Biology subjects

Zoldan, J.

Publications and source records attributed to Zoldan, J..

17 recordsLinked to original sources

bioRxiv2025

Cell Barcoding Reveals Lineage-dependent Outcomes in hiPSC Cardiac Differentiation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have potential applications in treating cardiovascular disease but are currently limited in their clinical translation. A primary limitation is the poor clinical scalability of hiPSC-CMs, with the heterogeneity of hiPSC cardiac differentiation significantly contributing to this limitation. We hypothesize that clinical scalability can be improved by tracking and controlling hiPSC clonal heterogeneity, a variable often overlooked in current differentiation approaches. "Fate priming", wherein clonal lineage identity determines differentiation fate, has been demonstrated in other stem cell differentiation pathways. We investigated fate priming in hiPSC cardiac differentiation using the ClonMapper cell barcoding platform to label, track, and isolate distinct hiPSC lineages from the same cell line. We show that certain hiPSC lineages preferentially differentiate into hiPSC-CMs or non-CMs. After isolating lineages with apparent fate priming, we found significant differences in cardiac differentiation outcomes between these single-clone populations and heterogeneous, multi-clone hiPSC populations. These findings indicate that lineage identity influences hiPSC cardiac differentiation outcomes. SIGNIFICANCE STATEMENTCardiovascular disease is a significant global health concern that can be addressed by engineering artificial tissues to develop new treatments for heart disease or to directly replace damaged heart tissue. Stem cells are a useful tool for engineering these tissues because of their ability to become cardiomyocytes. However, their clinical translation is limited by variability in the process of differentiating stem cells into cardiomyocytes. This article reports findings that show different lineages of genetically identical human induced pluripotent stem cells have different capacities for differentiating into cardiomyocytes, which may contribute to the variability observed.

bioengineering↗

bioRxiv2025

Dynamic Stiffening to Improve Vasculogenesis of hiPSC-Derived Endothelial Progenitors

Multiple groups have reported on the impact of hydrogel stiffness on vascular network formation in vitro, with overall findings indicating that less stiff hydrogels better support vasculogenesis. However, the majority of this research utilized hydrogels with static stiffness, even though vasculogenesis occurs in tandem with changes in extracellular matrix material properties. To that end, we hypothesize that dynamic modulation of hydrogel stiffness during the process of vasculogenesis, recapitulating changes observed during embryonic development, would improve vascular network formation. Using our Collagen I/Norbornene-modified hyaluronic acid hydrogel system, we swelled in additional crosslinker and photoinitiator and exposed the hydrogel to UV light, enabling hydrogel stiffening at user-defined time points with no significant effect on cell viability. We observed that in situ stiffening at early time points, prior to the onset of significant cell migration, resulted in more robust vascular network formation relative to unstiffened controls, while stiffening at later time points disrupted existing vascular networks. These trends continued in in vivo experiments in nude mice, with cell-laden hydrogels stiffened at early time points resulting in improved blood flow, while those stiffened at later time points had the opposite effect. We hypothesized that this was due to differential impacts of focal adhesion kinase (FAK) activation following in situ stiffening, as supplementation with a Rho kinase inhibitor, downstream of FAK, partially reversed the effects of in situ stiffening. Taken together, this research demonstrates the benefits of incorporating dynamic cues into hydrogel design to create more physiologically relevant vasculature.

bioengineering↗

bioRxiv2025

Tuning Scaffold Degradation with Non-Natural Peptidomimetics to Control Human Umbilical Vein Endothelial Cell Morphology and Vessel Formation

