In silico design and fabrication of an SFI chip-based microspheroid culture system.

The emergence of microfluidic devices and computational fluid dynamics (CFD) has propelled the need for next-generation biomimetic cell culture platforms that are flexible for monitoring and regulation. Therefore, this study evaluated a CFD application in an in silico-designed and spheroid-based flow integration 3D cell culture chip (SFI chip) to illustrate cell culture, drug screening, cytokine delivery, and differentiation of cells in a platform that partially recapitulates the natural environment. Our results show that a flow rate of 0.05 mL h-1 or less induced no physical stress in the SFI chip (15 mm), and uniform cell spheroids (approximately 200 µm) were formed across the platform. The cultured cells were tested in several experimental contexts (co-culture, drug screening, cytokine delivery, and differentiation), demonstrating the usefulness of computational simulation in expediting discovery and simple and effective means to scale the production of standardized cell spheroids cultured under dynamic and natural conditions. Advanced cell culture technologies can be used to accelerate research and discovery and the preclinical and clinical development of cell and cell-free therapies for urgent medical needs.

Application of co-culture technology of epithelial type cells and mesenchymal type cells using nanopatterned structures.

Various nanopatterning techniques have been developed to improve cell proliferation and differentiation efficiency. As we previously reported, nanopillars and pores are able to sustain human pluripotent stem cells and differentiate pancreatic cells. From this, the nanoscale patterns would be effective environment for the co-culturing of epithelial and mesenchymal cell types. Interestingly, the nanopatterning selectively reduced the proliferative rate of mesenchymal cells while increasing the expression of adhesion protein in epithelial type cells. Additionally, co-cultured cells on the nanopatterning were not negatively affected in terms of cell function metabolic ability or cell survival. This is in contrast to conventional co-culturing methods such as ultraviolet or chemical treatments. The nanopatterning appears to be an effective environment for mesenchymal co-cultures with typically low proliferative rates cells such as astrocytes, neurons, melanocytes, and fibroblasts without using potentially damaging treatments.

Functional Equivalency in Human Somatic Cell Nuclear Transfer-Derived Endothelial Cells.

The derivation of human embryonic stem cells (hESCs) by somatic cell nuclear transfer (SCNT) has prompted a re-emerging interest in using such cells for therapeutic cloning. Despite recent advancements in derivation protocols, the functional potential of CHA-NT4 derived cells is yet to be elucidated. For this reason, this study sought to differentiate CHA-NT4 cells toward an endothelial lineage in order to evaluate in vitro and in vivo functionality. To initial differentiation, embryoid body formation of CHA-NT4 was mediated by concave microwell system which was optimized for hESC-endothelial cell (EC) differentiation. The isolated CD31+ cells exhibited hallmark endothelial characteristics in terms of morphology, tubule formation, and ac-LDL uptake. Furthermore, CHA-NT4-derived EC (human nuclear transfer [hNT]-ESC-EC) transplantation in hind limb ischemic mice rescued the hind limb and restored blood perfusion. These findings suggest that hNT-ESC-EC are functionally equivalent to hESC-ECs, warranting further study of CHA-NT4 derivatives in comparison to other well established pluripotent stem cell lines. This revival of human SCNT-ESC research may lead to interesting insights into cellular behavior in relation to donor profile, mitochondrial DNA, and oocyte quality. Stem Cells 2019;37:623-630.

Mechanotransduction of human pluripotent stem cells cultivated on tunable cell-derived extracellular matrix.

Cell-derived matrices (CDM) are becoming an attractive alternative to conventional biological scaffolding platforms due to its unique ability to closely recapitulate a native extracellular matrix (ECM) de novo. Although cell-substrate interactions are recognized to be principal in regulating stem cell behavior, very few studies have documented the acclimation of human pluripotent stem cells (hPSCs) on pristine and altered cell-derived matrices. Here, we investigate crosslink-induced mechanotransduction of hPSCs cultivated on decellularized fibroblast-derived matrices (FDM) to explore cell adhesion, growth, migration, and pluripotency in various biological landscapes. The results showed either substrate-mediated induction or inhibition of the Epithelial-Mesenchymal-Transition (EMT) program, strongly suggesting that FDM stiffness can be a dominant factor in mediating hPSC plasticity. We further propose an optimal FDM substratum intended for long-term hPSC cultivation in a feeder-free niche-like microenvironment. This study carries significant implications for hPSC cultivation and encourages more in-depth studies towards the fundamentals of hPSC-CDM interactions.

From Bench to Market: Preparing Human Pluripotent Stem Cells Derived Cardiomyocytes for Various Applications.

