Kyung-Jin Jang

Engineered In Vitro Disease Models

The ultimate goal of most biomedical research is to gain greater insight into mechanisms of human disease or to develop new and improved therapies or diagnostics. Although great advances have been made in terms of developing disease models in animals, such as transgenic mice, many of these models fail to faithfully recapitulate the human condition. In addition, it is difficult to identify critical cellular and molecular contributors to disease or to vary them independently in whole-animal models. This challenge has attracted the interest of engineers, who have begun to collaborate with biologists to leverage recent advances in tissue engineering and microfabrication to develop novel in vitro models of disease. As these models are synthetic systems, specific molecular factors and individual cell types, including parenchymal cells, vascular cells, and immune cells, can be varied independently while simultaneously measuring system-level responses in real time. In this article, we provide some examples of these efforts, including engineered models of diseases of the heart, lung, intestine, liver, kidney, cartilage, skin and vascular, endocrine, musculoskeletal, and nervous systems, as well as models of infectious diseases and cancer. We also describe how engineered in vitro models can be combined with human inducible pluripotent stem cells to enable new insights into a broad variety of disease mechanisms, as well as provide a test bed for screening new therapies.

Detection of proteins using a colorimetric bio-barcode assay

The colorimetric bio-barcode assay is a red-to-blue color change-based protein detection method with ultrahigh sensitivity. This assay is based on both the bio-barcode amplification method that allows for detecting miniscule amount of targets with attomolar sensitivity and gold nanoparticle-based colorimetric DNA detection method that allows for a simple and straightforward detection of biomolecules of interest (here we detect interleukin-2, an important biomarker (cytokine) for many immunodeficiency-related diseases and cancers). The protocol is composed of the following steps: (i) conjugation of target capture molecules and barcode DNA strands onto silica microparticles, (ii) target capture with probes, (iii) separation and release of barcode DNA strands from the separated probes, (iv) detection of released barcode DNA using DNA-modified gold nanoparticle probes and (v) red-to-blue color change analysis with a graphic software. Actual target detection and quantification steps with premade probes take ∼3 h (whole protocol including probe preparations takes ∼3 days).

Two distinct filopodia populations at the growth cone allow to sense nanotopographical extracellular matrix cues to guide neurite outgrowth.

Background The process of neurite outgrowth is the initial step in producing the neuronal processes that wire the brain. Current models about neurite outgrowth have been derived from classic two-dimensional (2D) cell culture systems, which do not recapitulate the topographical cues that are present in the extracellular matrix (ECM) in vivo. Here, we explore how ECM nanotopography influences neurite outgrowth. Methodology/Principal Findings We show that, when the ECM protein laminin is presented on a line pattern with nanometric size features, it leads to orientation of neurite outgrowth along the line pattern. This is also coupled with a robust increase in neurite length. The sensing mechanism that allows neurite orientation occurs through a highly stereotypical growth cone behavior involving two filopodia populations. Non-aligned filopodia on the distal part of the growth cone scan the pattern in a lateral back and forth motion and are highly unstable. Filopodia at the growth cone tip align with the line substrate, are stabilized by an F-actin rich cytoskeleton and enable steady neurite extension. This stabilization event most likely occurs by integration of signals emanating from non-aligned and aligned filopodia which sense different extent of adhesion surface on the line pattern. In contrast, on the 2D substrate only unstable filopodia are observed at the growth cone, leading to frequent neurite collapse events and less efficient outgrowth. Conclusions/Significance We propose that a constant crosstalk between both filopodia populations allows stochastic sensing of nanotopographical ECM cues, leading to oriented and steady neurite outgrowth. Our work provides insight in how neuronal growth cones can sense geometric ECM cues. This has not been accessible previously using routine 2D culture systems.

Artificial Slanted Nanocilia Array as a Mechanotransducer for Controlling Cell Polarity

We present a method to induce cell directional behavior using slanted nanocilia arrays. NIH-3T3 fibroblasts demonstrated bidirectional polarization in a rectangular arrangement on vertical nanocilia arrays and exhibited a transition from a bidirectional to a unidirectional polarization pattern when the angle of the nanocilia was decreased from 90° to 30°. The slanted nanocilia guided and facilitated spreading by allowing the cells to contact the sidewalls of the nanocilia, and the directional migration of the cells opposed the direction of the slant due to the anisotropic bending stiffness of the slanted nanocilia. Although the cells recognized the underlying anisotropic geometry when the nanocilia were coated with fibronectin, collagen type I, and Matrigel, the cells lost their directionality when the nanocilia were coated with poly-d-lysine and poly-l-lysine. Furthermore, although the cells recognized geometrical anisotropy on fibronectin coatings, pharmacological perturbation of PI3K-Rac signaling hindered the directional elongation of the cells on both the slanted and vertical nanocilia. Furthermore, myosin light chain II was required for the cells to obtain polarized morphologies. These results indicated that the slanted nanocilia array provided anisotropic contact guidance cues to the interacting cells. The polarization of cells was controlled through two steps: the recognition of underlying geometrical anisotropy and the subsequent directional spreading according to the guidance cues.

Latent progenitor cells as potential regulators for tympanic membrane regeneration

Tympanic membrane (TM) perforation, in particular chronic otitis media, is one of the most common clinical problems in the world and can present with sensorineural healing loss. Here, we explored an approach for TM regeneration where the latent progenitor or stem cells within TM epithelial layers may play an important regulatory role. We showed that potential TM stem cells present highly positive staining for epithelial stem cell markers in all areas of normal TM tissue. Additionally, they are present at high levels in perforated TMs, especially in proximity to the holes, regardless of acute or chronic status, suggesting that TM stem cells may be a potential factor for TM regeneration. Our study suggests that latent TM stem cells could be potential regulators of regeneration, which provides a new insight into this clinically important process and a potential target for new therapies for chronic otitis media and other eardrum injuries.

Chapter 2:Kidney on a Chip

In this chapter, a general overview of progress made on the kidney on a chip will be introduced. We will focus on the understanding of kidney structure and function, and application of micro-engineered kidney-mimetic microsystems for the study of kidney function and disease in vitro. We will also discuss the potential use of these “kidney chips” for biomedical pharmaceutical and environmental safety testing applications as well as challenges for the field that must be overcome to translate these technologies into useful products in the future.