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Microfibrous substrate geometry as a critical trigger for organization, self-renewal, and differentiation of human embryonic stem cells within synthetic 3-dimensional microenvironments.

ABSTRACT: Substrates used to culture human embryonic stem cells (hESCs) are typically 2-dimensional (2-D) in nature, with limited ability to recapitulate in vivo-like 3-dimensional (3-D) microenvironments. We examined critical determinants of hESC self-renewal in poly-d-lysine-pretreated synthetic polymer-based substrates with variable microgeometries, including planar 2-D films, macroporous 3-D sponges, and microfibrous 3-D fiber mats. Completely synthetic 2-D substrates and 3-D macroporous scaffolds failed to retain hESCs or support self-renewal or differentiation. However, synthetic microfibrous geometries made from electrospun polymer fibers were found to promote cell adhesion, viability, proliferation, self-renewal, and directed differentiation of hESCs in the absence of any exogenous matrix proteins. Mechanistic studies of hESC adhesion within microfibrous scaffolds indicated that enhanced cell confinement in such geometries increased cell-cell contacts and altered colony organization. Moreover, the microfibrous scaffolds also induced hESCs to deposit and organize extracellular matrix proteins like laminin such that the distribution of laminin was more closely associated with the cells than the Matrigel treatment, where the laminin remained associated with the coated fibers. The production of and binding to laminin was critical for formation of viable hESC colonies on synthetic fibrous scaffolds. Thus, synthetic substrates with specific 3-D microgeometries can support hESC colony formation, self-renewal, and directed differentiation to multiple lineages while obviating the stringent needs for complex, exogenous matrices. Similar scaffolds could serve as tools for developmental biology studies in 3-D and for stem cell differentiation in situ and transplantation using defined humanized conditions.

SUBMITTER: Carlson AL

PROVIDER: S-EPMC3405262 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Microfibrous substrate geometry as a critical trigger for organization, self-renewal, and differentiation of human embryonic stem cells within synthetic 3-dimensional microenvironments.

Carlson Aaron L AL Florek Charles A CA Kim Joseph J JJ Neubauer Thomas T Moore Jennifer C JC Cohen Rick I RI Kohn Joachim J Grumet Martin M Moghe Prabhas V PV

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20120427 8

Substrates used to culture human embryonic stem cells (hESCs) are typically 2-dimensional (2-D) in nature, with limited ability to recapitulate in vivo-like 3-dimensional (3-D) microenvironments. We examined critical determinants of hESC self-renewal in poly-d-lysine-pretreated synthetic polymer-based substrates with variable microgeometries, including planar 2-D films, macroporous 3-D sponges, and microfibrous 3-D fiber mats. Completely synthetic 2-D substrates and 3-D macroporous scaffolds fai ...[more]

PMID: 22542683

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Project description:IntroductionHuman induced pluripotent stem cells (iPSCs) are a promising source of endothelial cells (iPSC-ECs) for engineering three-dimensional (3D) vascularized cardiac tissues. To mimic cardiac microvasculature, in which capillaries are oriented in parallel, we hypothesized that endothelial differentiation of iPSCs within topographically aligned 3D scaffolds would be a facile one-step approach to generate iPSC-ECs as well as induce aligned vascular organization.MethodsHuman iPSCs underwent endothelial differentiation within electrospun 3D polycaprolactone (PCL) scaffolds having either randomly oriented or parallel-aligned microfibers. Using transcriptional, protein, and endothelial functional assays, endothelial differentiation was compared between conventional two-dimensional (2D) films and 3D scaffolds having either randomly oriented or aligned microfibers. Furthermore, the role of parallel-aligned microfiber patterning on the organization of vessel-like networks was assessed.ResultsThe cells in both the randomly oriented and aligned 3D scaffolds demonstrated an 11-fold upregulation in gene expression of the endothelial phenotypic marker, CD31, compared to cells on 2D films. This upregulation corresponded to >3-fold increase in CD31 protein expression in 3D scaffolds, compared to 2D films. Concomitantly, other endothelial phenotypic markers including CD144 and endothelial nitric oxide synthase also showed significant transcriptional upregulation in 3D scaffolds by >7-fold, compared to 2D films. Nitric oxide production, which is characteristic of endothelial function, was produced 4-fold more abundantly in 3D scaffolds, compared to on 2D PCL films. Within aligned scaffolds, the iPSC-ECs displayed parallel-aligned vascular-like networks with 70% longer branch length, compared to cells in randomly oriented scaffolds, suggesting that fiber topography modulates vascular network-like formation and patterning.ConclusionTogether, these results demonstrate that 3D scaffold structure promotes endothelial differentiation, compared to 2D substrates, and that aligned topographical patterning induces anisotropic vascular network organization.

