Koh Meng Aw Yong

Hippo/YAP-mediated rigidity-dependent motor neuron differentiation of human pluripotent stem cells

Our understanding of the intrinsic mechanosensitive properties of human pluripotent stem cells (hPSCs), in particular the effects that the physical microenvironment has on their differentiation, remains elusive1. Here, we show that neural induction and caudalization of hPSCs can be accelerated by using a synthetic microengineered substrate system consisting of poly(dimethylsiloxane) micropost arrays (PMAs) with tunable mechanical rigidities. The purity and yield of functional motor neurons (MNs) derived from hPSCs within 23 days of culture using soft PMAs were improved more than 4- and 10-fold, respectively, compared to coverslips or rigid PMAs. Mechanistic studies revealed a multi-targeted mechanotransductive process involving Smad phosphorylation and nucleocytoplasmic shuttling, regulated by rigidity-dependent Hippo-YAP activities and actomyosin cytoskeleton integrity and contractility. Our findings suggest that substrate rigidity is an important biophysical cue influencing neural induction and subtype specification, and that microengineered substrates can thus serve as a promising platform for large-scale culture of hPSCs.

Microfluidic Blood Cell Sorting: Now and Beyond

Blood plays an important role in homeostatic regulation with each of its cellular components having important therapeutic and diagnostic uses. Therefore, separation and sorting of blood cells hasa been of a great interest to clinicians and researchers. However, while conventional methods of processing blood have been successful in generating relatively pure fractions, they are time consuming, labor intensive, and are not optimal for processing small volume blood samples. In recent years, microfluidics has garnered great interest from clinicians and researchers as a powerful technology for separating blood into different cell fractions. As microfluidics involves fluid manipulation at the microscale level, it has the potential for achieving high-resolution separation and sorting of blood cells down to a single-cell level, with an added benefit of integrating physical and biological methods for blood cell separation and analysis on the same single chip platform. This paper will first review the conventional methods of processing and sorting blood cells, followed by a discussion on how microfluidics is emerging as an efficient tool to rapidly change the field of blood cell sorting for blood-based therapeutic and diagnostic applications.

Probing the invasiveness of prostate cancer cells in a 3D microfabricated landscape

The metastatic invasion of cancer cells from primary tumors to distant ecological niches, rather than the primary tumors, is the cause of much cancer mortality [Zhang QB, et al. (2010) Int J Cancer 126:2534–2541; Chambers AF, Goss PE (2008) Breast Cancer Res 10:114]. Metastasis is a three-dimensional invasion process where cells spread from their site of origin and colonize distant microenvironmental niches. It is critical to be able to assess quantitatively the metastatic potential of cancer cells [Harma V, et al. (2010) PLoS ONE 5:e10431]. We have constructed a microfabricated chip with a three-dimensional topology consisting of lowlands and isolated square highlands (Tepuis), which stand hundreds of microns above the lowlands, in order to assess cancer cell metastatic potential as they invade the highlands. As a test case, the invasive ascents of the Tepui by highly metastatic PC-3 and noninvasive LNCaP prostate cancer cells were used. The vertical ascent by prostate cancer cells from the lowlands to the tops of the Tepui was imaged using confocal microscopy and used as a measure of the relative invasiveness. The less-metastatic cells (LNCaP) never populated all available tops, leaving about 15% of them unoccupied, whereas the more metastatic PC-3 cells occupied all available Tepuis. We argue that this distinct difference in invasiveness is due to contact inhibition.

Morphological effects on expression of growth differentiation factor 15 (GDF15), a marker of metastasis.

Cancer cells typically demonstrate altered morphology during the various stages of disease progression as well as metastasis. While much is known about how altered cell morphology in cancer is a result of genetic regulation, less is known about how changes in cell morphology affect cell function by influencing gene expression. In this study, we altered cell morphology in different types of cancer cells by disrupting the actin cytoskeleton or by modulating attachment and observed a rapid up‐regulation of growth differentiation factor 15 (GDF15), a member of the transforming growth factor‐beta (TGF‐β) super‐family. Strikingly, this up‐regulation was sustained as long as the cell morphology remained altered but was reversed upon allowing cell morphology to return to its typical configuration. The potential significance of these findings was examined in vivo using a mouse model: a small number of cancer cells grown in diffusion chambers that altered morphology increased mouse serum GDF15. Taken together, we propose that during the process of metastasis, cancer cells experience changes in cell morphology, resulting in the increased production and secretion of GDF15 into the surrounding environment. This indicates a possible relationship between serum GDF15 levels and circulating tumor cells may exist. Further investigation into the exact nature of this relationship is warranted. J. Cell. Physiol. 229: 362–373, 2014.

Identification of nuclear structural protein alterations associated with seminomas

Currently, there are no specific markers available for the early detection and for monitoring testicular cancer. Based upon an approach that targets nuclear structure, we have identified a set of proteins that are specific for seminomas, which may then have clinical utility for the disease. Utilizing samples obtained from men with no evidence of testicular cancer (n = 5) as well as those with seminomas (n = 6), nuclear matrix proteins were extracted and separated using a high‐resolution two‐dimensional electrophoresis gel system. The proteins were identified by mass spectrometry analysis. These analyses revealed seven nuclear matrix proteins associated with the normal testes, which did not appear in the seminomas. In the seminomas, four nuclear matrix proteins were identified to be associated with the disease that were absent in the normal testes. Mass spectrometric and immunoblot analyses of these proteins revealed that one of the proteins identified in the normal testes appears to be StAR‐related lipid transfer protein 7 (StARD7). In the non‐seminoma tissues, one of the identified proteins appears to be cell division protein kinase 10 (CDK10). Both StarD7 and CDK10 could potentially be involved in cell differentiation and growth, and thus may serve as potential targets for therapy of prognostication of seminomas. This is the first study to examine the role of nuclear structural proteins as potential biomarkers in testicular cancer. We are currently examining the roles of some of the identified proteins as potential biomarkers for the disease. J. Cell. Biochem. 108: 1274–1279, 2009.

