ratio. Studies of MVCs have used size exclusion chromatography-multiangle light scattering (SEC-MALS), which allows for the separate and individual analysis of the protein and biopolymer components based on their specific refractive index increment and UV extinction coefficient constants to determine the number of proteins bound per biopolymer molecule. In this work, we have applied traditional branching analysis to the SEC-MALS data, with the primary assumption that the polymer backbone can be used as the linear counterpart. We demonstrated good agreement between the branching values and the valency determined by traditional analysis, demonstrating that branching analysis can be used as an alternative technique to approximate the valency of MVCs. The branching analysis method also provides a more complete picture of the distribution of the measured values, provides important branching information about the molecules, and lowers the cost and complexity of the characterization. However, since MVC molecules are both conjugate molecules and branched molecules, the most powerful approach to their characterization would be to use both traditional multivalent conjugate analysis and branching analysis in conjunction.

Low Fouling Electrospun Scaffolds with Clicked Bioactive Peptides for Specific Cell Attachment.

While electrospun fibers are of interest as scaffolds for tissue engineering applications, nonspecific surface interactions such as protein adsorption often prevent researchers from controlling the exact interactions between cells and the underlying material. In this study we prepared electrospun fibers from a polystyrene-based macroinitiator, which were then grafted with polymer brushes using surface-initiated atom transfer radical polymerization (SI-ATRP). These brush coatings incorporated a trimethylsilyl-protected PEG-alkyne monomer, allowing azide functional molecules to be covalently attached, while simultaneously reducing nonspecific protein adsorption on the fibers. Cells were able to attach and spread on fibrous substrates functionalized with a pendant RGD-containing peptide, while spreading was significantly reduced on nonfunctionalized fibers and those with the equivalent RGE control peptide. This effect was observed both in the presence and absence of serum in the culture media, indicating that protein adsorption on the fibers was minimal and cell adhesion within the fibrous scaffold was mediated almost entirely through the cell-adhesive RGD-containing peptide.

Do calcium channel blockers applied to cardiomyocytes cause increased channel expression resulting in reduced efficacy?

In the initial hours following the application of the calcium channel blocker (CCB) nifedipine to microtissues consisting of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we observe notable variations in the drug's efficacy. Here, we investigate the possibility that these temporal changes in CCB effects are associated with adaptations in the expression of calcium ion channels in cardiomyocyte membranes. To explore this, we employ a recently developed mathematical model that delineates the regulation of calcium ion channel expression by intracellular calcium concentrations. According to the model, a decline in intracellular calcium levels below a certain target level triggers an upregulation of calcium ion channels. Such an upregulation, if instigated by a CCB, would then counteract the drug's inhibitory effect on calcium currents. We assess this hypothesis using time-dependent measurements of hiPSC-CMs dynamics and by refining an existing mathematical model of myocyte action potentials incorporating the dynamic nature of the number of calcium ion channels. The revised model forecasts that the CCB-induced reduction in intracellular calcium concentrations leads to a subsequent increase in calcium ion channel expression, thereby attenuating the drug's overall efficacy. The data and fit models suggest that dynamic changes in cardiac cells in the presence of CCBs may be explainable by induced changes in protein expression, and that this may lead to challenges in understanding calcium based drug effects on the heart unless timings of applications are carefully considered.

Molecular characterization of multivalent bioconjugates by size-exclusion chromatography with multiangle laser light scattering.

The degree of substitution and valency of bioconjugate reaction products are often poorly judged or require multiple time- and product-consuming chemical characterization methods. These aspects become critical when analyzing and optimizing the potency of costly polyvalent bioactive conjugates. In this study, size-exclusion chromatography with multiangle laser light scattering was paired with refractive index detection and ultraviolet spectroscopy (SEC-MALS-RI-UV) to characterize the reaction efficiency, degree of substitution, and valency of the products of conjugation of either peptides or proteins to a biopolymer scaffold, i.e., hyaluronic acid (HyA). Molecular characterization was more complete compared to estimates from a protein quantification assay, and exploitation of this method led to more accurate deduction of the molecular structures of polymer bioconjugates. Information obtained using this technique can improve macromolecular engineering design principles and help to better understand multivalent macromolecular interactions in biological systems.

Multiplexed microfluidic platform for stem-cell derived pancreatic islet ß cells.

