Substrate topography and size determine the fate of human embryonic stem cells to neuronal or glial lineage.

Efficient derivation of neural cells from human embryonic stem cells (hESCs) remains an unmet need for the treatment of neurological disorders. The limiting factors for current methods include being labor-intensive, time-consuming and expensive. In this study, we hypothesize that the substrate topography, with optimal geometry and dimension, can modulate the neural fate of hESCs and enhance the efficiency of differentiation. A multi-architectural chip (MARC) containing fields of topographies varying in geometry and dimension was developed to facilitate high-throughput analysis of topography-induced neural differentiation in vitro. The hESCs were subjected to "direct differentiation", in which small clumps of undifferentiated hESCs were cultured directly without going through the stage of embryoid body formation, on the MARC with N2 and B27 supplements for 7 days. The gene and protein expression analysis indicated that the anisotropic patterns like gratings promoted neuronal differentiation of hESCs while the isotropic patterns like pillars and wells promoted the glial differentiation of hESCs. This study showed that optimal combination of topography and biochemical cues could shorten the differentiation period and allowed derivation of neurons bearing longer neurites that were aligned along the grating axis. The MARC platform would enable high-throughput screening of topographical substrates that could maximize the efficiency of neuronal differentiation from pluripotent stem cells.

Normalized median fluorescence: an alternative flow cytometry analysis method for tracking human embryonic stem cell states during differentiation.

Human embryonic stem cells (hESCs) are a promising cell source for tissue engineering and regenerative medicine, but before they can be used in therapies, we must be able to accurately identify the state and progeny of hESCs. One of the most commonly used methods for identification is flow cytometry. Many flow cytometry applications use antibodies to detect the amount of antigen present on/in a cell. This allows for the identification of unique cell populations or the tracking of expression changes within a population during differentiation. The results are typically presented as a percentage of positively expressing cells (%Pos) for a marker of choice, relative to a negative control. However, this reporting term is vulnerable to distortion from outliers and inaccuracy from loss of information about the population's fluorescence intensity. In this article, we describe an alternate strategy that uses the normalized median fluorescence intensity (nMFI), in which the MFI of the stained sample is normalized to the MFI of the negative control, as the reporting term to more accurately describe a population of cells in culture. We observed that nMFI provides a more accurate representation for the quality of a starting population and comparing data of different experimental runs. In addition, we demonstrated that the nMFI is a more sensitive measure of pluripotent and differentiation markers expression changes during hESC differentiation into three germ layer lineages.

Temporal application of topography to increase the rate of neural differentiation from human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) are a promising cell source for tissue engineering and regenerative medicine, especially in the field of neurobiology. Neural differentiation protocols have been developed to differentiate hPSCs into specific neural cells, but these predominantly rely on biochemical cues. Recently, differentiation protocols have incorporated topographical cues to increase the total neuronal yield. However, the means by which these topographical cues improve neuronal yield remains unknown. In this study, we explored the effect of topography on the neural differentiation of hPSC by quantitatively studying the changes in marker expression at a transcript and protein level. We found that 2 µm gratings increase the rate of neural differentiation, and that an additional culture period of 2 µm gratings in the absence of neurotrophic signals can improve the neural differentiation of hPSCs. We envisage that this work can be incorporated into future differentiation protocols to decrease the differentiation period as well as the biochemical signals added, thus generating hPSC-derived neural cells in a more cost effective and efficient manner.

Nanotopography/mechanical induction of stem-cell differentiation.

The interplay of biophysical and biochemical cues in the extracellular microenvironment regulate and control the cell fate of stem cells. Understanding the interaction between stem cells and the extracellular substrate will be crucial in controlling stem cell differentiation for regenerative medicine applications. One of the biophysical properties of the microenvironment is substrate topology, which has been demonstrated to be an important mediator of stem cell lineage regulation. Biomimetic microenvironment topology can be engineered by chemical patterning or physical patterning. The rapid advancements in nanofabrication techniques have enabled versatility in patterning types with controlled chemistries, geometries and sizes. The chapter will focus on discussing the effect on physical nanotopography on stem cell differentiation and the current theories on the topography/ mechanical force induction of stem cell differentiation possibly through integrin clustering, focal adhesion, cytoskeleton organization and the nuclear mechanosensing to sense and integrate these biophysical signals from the extracellular microenvironment.