Mechanics-guided embryonic patterning of neuroectoderm tissue from human pluripotent stem cells

Classic embryological studies have successfully applied genetics and cell biology principles to understand embryonic development. However, it remains unresolved how mechanics, as an integral driver of development, is involved in controlling tissue-scale cell fate patterning. Here we report a micropatterned human pluripotent stem (hPS)-cell-based neuroectoderm developmental model, in which pre-patterned geometrical confinement induces emergent patterning of neuroepithelial and neural plate border cells, mimicking neuroectoderm regionalization during early neurulation in vivo. In this hPS-cell-based neuroectoderm patterning model, two tissue-scale morphogenetic signals—cell shape and cytoskeletal contractile force—instruct neuroepithelial/neural plate border patterning via BMP-SMAD signalling. We further show that ectopic mechanical activation and exogenous BMP signalling modulation are sufficient to perturb neuroepithelial/neural plate border patterning. This study provides a useful microengineered, hPS-cell-based model with which to understand the biomechanical principles that guide neuroectoderm patterning and hence to study neural development and disease.Mechanical cues play critical roles in embryonic development. A micropatterned neuroectoderm developmental model based on human pluripotent stem cells now reveals how morophogenetic signals such as cell shape and contractility regulate neural tissue development.

Microfluidic modeling of cancer metastasis

The tumor microenvironment is increasingly recognized as an important contributor to the processes of growth, invasion, and metastasis. Molecular and cellular targets within the microenvironment may thus offer new and fruitful targets for therapeutic intervention. However, progress in understanding and clinically leveraging this regulation has been limited by the complexity of the in vivo microenvironment, which does not easily allow clear dissection of specific regulatory effects. This in turn has created a strong need for in vitro engineered model systems that offer highly precise and independent control of a variety of extracellular parameters that can faithfully recapitulate the tumor microenvironment. In this chapter, we review recent progress in the development of such systems. We discuss microenvironmental signals that regulate tumor growth in vivo, focusing on the extracellular signals a cell receives in the most critical steps in Andrew Rape and Sanjay KumarThe tumor microenvironment is increasingly recognized as an important contributor to the processes of growth, invasion, and metastasis. Molecular and cellular targets within the microenvironment may thus offer new and fruitful targets for therapeutic intervention. However, progress in understanding and clinically leveraging this regulation has been limited by the complexity of the in vivo microenvironment, which does not easily allow clear dissection of specific regulatory effects. This in turn has created a strong need for in vitro engineered model systems that offer highly precise and independent control of a variety of extracellular parameters that can faithfully recapitulate the tumor microenvironment. In this chapter, we review recent progress in the development of such systems. We discuss microenvironmental signals that regulate tumor growth in vivo, focusing on the extracellular signals a cell receives in the most critical steps in

Mechanotransduction-Induced Reversible Phenotypic Switching in Prostate Cancer Cells

Phenotypic plasticity is posed to be a vital trait of cancer cells such as circulating tumor cells, allowing them to undergo reversible or irreversible switching between phenotypic states important for tumorigenesis and metastasis. While irreversible phenotypic switching can be detected by studying the genome, reversible phenotypic switching is often difficult to examine due to its dynamic nature and the lack of knowledge about its contributing factors. In this study, we demonstrate that culturing cells in different physical environments, stiff, soft, or suspension, induced a phenotypic switch in prostate cancer cells via mechanotransduction. The mechanosensitive phenotypic switching in prostate cancer cells was sustainable yet reversible even after long-term culture, demonstrating the impact of mechanical signals on prostate cancer cell phenotypes. Importantly, such a mechanotransduction-mediated phenotypic switch in prostate cancer cells was accompanied by decreased sensitivity of the cells to paclitaxel, suggesting a role of mechanotransduction in the evolution of drug resistance. Multiple signaling pathways such as p38MAPK, ERK, and Wnt were found to be involved in the mechanotransduction-induced phenotypic switching of prostate cancer cells. Given that cancer cells experience different physical environments during disease progression, this study provides useful information about the important role of mechanotransduction in cancer, and how circulating tumor cells may be capable of continuously changing their phenotypes throughout the disease process.

Tracking the tumor invasion front using long-term fluidic tumoroid culture

The analysis of invading leader cells at the tumor invasion front is of significant interest as these cells may possess a coordinated functional and molecular phenotype which can be targeted for therapy. However, such analyses are currently limited by available technologies. Here, we report a fluidic device for long-term three-dimensional tumoroid culture which recapitulated the tumor invasion front, allowing for both quantification of invasive potential and molecular characterization of invasive leader cells. Preliminary analysis of the invasion front indicated an association with cell proliferation and higher expression of growth differentiation factor 15 (GDF15). This device makes real-time tracking of invading leader cell phenotypes possible and has potential for use with patient material for clinical risk stratification and personalized medicine.

Synthetic micro/nanoengineered tools to study mechanobiology and its regulatory role for human pluripotent stem cells

In this talk, I will discuss our recent effort using synthetic micro/nanoengineered tools to study mechano-sensitive and -responsive behaviors of human pluripotent stem cells (hPSCs) and the underlying molecular and cellular mechanisms.