The in vitro vascularization of 3D tissue constructs, such as hydrogels, remains a paramount challenge in tissue engineering. Extracellular matrix degradation and remodeling are key parts of the vascularization process; however, it is difficult to isolate the effects of degradability in both natural and synthetic matrix models. Naturally-derived matrices typically couple degradability to other material properties, whereas synthetic matrices rely on short peptide sequences to impart degradability, which typically exhibit substrate overlap to many proteases. Here, we present a method to independently and broadly tune 3D hydrogel degradation using crosslinkers with non-natural peptoid (N-substituted glycine) substitutions. Increased peptoid substitutions reduced hydrogel degradability to collagenases without altering hydrogel modulus, swelling ratio, or crosslinker length. Using this approach, human umbilical vein endothelial cells (HUVECs) encapsulated in more degradable hydrogels proliferated more, formed more vessels, exhibited higher metabolic activity, and secreted more extracellular matrix than HUVECs encapsulated in less degradable or non-degradable hydrogels. Interestingly, HUVECs encapsulated in the least degradable hydrogels secreted significantly higher matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) than HUVECs encapsulated in the most degradable hydrogels, suggesting higher MMP secretion to compensate for the reduced matrix degradability. Overall, this work highlights the importance of protease-mediated remodeling on vascularization and suggests that peptoid substitutions are effective for tuning hydrogel degradability for a variety of 3D cell applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/684851v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@c22b3corg.highwire.dtl.DTLVardef@1a4ae14org.highwire.dtl.DTLVardef@a0ad1forg.highwire.dtl.DTLVardef@c5def5_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Donor Age Impairs Vasculogenic Potential of hiPSC-Derived Endothelial Progenitors

BackgroundHuman induced pluripotent stem cells (hiPSCs) hold promise for vascular regeneration, but preliminary research often relies on neonatal donors, whereas clinical applications will use cells derived from older adults. Although the impact of donor age on reprogramming efficiency has been studied, its effect on the functionality of hiPSC-derived endothelial progenitors (hiPSC-EPs) remains unclear. This question is the focus of the current study. Methods and ResultsWe derived EPs from iPSCs sourced from three neonatal donors (ND) and three mature donors (MD) matched 1:1 for sex and somatic cell origin. We assessed their functional, epigenetic, and transcriptomic characteristics. Despite higher CD34 yields from MD-iPSCs, MD-hiPSC-EPs formed poorly interconnected and non-lumenized vascular structures in 3D hydrogels, compared to neonatal donor (ND) lines. In 2D culture, MD-hiPSC-EPs exhibited reduced cell density and aberrant VE-Cadherin localization. DNA methylation analysis revealed that somatic cell origin was the dominant driver of variance, but consistent differences in methylation of mesoderm commitment, angiogenesis, ECM remodeling, and cytoskeleton-related genes were observed between age groups. Epigenetic age prediction showed MD-hiPSC-EPs had more developmentally advanced signatures, potentially explaining their shift away from vasculogenic competence. Our RNA-sequencing findings confirm trends seen in the DNA methylation data and show differential expression of pathways linked to mitochondrial regulation and nitric oxide signaling. ConclusionsDonor age significantly alters the vasculogenic function of hiPSC-EPs. These findings underscore the necessity of donor-specific considerations in hiPSC-based vascular engineering and highlight potential barriers to translating hiPSC-derived therapeutics into older patient populations.

bioengineering↗

bioRxiv2025

Multicellularity, Culture Duration, and Hydrogel Stiffness Guide Induced Pluripotent Stem Cell-Derived Endothelial Progenitor Cell Contractility

Human induced pluripotent stem cells (hiPSCs) offer immense potential for tissue engineering, yet poor vascularization remains a significant hurdle. Understanding how hiPSC-derived endothelial progenitor cells (hiPSC-EPs) form networks is essential for therapeutic progress. This study investigates extracellular matrix (ECM) remodeling and cellular contractility during the early self-assembly of hiPSC-EPs within 3D hyaluronic acid-based hydrogels. By tracking microsphere displacements before and after cytochalasin-D treatment, we quantified contractile forces in single cells and clusters at days 4 and 7. We then applied a novel inverse modeling approach using a compressible material model to determine spatially varying changes in hydrogel modulus caused by enzymatic degradation and ECM deposition. Our findings reveal that basal contractility and remodeling are nonlinearly influenced by multicellularity, culture duration, and initial stiffness. Increased hydrogel stiffness, paired with synergistic rises in strain energy, resulted in high traction forces in longer culture times. These results provide critical mechanical insights into hiPSC-EP self-assembly, advancing our ability to engineer functional vascular networks.

bioengineering↗

bioRxiv2025

Modulating Hydrogel Stiffness Through Light-Based 3D Printing to Mimic Cardiac Fibrosis and Cardiomyocyte Dysfunction Using hiPSC-Derived Cells