Stem cell-derived ß cells offer an alternative to primary islets for biomedical discoveries as well as a potential surrogate for islet transplantation. The expense and challenge of obtaining and maintaining functional stem cell-derived ß cells calls for a need to develop better high-content and high-throughput culture systems. Microphysiological systems (MPS) are promising high-content in vitro platforms, but scaling for high-throughput screening and discoveries remain a challenge. Traditionally, simultaneous multiplexing of liquid handling and cell loading poses a challenge in the design of high-throughput MPS. Furthermore, although MPS for islet ß culture/testing have been developed, studies on multi-day culture of stem-cell derived ß cells in MPS have been limited. We present a scalable, multiplexed islet ß MPS device that incorporates microfluidic gradient generators to parallelize fluid handling for culture and test conditions. We demonstrated the viability and functionality of the stem cell-derived enriched ß clusters (eBCs) for a week, as assessed by the ∼2 fold insulin release by the clusters to glucose challenge. To show the scalable multiplexing for drug testing, we demonstrated the loss of stimulation index after long-term exposure to logarithmic concentration range of glybenclamide. The MPS cultured eBCs also confirmed a glycolytic bottleneck as inferred by insulin secretion responses to metabolites methyl succinate and glyceric acid. Thus, we present an innovative culture platform for eBCs with a balance of high-content and high-throughput characteristics.

Validating the Arrhythmogenic Potential of High-, Intermediate-, and Low-Risk Drugs in a Human-Induced Pluripotent Stem Cell-Derived Cardiac Microphysiological System.

Evaluation of arrhythmogenic drugs is required by regulatory agencies before any new compound can obtain market approval. Despite rigorous review, cardiac disorders remain the second most common cause for safety-related market withdrawal. On the other hand, false-positive preclinical findings prohibit potentially beneficial candidates from moving forward in the development pipeline. Complex in vitro models using cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) have been identified as a useful tool that allows for rapid and cost-efficient screening of proarrhythmic drug risk. Currently available hiPSC-CM models employ simple two-dimensional (2D) culture formats with limited structural and functional relevance to the human heart muscle. Here, we present the use of our 3D cardiac microphysiological system (MPS), composed of a hiPSC-derived heart micromuscle, as a platform for arrhythmia risk assessment. We employed two different hiPSC lines and tested seven drugs with known ion channel effects and known clinical risk: dofetilide and bepridil (high risk); amiodarone and terfenadine (intermediate risk); and nifedipine, mexiletine, and lidocaine (low risk). The cardiac MPS successfully predicted drug cardiotoxicity risks based on changes in action potential duration, beat waveform (i.e., shape), and occurrence of proarrhythmic events of healthy patient hiPSC lines in the absence of risk cofactors. We showcase examples where the cardiac MPS outperformed existing hiPSC-CM 2D models.

Metabolically driven maturation of human-induced-pluripotent-stem-cell-derived cardiac microtissues on microfluidic chips.

The immature physiology of cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) limits their utility for drug screening and disease modelling. Here we show that suitable combinations of mechanical stimuli and metabolic cues can enhance the maturation of hiPSC-derived cardiomyocytes, and that the maturation-inducing cues have phenotype-dependent effects on the cells' action-potential morphology and calcium handling. By using microfluidic chips that enhanced the alignment and extracellular-matrix production of cardiac microtissues derived from genetically distinct sources of hiPSC-derived cardiomyocytes, we identified fatty-acid-enriched maturation media that improved the cells' mitochondrial structure and calcium handling, and observed divergent cell-source-dependent effects on action-potential duration (APD). Specifically, in the presence of maturation media, tissues with abnormally prolonged APDs exhibited shorter APDs, and tissues with aberrantly short APDs displayed prolonged APDs. Regardless of cell source, tissue maturation reduced variabilities in spontaneous beat rate and in APD, and led to converging cell phenotypes (with APDs within the 300-450 ms range characteristic of human left ventricular cardiomyocytes) that improved the modelling of the effects of pro-arrhythmic drugs on cardiac tissue.

Chemical patterning of ultrathin polymer films by direct-write multiphoton lithography.

