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The expansion of pluripotent stem cells (PSCs) in vitro remains a critical barrier to their use in tissue engineering and regenerative medicine. Biochemical methods for PSC expansion are known to produce heterogeneous cell populations with varying states of pluripotency and are cost-intensive, hindering their clinical translation. Engineering biomaterials to physically control PSC fate offers an alternative approach. Surface or substrate topography is a promising design parameter for engineering biomaterials. Topographical cues have been shown to elicit profound effects on stem cell differentiation and proliferation. Previous reports have shown isotropic substrate topographies to be promising in expanding PSCs. However, the optimal feature to promote PSC proliferation and the pluripotent state has not yet been determined. In this work, the MultiARChitecture (MARC) plate is developed to conduct a high-throughput analysis of topographical cues in a 96-well plate format. The MARC plate is a reproducible and customizable platform for the analysis of multiple topographical patterns and features and is compatible with both microscopic assays and molecular biology techniques. The MARC plate is used to evaluate the expression of pluripotency markers Oct4, Nanog , and Sox2 and the differentiation marker LmnA as well as the proliferation of murine embryonic stem (mES) cells. Our systematic analyses identified three topographical patterns that maintain pluripotency in mES cells after multiple passages: 1 µ m pillars (1 µ m spacing, square arrangement), 2 µ m wells (c-c ( x, y ) = 4, 4 µ m), and 5 µ m pillars (c-c ( x, y ) = 7.5, 7.5 µ m). This study represents a step towards developing a biomaterial platform for controlled murine PSC expansion., (Creative Commons Attribution license.)

Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) present an attractive alternative to primary EC sources for vascular grafting. However, there is a need to mature them towards either an arterial or venous subtype. A vital environmental factor involved in the arteriovenous specification of ECs during early embryonic development is fluid shear stress; therefore, there have been attempts to employ adult arterial shear stress conditions to mature hPSC-ECs. However, hPSC-ECs are naïve to fluid shear stress, and their shear responses are still not well understood. Here, we used a multiplex microfluidic platform to systematically investigate the dose-time shear responses on hPSC-EC morphology and arterial-venous phenotypes over a range of magnitudes coincidental with physiological levels of embryonic and adult vasculatures. The device comprised of six parallel cell culture chambers that were individually linked to flow-setting resistance channels, allowing us to simultaneously apply shear stress ranging from 0.4 to 15 dyne/cm 2 . We found that hPSC-ECs required up to 40 hr of shear exposure to elicit a stable phenotypic change. Cell alignment was visible at shear stress <1 dyne/cm 2 , which was independent of shear stress magnitude and duration of exposure. We discovered that the arterial markers NOTCH1 and EphrinB2 exhibited a dose-dependent increase in a similar manner beyond a threshold level of 3.8 dyne/cm 2 , whereas the venous markers COUP-TFII and EphB4 expression remained relatively constant across different magnitudes. These findings indicated that hPSC-ECs were sensitive to relatively low magnitudes of shear stress, and a critical level of ~4 dyne/cm 2 was sufficient to preferentially enhance their maturation into an arterial phenotype for future vascular tissue engineering applications., (© 2018 Wiley Periodicals, Inc.)

Studies of electrical stimulation therapies for the treatment of neurological disorders, such as deep brain stimulation, have almost exclusively been performed using animal-models. However, because animal-models can only approximate human brain disorders, these studies should be supplemented with an in vitro human cell-culture based model to substantiate the results of animal-based studies and further investigate therapeutic benefit in humans. This study presents a novel approach to analyze the effect of electrical stimulation on the neurogenesis of patient-induced pluripotent stem cell (iPSC) derived neural progenitor cell (NPC) lines, in vitro using a 3D graphene scaffold system. The iPSC-derived hNPCs used to demonstrate the system were collected from patients with Rett syndrome, a debilitating neurodevelopmental disorder. The graphene scaffold readily supported both the wild-type and Rett NPCs. Electrical stimulation parameters were optimized to accommodate both wild-type and Rett cells. Increased cell maturation and improvements in cell morphology of the Rett cells was observed after electrical stimulation. The results of the pilot study of electrical stimulation to enhance Rett NPCs neurogenesis were promising and support further investigation of the therapy. Overall, this system provides a valuable tool to study electrical stimulation as a potential therapy for neurological disorders using patient-specific cells.

Cell therapy for vascular damage has been showing promises as alternative therapy for endothelial dysfunctions since the discovery of the endothelial progenitor cells (EPCs). However, isolated EPCs from peripheral blood yield low cell amounts and alternative cell source must be explored. The aim of this study was to investigate the influence of topography on the endothelial differentiation of an alternative cell source - human mesenchymal stem cells (hMSCs) from bone marrow. Utilizing the MultiARChitecture (MARC) chip, a systematic screening of variety of patterned surfaces and different medium compositions was performed. While topographical patterns alone induce endothelial differentiation, a synergistic enhancement was observed when topography was combined with a medium enriched with vascular endothelial growth factor (VEGF). The 1.8 μm diameter convex microlens pattern in combination with the VEGF enriched medium was shown to be the most efficient on the endothelial differentiation, yielding up to 10% of CD34 + CD133 + KDR + marker expressing differentiated hMSCs as analyzed by flow cytometry. The quantified tube-like structures in the Matrigel assay in vitro indicated a vasculogenic potential of these endothelial progenitor-like differentiated hMSCs that was investigated further in a Matrigel plug assay in vivo in a rat for seven days. Explanted Matrigel plugs were processed with hematoxylin-eosin (H&E) and anti-Ulex Europaeus agglutinin (UEA-1) staining to visualize the capillaries and to identify the presence of human cells. The hMSCs cultured on the 1.8 μm diameter convex microlens in a medium enriched with VEGF, implanted in a Matrigel plug in a rat, showed the highest capillary density, the highest UEA-1 + capillary density, as well as the highest UEA-1 + cell survival density that were not included in the vasculogenesis. These findings indicate the active participation of the vasculogenic hMSCs in the vasculogenesis. The endothelial differentiation of hMSCs using this synergistic combination of microlens and VEGF enriched medium was also demonstrated in hMSCs from different male and female donors. The culture platform with combination of topography and biochemical cues could generate vasculogenic cell populations that may prove useful in vascular damage or other clinical applications., (Copyright © 2017 Elsevier Ltd. All rights reserved.)