The human hearts limited regenerative capacity is a significant barrier to addressing cardiovascular disease (CVD). This is particularly true for cardiac fibrosis, a form of CVD wherein the wound healing process has gone awry. In cardiac fibrosis, excessive scar tissue formation due to dysregulated remodeling of the hearts extracellular matrix (ECM) results in increased stiffness that reduces cardiac output and can lead to heart failure. This dysregulated ECM deposition is driven by activated cardiac fibroblasts, where cell substrate stiffness is known to play a role in cardiac fibroblast activation. New preclinical models that accurately recapitulate the behavior of activated cardiac fibroblasts are needed to better understand and treat cardiac fibrosis. To this end, we describe a model wherein human induced pluripotent stem cell (hiPSC)-derived cardiac fibroblasts (HCFs) are cultured on 3D printed hydrogels of tunable stiffness, fabricated using dosage controlled digital light processing (DLP). We demonstrate that our model can induce HCF activation in the absence of TGF{beta}, a key mediator of fibroblast activation, surpassing the activation levels seen with HCFs activated with TGF{beta} on protein-coated tissue culture plates. Furthermore, combining stiffer hydrogels with TGF{beta} recapitulates fibroblast activation similar to what is observed in native cardiac tissue. Lastly, by indirectly coculturing HCFs seeded and activated on these stiff hydrogels with hiPSC-derived cardiomyocytes, we demonstrate that the activated HCFs in our cardiac fibrosis model can impair cardiomyocyte function, mimicking the deleterious effects of cardiac fibrosis.

bioengineering↗

bioRxiv2025

Optimizing cardiac organoid culture to enhance maturation, viability, and cardiotoxicity assessments

Development of relevant, human induced pluripotent stem cell-derived cardiac organoids is essential to recapitulate myocardium physiology and functionality for assessment of drug-induced toxicity evaluations. However, the optimal conditions for culturing self-aggregating multicellular cardiac organoids are not well-elucidated, particularly the impact of noncardiomyocytes. In this study, we generated cardiac organoids at varying seeding densities to formulate organoids that meet or exceed the biological diffusion limit. We assessed their morphology, gene expression profiles, beating functionality, viability, and mitochondrial activity over time. Our results show that organoid sizes stabilize by seven days of culture, regardless of seeding density. However, organoids seeded with 20,000 cells retained an optimal cardiac signature that promotes cardiac maturity and minimizes fibrotic tendencies, especially when culturing longer than seven days. While all organoid populations maintained their beating functionalities, those seeded with 80,000 cells exhibited greater cell shedding and increased apoptosis at long term culture. In contrast, minimal apoptosis was observed in organoids seeded with 20,000 cells after seven days. Mitochondrial staining further revealed that organoids seeded with 20,000 cells consistently demonstrated higher metabolic activity. Taken together, organoids seeded with 20,000 cells and cultured for seven days yielded the healthiest morphology, transcriptional signature, and viability, while maintaining robust beating kinetics. Importantly, compared to 2D cultures, these optimized organoids demonstrate improved sensitivity to clinically relevant doxorubicin-induced cardiotoxicity, enabling more accurate dose-response evaluations that better reflect therapeutic conditions. Impact StatementThis work highlights key tissue engineering considerations for generating self-assembling cardiac organoids suitable for scalable, high-throughput drug screening and discovery. Understanding the effect of seeding density and culture duration on organoid size and, consequently on gene expression, beating functionalities, apoptosis, and metabolic activity, has broader implications for establishing optimal organoid culture conditions. These insights enable the production of large quantities of cardiac organoids capable of modeling drug-induced toxicity effects on a clinically relevant timescale.

bioengineering↗

bioRxiv2025

Matrix Stiffness Regulates Mechanotransduction and Vascular Network Formation of hiPSC-Derived Endothelial Progenitors Encapsulated in 3D Hydrogels