We applied 2-photon laser ablation to write subdiffraction nanoscale chemical patterns into ultrathin polymer films under ambient conditions. Poly(ethylene glycol) methacrylate brush layers were prepared on quartz substrates via surface-initiated atom-transfer radical polymerization and ablated to expose the underlying substrate using the nonlinear 2-photon absorbance of a frequency-doubled Ti:sapphire femtosecond laser. Single-shot ablation thresholds of polymer films were ~1.5 times smaller than that of a quartz substrate, which allowed patterning of nanoscale features without damage to the underlying substrate. At a 1/e(2) laser spot diameter of 0.86 µm, the features of exposed substrate approached ~80 nm, well below the diffraction limit for 400 nm light. Ablated features were chemically distinct and amenable to chemical modification.

Characterization of integrin engagement during defined human embryonic stem cell culture.

Human embryonic stem (hES) cells are pluripotent, capable of differentiating into any cell type of the body, and therefore have the ability to provide insights into mechanisms of human development and disease, as well as to provide a potentially unlimited supply of cells for cell-based therapy and diagnostics. Knowledge of the adhesion receptors that hES cells employ to engage extracellular matrix (ECM) proteins is of basic biological interest and can enhance the design of cell culture and implantation systems to enable these biomedical applications. Although hES cells express a variety of cell surface receptors, little is known about which integrins are involved during subculture and passage. Matrigel is broadly used as a cell adhesive matrix for hES cell culture. Here, we sought to identify which integrins hES cells exploit for adhesion to Matrigel-coated surfaces in defined medium conditions. Using RT-PCR, flow cytometry, and fluorescence immunochemistry, we found that numerous integrins were expressed by H1 hES cells; however, antibody blocking assays indicated that only alpha(v)beta(3), alpha(6), beta(1), and alpha(2)beta(1) played a significant role in the initial adhesion of the hES cells to Matrigel in defined medium conditions. We subsequently identified a cohort of synthetic peptides that, when adsorbed to the culture surface, promoted H1 hES cell attachment and proliferation, as well as maintained a pluripotent phenotype. Peptides designed to engage with alpha(v)beta(3), alpha(6), beta(1), and alpha(2)beta(1) integrins and syndecan-1 were tested both individually and in various combinations. A combination of two integrin-engaging peptides (AG-10, C-16) and one syndecan-engaging peptide (AG-73) was sufficient to promote hES cell adhesion, maintenance, and proliferation. We propose that a specific integrin "fingerprint" is necessary for maintenance of hES cell self-renewal, and synthetic culture systems must capture this engagement profile for hES cells to remain undifferentiated.-Meng, Y., Eshghi, S., Li, Y. J., Schmidt, R., Schaffer, D. V., Healy, K. E. Characterization of integrin engagement during defined human embryonic stem cell culture.

Stem cell-based vascularization of microphysiological systems.

Microphysiological systems (MPSs) (i.e., tissue or organ chips) exploit microfluidics and 3D cell culture to mimic tissue and organ-level physiology. The advent of human induced pluripotent stem cell (hiPSC) technology has accelerated the use of MPSs to study human disease in a range of organ systems. However, in the reduction of system complexity, the intricacies of vasculature are an often-overlooked aspect of MPS design. The growing library of pluripotent stem cell-derived endothelial cell and perivascular cell protocols have great potential to improve the physiological relevance of vasculature within MPS, specifically for in vitro disease modeling. Three strategic categories of vascular MPS are outlined: self-assembled, interface focused, and 3D biofabricated. This review discusses key features and development of the native vasculature, linking that to how hiPSC-derived vascular cells have been generated, the state of the art in vascular MPSs, and opportunities arising from interdisciplinary thinking.

Isochoric supercooled preservation and revival of human cardiac microtissues.

Low-temperature biopreservation and 3D tissue engineering present two differing routes towards eventual on-demand access to transplantable biologics, but recent advances in both fields present critical new opportunities for crossover between them. In this work, we demonstrate sub-zero centigrade preservation and revival of autonomously beating three-dimensional human induced pluripotent stem cell (hiPSC)-derived cardiac microtissues via isochoric supercooling, without the use of chemical cryoprotectants. We show that these tissues can cease autonomous beating during preservation and resume it after warming, that the supercooling process does not affect sarcomere structural integrity, and that the tissues maintain responsiveness to drug exposure following revival. Our work suggests both that functional three dimensional (3D) engineered tissues may provide an excellent high-content, low-risk testbed to study complex tissue biopreservation in a genetically human context, and that isochoric supercooling may provide a robust method for preserving and reviving engineered tissues themselves.