Human cardiomyocytes (CMs) cease to proliferate and remain terminally differentiated thereafter, when humans reach the mid-20s. Thus, any damages sustained by myocardium tissue are irreversible, and they require medical interventions to regain functionality. To date, new surgical procedures and drugs have been developed, albeit with limited success, to treat various heart diseases including myocardial infarction. Hence, there is a pressing need to develop more effective treatment methods to address the increasing mortality rate of the heart diseases. Functional CMs are not only an important in vitro cellular tool to model various types of heart diseases for drug development, but they are also a promising therapeutic agent for cell therapy. However, the limited proliferative capacity entails difficulties in acquiring functional CMs in the scale that is required for pathological studies and cell therapy development. Stem cells, human pluripotent stem cells (hPSCs) in particular, have been considered as an unlimited cellular source for providing functional CMs for various applications. Notable progress has already been made: the first clinical trials of hPSCs derived CMs (hPSC-CMs) for treating myocardial infarction was approved in 2015, and their potential use in disease modeling and drug discovery is being fully explored. This concise review gives an account of current development of differentiation, purification and maturation techniques for hPSC-CMs, and their application in cell therapy development and pharmaceutical industries will be discussed with the latest experimental evidence.

Physiological Effects of Ac4ManNAz and Optimization of Metabolic Labeling for Cell Tracking.

Metabolic labeling techniques are powerful tools for cell labeling, tracking and proteomic analysis. However, at present, the effects of the metabolic labeling agents on cell metabolism and physiology are not known. To address this question, in this study, we analyzed the effects of cells treated with Ac4ManNAz through microarray analysis and analyses of membrane channel activity, individual bio-physiological properties, and glycolytic flux. According to the results, treatment with 50 µM Ac4ManNAz led to the reduction of major cellular functions, including energy generation capacity, cellular infiltration ability and channel activity. Interestingly, 10 µM Ac4ManNAz showed the least effect on cellular systems and had a sufficient labeling efficiency for cell labeling, tracking and proteomic analysis. Based on our results, we suggest 10 µM as the optimum concentration of Ac4ManNAz for in vivo cell labeling and tracking. Additionally, we expect that our approach could be used for cell-based therapy for monitoring the efficacy of molecule delivery and the fate of recipient cells.

Effect of BMP-2 Delivery Mode on Osteogenic Differentiation of Stem Cells.

Differentiation of stem cells is an important strategy for regeneration of defective tissue in stem cell therapy. Bone morphogenetic protein-2 (BMP-2) is a well-known osteogenic differentiation factor that stimulates stem cell signaling pathways by activating transmembrane type I and type II receptors. However, BMPs have a very short half-life and may rapidly lose their bioactivity. Thus, a BMP delivery system is required to take advantage of an osteoinductive effect for osteogenic differentiation. Previously, BMP delivery has been designed and evaluated for osteogenic differentiation, focusing on carriers and sustained release system for delivery of BMPs. The effect of the delivery mode in cell culture plate on osteogenic differentiation potential was not evaluated. Herein, to investigate the effect of delivery mode on osteogenic differentiation of BM-MSCs in this study, we fabricated bottom-up release and top-down release systems for culture plate delivery of BMP-2. And also, we selected Arg-Gly-Asp- (RGD-) conjugated alginate hydrogel for BMP-2 delivery because alginate is able to release BMP-2 in a sustained manner and it is a biocompatible material. After 7 days of culture, the bottom-up release system in culture plate significantly stimulated alkaline phosphate activity of human bone marrow-mesenchymal stem cells. The present study highlights the potential value of the tool in stem cell therapy.

Nanotopographical control for maintaining undifferentiated human embryonic stem cell colonies in feeder free conditions.

Recently emerging evidence has indicated surface nanotopography as an important physical parameter in the stem cell niche for regulating cell fate and behaviors for various types of stem cells. In this study, a substrate featuring arrays of increasing nanopillar diameter was devised to investigate the effects of varying surface nanotopography on the maintenance of undifferentiated human embryonic stem cells (hESC) colonies in the absence of feeder cells. Single hESCs cultured across gradient nanopattern (G-Np) substrate were generally organized into compact colonies, and expressed higher levels of undifferentiated markers compared to those cultured on the unstructured control substrate. In particular, hESC demonstrates a propensity to organize into more compact colonies expressing higher levels of undifferentiated markers towards a smaller nanopillar diameter range (D = 120-170 nm). Cell-nanotopography interactions modulated the formation of focal adhesions and cytoskeleton reorganization to restrict colony spreading, which reinforced E-cadherin mediated cell-cell adhesions in hESC colonies. Maintaining compact hESC colony integrity revealed to be indispensable for hESC undifferentiated state as the loss of cell-cell adhesions within spreading hESC on the control substrate exhibited morphological and gene expression signatures of epithelial-to-mesenchymal transition-like processes. Findings in this study demonstrated a feasible approach to screen the optimal nanotopographical cues for maintaining undifferentiated hESC colonies in feeder free conditions, which provides a platform for further investigations into developing hESC feeder free culture systems for the purpose of regenerative medicine.