The mechanical properties of the extracellular matrix (ECM), particularly stiffness, regulate endothelial progenitor responses during vascular development, yet their behavior in physiologically compliant matrices (<1 kPa) remains underexplored. Using norbornene-modified hyaluronic acid (NorHA) hydrogels with tunable stiffness (190-884 Pa), we investigated how hydrogel stiffness influences cell morphology, endothelial maturation, mechanotransduction, and microvascular network formation in human induced pluripotent stem cell-derived endothelial progenitors (hiPSC-EPs). Our findings reveal a stiffness-dependent tradeoff between mechanotransduction and vascular network formation. At intermediate stiffness (551 Pa), cells exhibited the greatest increase in endothelial marker CD31 expression and Yes-associated protein (YAP)/ transcriptional coactivator with PDZ-binding motif (TAZ) nuclear translocation, indicating enhanced mechanotransduction and endothelial maturation. However, this did not translate to superior plexus formation. Instead, the most compliant matrix (190 Pa) supported greater vascular connectivity, characterized by longer branches ([~]0.03/volume vs. 0.015 at 551 Pa) and enhanced actin remodeling. 3D cell contraction measurements revealed a 15.6-fold higher basal displacement in compliant hydrogels, suggesting that cell-generated forces and matrix deformability collectively drive vascular morphogenesis. Unlike prior studies focusing on pathological stiffness ranges (>10 kPa), our results emphasize that vascularization is not solely driven by the most mechanotransductive environment but rather by a balance of compliance, contractility, and cell-induced remodeling. These findings underscore the need to design hydrogels that provide sufficient mechanotransduction for endothelial maturation while maintaining compliance to support dynamic vascular morphogenesis. This work provides a mechanically tuned framework for optimizing microenvironments to balance endothelial differentiation and vascular network formation in tissue engineering and regenerative medicine.

bioengineering↗

bioRxiv2024

Differentiation Protocol-Dependent Variability in hiPSC-Derived Endothelial Progenitor Functionality

PurposeWith multiple groups generating methods for differentiating induced pluripotent stem cells (hiPSCs) into endothelial cells/endothelial progenitors (ECs/EPs), there is a need to better understand the specific endothelial subtypes that different differentiation protocols produce. This is especially important for accurate tissue modeling as researchers continue to incorporate the endothelium into engineered tissues. MethodsWe illustrated the heterogeneity of cells produced from different differentiation protocols by focusing on two selected protocols, one driving differentiation exclusively using small molecules (SM Protocol) and one driving differentiation primarily using growth factors (GF Protocol). We characterized the cells through a combination of vasculogenic computational analysis following encapsulation in 3D hydrogels, RNA sequencing, and measurement of soluble factor release. ResultsVasculogenic computational analysis indicates that cells from the GF protocol formed more dense and interconnected vasculature compared to cells from the SM protocol. Likewise, RNA-seq analysis showed GF-derived cell enrichment in pathways involved in cell migration and angiogenesis. In addition, GF-derived cells favor differentiation to arterial endothelial cells as well as predisposition to undergoing a partial endothelial-to-mesenchymal transition, whereas SM-derived cells resemble immature progenitors based on higher proliferation rates as well as ECM remodeling. These trends also persisted following extended 3D culture. ConclusionsThe results demonstrate that despite using the same starting hiPSC population and isolating EPs using the same surface marker, the two differentiation protocols yield highly distinct cell populations. This work highlights the importance of understanding the specific endothelial subtypes produced by different differentiation protocols for the creation of more accurate tissue models. Lay SummaryAlthough multiple groups have created protocols for differentiating human induced pluripotent stem cells (hiPSCs) to endothelial progenitors (EPs), it is not clear if different differentiation protocols generate different endothelial subtypes, matching the variance seen in vivo To that end, we performed an in-depth characterization of two selected hiPSC-EP differentiation protocols, primarily focusing on cells cultured in 3D hydrogels, to better understand the specific endothelial subtypes produced. We found significant differences in both cell maturity and cell phenotype that persisted even after extended culture. Future WorkFuture work will involve extending the existing study to include additional hiPSC-EP differentiation protocols as well as conducting single-cell RNAseq to better understand the makeup of different cell populations both before and after isolating CD34+ cells. Because smooth muscle cells play an important role in vessel stabilization, we will also incorporate hiPSC-derived smooth muscle cells into the system.

bioengineering↗

bioRxiv2024

Dynamic Electrical Stimulation Promotes hiPSC-CM Differentiation and Functionality