Semisynthetic Hyaluronic Acid-Based Hydrogel Promotes Recovery of the Injured Tibialis Anterior Skeletal Muscle Form and Function.

Volumetric muscle loss (VML) injuries are characterized by a degree of tissue loss that exceeds the endogenous regenerative capacity of muscle, resulting in permanent structural and functional deficits. Such injuries are a consequence of trauma, as well as a host of congenital and acquired diseases and disorders. Despite significant preclinical research with diverse biomaterials, as well as early clinical studies with implantation of decellularized extracellular matrices, there are still significant barriers to more complete restoration of muscle form and function following repair of VML injuries. In fact, identification of novel biomaterials with more advantageous regenerative profiles is a critical limitation to the development of improved therapeutics. As a first step in this direction, we evaluated a novel semisynthetic hyaluronic acid-based (HyA) hydrogel that embodies material features more favorable for robust muscle regeneration. This HyA-based hydrogel is composed of an acrylate-modified HyA (AcHyA) macromer, an AcHyA macromer conjugated with the bsp-RGD(15) peptide sequence to enhance cell adhesion, a high-molecular-weight heparin to sequester growth factors, and a matrix metalloproteinase-cleavable cross-linker to allow for cell-dependent remodeling. In a well-established, clinically relevant rat tibialis anterior VML injury model, we report observations of robust functional recovery, accompanied by volume reconstitution, muscle regeneration, and native-like vascularization following implantation of the HyA-based hydrogel at the site of injury. These findings have important implications for the development and clinical application of the improved biomaterials that will be required for stable and complete functional recovery from diverse VML injuries.

High-Throughput Discovery of Targeted, Minimally Complex Peptide Surfaces for Human Pluripotent Stem Cell Culture.

Human pluripotent stem cells harbor an unlimited capacity to generate therapeutically relevant cells for applications in regenerative medicine. However, to utilize these cells in the clinic, scalable culture systems that activate defined receptors and signaling pathways to sustain stem cell self-renewal are required; and synthetic materials offer considerable promise to meet these needs. De novo development of materials that target novel pathways has been stymied by a limited understanding of critical receptor interactions maintaining pluripotency. Here, we identify peptide agonists for the human pluripotent stem cell (hPSC) laminin receptor and pluripotency regulator, α6-integrin, through unbiased, library-based panning strategies. Biophysical characterization of adhesion suggests that identified peptides bind hPSCs through α6-integrin with sub-µM dissociation constants similar to laminin. By harnessing a high-throughput microculture platform, we developed predictive guidelines for presenting these integrin-targeting peptides alongside canonical binding motifs at optimal stoichiometries to generate nascent culture surfaces. Finally, when presented as self-assembled monolayers, predicted peptide combinations supported hPSC expansion, highlighting how unbiased screens can accelerate the discovery of targeted biomaterials.

In vitro safety "clinical trial" of the cardiac liability of drug polytherapy.

Only a handful of US Food and Drug Administration (FDA) Emergency Use Authorizations exist for drug and biologic therapeutics that treat severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infection. Potential therapeutics include repurposed drugs, some with cardiac liabilities. We report on a chronic preclinical drug screening platform, a cardiac microphysiological system (MPS), to assess cardiotoxicity associated with repurposed hydroxychloroquine (HCQ) and azithromycin (AZM) polytherapy in a mock phase I safety clinical trial. The MPS contained human heart muscle derived from induced pluripotent stem cells. The effect of drug response was measured using outputs that correlate with clinical measurements, such as QT interval (action potential duration) and drug-biomarker pairing. Chronic exposure (10 days) of heart muscle to HCQ alone elicited early afterdepolarizations and increased QT interval past 5 days. AZM alone elicited an increase in QT interval from day 7 onward, and arrhythmias were observed at days 8 and 10. Monotherapy results mimicked clinical trial outcomes. Upon chronic exposure to HCQ and AZM polytherapy, we observed an increase in QT interval on days 4-8. Interestingly, a decrease in arrhythmias and instabilities was observed in polytherapy relative to monotherapy, in concordance with published clinical trials. Biomarkers, most of them measurable in patients' serum, were identified for negative effects of monotherapy or polytherapy on tissue contractile function, morphology, and antioxidant protection. The cardiac MPS correctly predicted clinical arrhythmias associated with QT prolongation and rhythm instabilities. This high content system can help clinicians design their trials, rapidly project cardiac outcomes, and define new monitoring biomarkers to accelerate access of patients to safe coronavirus disease 2019 (COVID-19) therapeutics.