Project description:Successful realization of the enormous potential of pluripotent stem cells in regenerative medicine demands the development of well-defined culture conditions. Maintenance of embryonic stem cells (ESCs) typically requires co-culture with feeder layer cells, generally mouse embryonic fibroblasts (MEFs). Concerns about xenogeneic pathogen contamination and immune reaction to feeder cells underlie the need for ensuring the safety and efficacy of future stem cell-based products through the development of a controlled culture environment. To gain insight into the effectiveness of MEF layers, here we have developed a biomimetic synthetic feeder layer (BSFL) that is acellular and replicates the stiffness and topography of MEFs. The mechanical properties of MEFs were measured using atomic force microscopy. The average Young's modulus of the MEF monolayers was replicated using tunable polyacrylamide (PA) gels. BSFLs replicated topographical features of the MEFs, including cellular, subcellular, and cytoskeletal features. On BSFLs, mouse ESCs adhered and formed compact round colonies; similar to on MEF controls but not on Flat PA. ESCs on BSFLs maintained their pluripotency and self-renewal across passages, formed embryoid bodies and differentiated into progenitors of the three germ layers. This acellular biomimetic synthetic feeder layer supports stem cell culture without requiring co-culture of live xenogeneic feeder cells, and provides a versatile, tailorable platform for investigating stem cell growth.Embryonic stem cells have enormous potential to aid therapeutics, because they can renew themselves and become different cell types. This study addresses a key challenge for ESC use - growing them safely for human patients. ESCs typically grow with a feeder layer of mouse fibroblasts. Since patients have a risk of immune response to feeder layer cells, we have developed a material to mimic the feeder layer and eliminate this risk. We investigated the influence of feeder layer topography and stiffness on mouse ESCs. While the biomimetic synthetic feeder layer contains no live cells, it replicates the stiffness and topography of feeder layer cells. Significantly, ESCs grown on BSFLs retain their abilities to grow and become multiple cell types.

Project description:Background: Prolongation of the QT interval of the electrocardiogram (ECG), underlain by prolongation of the action potential duration (APD) at the cellular level, is linked to increased vulnerability to cardiac arrhythmia. Pharmacological management of arrhythmia associated with QT prolongation is typically achieved through attempting to restore APD to control ranges, reversing the enhanced vulnerability to Ca2+-dependent afterdepolarisations (arrhythmia triggers) and increased transmural dispersion of repolarisation (arrhythmia substrate) associated with APD prolongation. However, such pharmacological modulation has been demonstrated to have limited effectiveness. Understanding the integrative functional impact of pharmacological modulation requires simultaneous investigation of both the trigger and substrate. Methods: We implemented a multi-scale (cell and tissue) in silico approach using a model of the human ventricular action potential, integrated with a model of stochastic 3D spatiotemporal Ca2+ dynamics, and parameter modification to mimic prolonged QT conditions. We used these models to examine the efficacy of the hERG activator MC-II-157c in restoring APD to control ranges, examined its effects on arrhythmia triggers and substrates, and the interaction of these arrhythmia triggers and substrates. Results: QT prolongation conditions promoted the development of spontaneous release events underlying afterdepolarisations during rapid pacing. MC-II-157c applied to prolonged QT conditions shortened the APD, inhibited the development of afterdepolarisations and reduced the probability of afterdepolarisations manifesting as triggered activity in single cells. In tissue, QT prolongation resulted in an increased transmural dispersion of repolarisation, which manifested as an increased vulnerable window for uni-directional conduction block. In some cases, MC-II-157c further increased the vulnerable window through its effects on INa. The combination of stochastic release event modulation and transmural dispersion of repolarisation modulation by MC-II-157c resulted in an integrative behavior wherein the arrhythmia trigger is reduced but the arrhythmia substrate is increased, leading to variable and non-linear overall vulnerability to arrhythmia. Conclusion: The relative balance of reduced trigger and increased substrate underlies a multi-dimensional role of MC-II-157c in modulation of cardiac arrhythmia vulnerability associated with prolonged QT interval.

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Microfibrous substrate geometry as a critical trigger for organization, self-renewal, and differentiation of human embryonic stem cells within synthetic 3-dimensional microenvironments.

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Microfibrous substrate geometry as a critical trigger for organization, self-renewal, and differentiation of human embryonic stem cells within synthetic 3-dimensional microenvironments.

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