Human induced pluripotent stem cell differentiated cardiomyocytes (hiPSC-CMs) hold great potential to resolve cardiovascular disease but are stymied by their functional immaturity. The complex electric potentials measured during cardiogenesis point to the potential of exogenous electrical stimulation in improving cardiac differentiation and functionality. Herein, we create, validate, and implement a low-cost electrical stimulation device to stimulate hiPSCs during cardiac differentiation. Notably, our open-source device enables the generation of complex electrical stimulation regimes that may vary in frequency and pulse duration over time. Our results demonstrate that dynamic stimulation during differentiation improves cardiac differentiation efficiency, calcium handling, and flow velocity, and promotes significant transcriptomic pathway enrichment compared to static stimulation or no stimulation controls. Moreover, dynamic stimulation enhances electrochemical coupling and promotes the expression of cardiogenic pathways, potentially via sarcomere development. We anticipate that more complex dynamic electrical stimulation regimens may be generated to further optimize hiPSC-CM functionality and maturity.

bioengineering↗

bioRxiv2022

Endometrial decidualization status modulates endometrial perivascular complexity and trophoblast outgrowth in gelatin hydrogels

The endometrium undergoes rapid cycles of vascular growth, remodeling, and breakdown during the menstrual cycle and pregnancy. Decidualization is an endometrial differentiation process driven by steroidal sex hormones that is critical for blastocyst-uterine interfacing and blastocyst implantation. Certain pregnancy disorders may be linked to decidualization processes. However, much remains unknown regarding the role of decidualization and reciprocal trophoblast-endometrial interactions on endometrial angiogenesis and trophoblast invasion. Here, we report an artificial endometrial perivascular niche embedded in gelatin methacrylol hydrogels that displays morphological and functional patterns of decidualization. We show vessel complexity and soluble factor secretion are sensitive to decidualization and affect trophoblast motility. Importantly, we demonstrate the engineered perivascular niche can be combined with epithelial cultures to form a stratified endometrial model. This artificial perivascular niche provides a well-characterized platform to investigate dynamic changes in angiogenesis in response to pathological and physiological endometrial states. TeaserWe describe an endometrial vessel model to understand endometrial vasculature in the menstrual cycle and pregnancy.

bioengineering↗

bioRxiv2022

SARS-CoV-2 spike protein induces endothelial dysfunction in 3D engineered vascular networks

With new daily discoveries about the long-term impacts of COVID-19 there is a clear need to develop in vitro models that can be used to better understand the pathogenicity and impact of COVID-19. Here we demonstrate the utility of developing a model of endothelial dysfunction that utilizes induced pluripotent stem cell-derived endothelial progenitors encapsulated in collagen hydrogels to study the effects of COVID-19 on the endothelium. We found that treating these cell-laden hydrogels with SARS-CoV-2 spike protein resulted in a significant decrease in the number of vessel-forming cells as well as vessel network connectivity. Following treatment with the anti-inflammatory drug dexamethasone, we were able to prevent SARS-CoV-2 spike protein-induced endothelial dysfunction. In addition, we confirmed release of inflammatory cytokines associated with the COVID-19 cytokine storm. In conclusion, we have demonstrated that even in the absence of immune cells, we are able to use this 3D in vitro model for angiogenesis to reproduce COVID-19 induced endothelial dysfunction seen in clinical settings.

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bioRxiv2022

Engineering Alignment has Mixed Effects on hiPSC-CM Maturation

The potential of human induced pluripotent stem cell differentiated cardiomyocytes (hiPSC-CMs) is greatly limited by their functional immaturity. Strong relationships exist between CM structure and function, leading many in the field to seek ways to mature hiPSC-CMs by culturing on biomimetic substrates, specifically those that promote alignment. However, these in vitro models have so far failed to replicate the alignment that occurs during cardiac differentiation. We show that engineered alignment, incorporated before and during cardiac differentiation, affects hiPSC-CM electrochemical coupling and mitochondrial morphology. We successfully engineer alignment in differentiating hiPSCs as early as Day 4. We uniquely apply optical redox imaging to monitor the metabolic changes occurring during cardiac differentiation. We couple this modality with cardiac-specific markers, which allows us to assess cardiac metabolism in heterogeneous cell populations. The engineered alignment drives hiPSC-CM differentiation towards the ventricular compact CM subtype and improves electrochemical coupling in the short term. Moreover, we observe glycolysis to oxidative phosphorylation switch throughout differentiation and CM development. On the subcellular scale, we note changes in mitochondrial morphology in the long term. Our results demonstrate that cellular alignment accelerates hiPSC-CM maturity and emphasizes the interrelation of structure and function in cardiac development. We anticipate that combining engineered alignment with additional maturation strategies will result in improved development of mature CMs from hiPSC and strongly improve cardiac tissue engineering. Impact StatementThis work demonstrates the mixed effect of engineered structure in inducing matured function of human induced pluripotent stem cell -derived cardiomyocytes. Isolating the impacts of hiPSC-CM alignment on functionality is a necessary step in optimizing the culture conditions to develop cardiac cell therapies. Furthermore, our work has broader implications concerning how we understand the impact of mechanical microenvironments on stem cell differentiation and development.