Heart Muscle Microphysiological System for Cardiac Liability Prediction of Repurposed COVID-19 Therapeutics.

Despite global efforts, it took 7 months between the proclamation of global SARS-CoV-2 pandemic and the first FDA-approved treatment for COVID-19. During this timeframe, clinicians focused their efforts on repurposing drugs, such as hydroxychloroquine (HCQ) or azithromycin (AZM) to treat hospitalized COVID-19 patients. While clinical trials are time-consuming, the exponential increase in hospitalizations compelled the FDA to grant an emergency use authorization for HCQ and AZM as treatment for COVID-19, although there was limited evidence of their combined efficacy and safety. The authorization was revoked 4 months later, giving rise to controversial political and scientific debates illustrating important challenges such as premature authorization of potentially ineffective or unsafe therapeutics, while diverting resources from screening of effective drugs. Here we report on a preclinical drug screening platform, a cardiac microphysiological system (MPS), to rapidly identify clinically relevant cardiac liabilities associated with HCQ and AZM. The cardiac MPS is a microfabricated fluidic system in which cardiomyocytes derived from human induced pluripotent stem cells self-arrange into a uniaxially beating tissue. The drug response was measured using outputs that correlate with clinical measurements such as action potential duration (proxy for clinical QT interval) and drug-biomarker pairing. The cardiac MPS predicted clinical arrhythmias associated with QT prolongation and rhythm instabilities in tissues treated with HCQ. We found no change in QT interval upon acute exposure to AZM, while still observing a significant increase in arrhythmic events. These results suggest that this MPS can not only predict arrhythmias, but it can also identify arrhythmias even when QT prolongation is absent. When exposed to HCQ and AZM polytherapy, this MPS faithfully reflected clinical findings, in that the combination of drugs synergistically increased QT interval when compared to single drug exposure, while not worsening the overall frequency of arrhythmic events. The high content cardiac MPS can rapidly evaluate the cardiac safety of potential therapeutics, ultimately accelerating patients' access to safe and effective treatments.

Integrated Isogenic Human Induced Pluripotent Stem Cell-Based Liver and Heart Microphysiological Systems Predict Unsafe Drug-Drug Interaction.

Three-dimensional (3D) microphysiological systems (MPSs) mimicking human organ function in vitro are an emerging alternative to conventional monolayer cell culture and animal models for drug development. Human induced pluripotent stem cells (hiPSCs) have the potential to capture the diversity of human genetics and provide an unlimited supply of cells. Combining hiPSCs with microfluidics technology in MPSs offers new perspectives for drug development. Here, the integration of a newly developed liver MPS with a cardiac MPS-both created with the same hiPSC line-to study drug-drug interaction (DDI) is reported. As a prominent example of clinically relevant DDI, the interaction of the arrhythmogenic gastroprokinetic cisapride with the fungicide ketoconazole was investigated. As seen in patients, metabolic conversion of cisapride to non-arrhythmogenic norcisapride in the liver MPS by the cytochrome P450 enzyme CYP3A4 was inhibited by ketoconazole, leading to arrhythmia in the cardiac MPS. These results establish integration of hiPSC-based liver and cardiac MPSs to facilitate screening for DDI, and thus drug efficacy and toxicity, isogenic in the same genetic background.

Surface creasing instability of soft polyacrylamide cell culture substrates.

Efforts to understand and engineer cell behavior in mechanically soft environments frequently employ two-dimensional cell culture substrates consisting of thin hydrogel layers with low elastic modulus supported on rigid substrates to facilitate culturing, imaging, and analysis. Here we characterize how an elastic creasing instability of the gel surface may occur for the most widely used soft cell culture substrate, polyacrylamide hydrogels, and show that stem cells respond to and change their behavior due to these surface features. The regions of stability and corresponding achievable ranges of modulus are elucidated in terms of the monomer and cross-linker concentrations, providing guidance for the synthesis of both smooth and creased soft cell substrates for basic and applied cell engineering efforts.