bioengineering↗

bioRxiv2022

Biomechanical dependence of SARS-CoV-2 infections

Older people have been disproportionately vulnerable to the current SARS-CoV-2 pandemic, with an increased risk of severe complications and death compared to other age groups. A mix of underlying factors has been speculated to give rise to this differential infection outcome, including changes in lung physiology, weakened immunity, and severe immune response. Our study focuses on the impact of biomechanical changes in lungs that occur as individuals age, i.e., the stiffening of the lung parenchyma and increased matrix fiber density. We used hydrogels with an elastic modulus of 0.2 and 50 kPa and conventional tissue culture surfaces to investigate how infection rate changes with parenchymal tissue stiffness in lung epithelial cells challenged with SARS-CoV-2 Spike (S) protein pseudotyped lentiviruses. Further, we employed electrospun fiber matrices to isolate the effect of matrix density. Given the recent data highlighting the importance of alternative virulent strains, we included both the native strain identified in early 2020 and an early S protein variant (D614G) that was shown to increase the viral infectivity markedly. Our results show that cells on softer and sparser scaffolds, closer resembling younger lungs, exhibit higher infection rates by the WT and D614G variant. This suggests that natural changes in lung biomechanics do not increase the propensity for SARS-CoV-2 infection and that other factors, such as a weaker immune system, may contribute to increased disease burden in the elderly.

bioengineering↗

bioRxiv2021

Towards prevention of re-entrant arrhythmias: Injectable hydrogel electrodes enable direct capture of previously inaccessible cardiac tissues

Re-entrant arrhythmias--the leading cause of sudden cardiac death--are caused by diseased myocardial tissue and consequent delayed myocardial conduction. Access to the coronary veins that cross the "culprit" scar regions where re-entry originates can provide improved pacing to these delayed regions, offering a novel opportunity to prevent ventricular arrhythmias and circumvent the need for painful defibrillation, risky cardiac ablation, or toxic and often ineffective antiarrhythmic medications. However, there are no pacing electrodes which are small or focal enough to navigate these tributaries. To address this need, we have developed an injectable conductive hydrogel that can fill the epicardial coronary veins and their mid-myocardial tributaries. When connected to a standard pacing lead, these injected hydrogels can be converted into flexible electrodes that directly pace the previously inaccessible mid-myocardial tissue. In our two-component system, hydrogel precursor solutions can be injected through a dual lumen catheter in a minimally invasive deployment strategy to provide direct access to the diseased regions with precision and ease. Mixing of the two solutions upon injection into the vein activates redox-initiated crosslinking of the gel for rapid in situ cure without an external stimulus. An ex vivo porcine model was used to identify the requisite viscosity and cure rate for gel retention and homogeneity. Ionic species added to the hydrogel precursor solutions conferred conductivity above target myocardium values that was retained after implantation. Successful in vivo deployment demonstrated that the hydrogel electrode filled the anterior interventricular vein with extension into the septal (mid-myocardial) venous tributaries to depths far more distal and refined than any current technologies allow. In addition to successful capture and pacing of the heart, analysis of surface ECG tracings revealed a novel pacing observation highly specific for and suggestive of capture of extensive swaths of septal myocardial tissue. This is the first report of an injectable electrode used to successfully pace the mid-myocardium and mimic physiologic conduction. Furthermore, in vivo cardiac electroanatomical mapping studies in an ablation scar model showed uniform capture along the hydrogel in the vessels as well as increased capture area compared to point pacing. Collectively, these findings demonstrate that this injectable hydrogel electrode can be deployed to scarred regions of the heart to provide a reliable pacing modality that most closely resembles native conduction with the potential to eliminate delayed myocardial conduction and associated re-entry. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/467102v2_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1577526org.highwire.dtl.DTLVardef@cc510dorg.highwire.dtl.DTLVardef@1dd426eorg.highwire.dtl.DTLVardef@1346aab_HPS_FORMAT_FIGEXP M_FIG C_FIG One Sentence SummaryInjectable hydrogel electrodes achieve pacing that mimics physiologic conduction by capturing midmyocardial tissue

bioengineering↗

bioRxiv2020

Quantification of iPSC-derived vascular networks in novel phototunable angiogenic hydrogels

Vascularization of engineered scaffolds remains a critical obstacle hindering the translation of tissue engineering from the bench to the clinic. Previously, we demonstrated the robust micro-vascularization of collagen hydrogels with induced pluripotent stem cell (iPSC)-derived endothelial progenitors; however, physically cross-linked collagen hydrogels compact rapidly and exhibit limited strength. To address these challenges, we synthesized an interpenetrating polymer network (IPN) hydrogel comprised of collagen and norbornene-modified hyaluronic acid (NorHA). This dual-network hydrogel combines the natural cues presented by collagens binding sites and extracellular matrix (ECM)-mimicking fibrous architecture with the in situ modularity and chemical cross-linking of NorHA. We modulated the stiffness and degradability of this novel IPN hydrogel by varying the concentration and sequence, respectively, of the NorHA peptide cross-linker. Rheological characterization of the photo-mediated gelation process revealed that the stiffness of the IPN hydrogel increased with cross-linker concentration and was decoupled from the bulk NorHA content. Conversely, the swelling of the IPN hydrogel decreased linearly with increasing cross-linker concentration. Collagen microarchitecture remained relatively unchanged across cross-linking conditions, although the mere addition of NorHA delayed collagen fibrillogenesis. Upon iPSC-derived endothelial progenitor encapsulation, robust, lumenized microvascular networks developed in IPN hydrogels over two weeks. Subsequent computational analysis showed that an initial rise in stiffness increased the number of branch points and vessels, but vascular growth was suppressed in high stiffness IPN hydrogels. These results suggest that an IPN hydrogel consisting of collagen and NorHA is highly tunable, compaction resistant, and capable of stimulating angiogenesis. STATEMENT OF SIGNIFICANCEWe have synthesized the first tunable collagen and norbornene functionalized hyaluronic acid (NorHA) interpenetrating polymer network hydrogel. This unique biomaterial allows for control over hydrogel stiffness, independent of the total polymer concentration, by varying the concentration of a peptide cross-linker and was specifically designed to produce a biomimetic vasculogenic microenvironment. Using the system, we performed a detailed study of the vasculogenesis of induced pluripotent stem cell-derived (iPSC) endothelial progenitors, a poorly studied cell source with considerable therapeutic potential. Our results show that vascular growth can be tuned by altering the stiffness and degradability of the scaffolds independently. Finally, we improved upon our open-source computational pipeline programmed in ImageJ and MATLAB to further quantify vascular topologies in three dimensions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/259630v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1d3ee04org.highwire.dtl.DTLVardef@af739aorg.highwire.dtl.DTLVardef@cec470org.highwire.dtl.DTLVardef@c2ae18_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Perivascular Secretome Influences Hematopoietic Stem Cell Maintenance in a Gelatin Hydrogel

Adult hematopoietic stem cells (HSCs) produce the bodys full complement of blood and immune cells. They reside in specialized microenvironments, or niches, within the bone marrow. The perivascular niche near blood vessels is believed to help maintain primitive HSCs in an undifferentiated state but demonstration of this effect is difficult. In vivo studies make it challenging to determine the direct effect of the endosteal and perivascular niches as they can be in close proximity, and two-dimensional in vitro cultures often lack an instructive extracellular matrix environment. We describe a tissue engineering approach to develop and characterize a three-dimensional perivascular tissue model to investigate the influence of the perivascular secretome on HSC behavior. We generate 3D endothelial networks in methacrylamide-functionalized gelatin hydrogels using human umbilical vein endothelial cells (HUVECs) and mesenchymal stromal cells (MSCs). We identify a subset of secreted factors important for HSC function, and examine the response of primary murine HSCs in hydrogels to the perivascular secretome. Within 4 days of culture, perivascular conditioned media promoted maintenance of a greater fraction of hematopoietic stem and progenitor cells. This work represents an important first-generation perivascular model to investigate the role of niche secreted factors on the maintenance of primary HSCs.

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