A synthetic, closed-looped gene circuit for the autonomous regulation of RUNX2 activity during chondrogenesis.

The transcription factor RUNX2 is a key regulator of chondrocyte phenotype during development, making it an ideal target for prevention of undesirable chondrocyte maturation in cartilage tissue-engineering strategies. Here, we engineered an autoregulatory gene circuit (cisCXp-shRunx2) that negatively controls RUNX2 activity in chondrogenic cells via RNA interference initiated by a tunable synthetic Col10a1-like promoter (cisCXp). The cisCXp-shRunx2 gene circuit is designed based on the observation that induced RUNX2 silencing after early chondrogenesis enhances the accumulation of cartilaginous matrix in ATDC5 cells. We show that the cisCXp-shRunx2 initiates RNAi of RUNX2 in maturing chondrocytes in response to the increasing intracellular RUNX2 activity without interfering with early chondrogenesis. The induced loss of RUNX2 activity in turn negatively regulates the gene circuit itself. Moreover, the efficacy of RUNX2 suppression from cisCXp-shRunx2 can be controlled by modifying the sensitivity of cisCXp promoter. Finally, we show the efficacy of inhibiting RUNX2 in preventing matrix loss in human mesenchymal stem cell-derived (hMSC-derived) cartilage under conditions that induce chondrocyte hypertrophic differentiation, including inflammation. Overall, our results demonstrated that the negative modulation of RUNX2 activity with our autoregulatory gene circuit enhanced matrix synthesis and resisted ECM degradation by reprogrammed MSC-derived chondrocytes in response to the microenvironment of the degenerative joint.

LARGE glycans on dystroglycan function as a tunable matrix scaffold to prevent dystrophy.

The dense glycan coat that surrounds every cell is essential for cellular development and physiological function, and it is becoming appreciated that its composition is highly dynamic. Post-translational addition of the polysaccharide repeating unit [-3-xylose-α1,3-glucuronic acid-ß1-]n by like-acetylglucosaminyltransferase (LARGE) is required for the glycoprotein dystroglycan to function as a receptor for proteins in the extracellular matrix. Reductions in the amount of [-3-xylose-α1,3-glucuronic acid-ß1-]n (hereafter referred to as LARGE-glycan) on dystroglycan result in heterogeneous forms of muscular dystrophy. However, neither patient nor mouse studies has revealed a clear correlation between glycosylation status and phenotype. This disparity can be attributed to our lack of knowledge of the cellular function of the LARGE-glycan repeat. Here we show that coordinated upregulation of Large and dystroglycan in differentiating mouse muscle facilitates rapid extension of LARGE-glycan repeat chains. Using synthesized LARGE-glycan repeats we show a direct correlation between LARGE-glycan extension and its binding capacity for extracellular matrix ligands. Blocking Large upregulation during muscle regeneration results in the synthesis of dystroglycan with minimal LARGE-glycan repeats in association with a less compact basement membrane, immature neuromuscular junctions and dysfunctional muscle predisposed to dystrophy. This was consistent with the finding that patients with increased clinical severity of disease have fewer LARGE-glycan repeats. Our results reveal that the LARGE-glycan of dystroglycan serves as a tunable extracellular matrix protein scaffold, the extension of which is required for normal skeletal muscle function.

Phosphate regulates chondrogenesis in a biphasic and maturation-dependent manner.

Inorganic phosphate (Pi) has been recognized as an important signaling molecule that modulates chondrocyte maturation and cartilage mineralization. However, conclusive experimental evidence for its involvement in early chondrogenesis is still lacking. Here, using high-density monolayer (2D) and pellet (3D) culture models of chondrogenic ATDC5 cells, we demonstrate that the cell response to Pi does not correlate with the Pi concentration in the culture medium but is better predicted by the availability of Pi on a per cell basis (Pi abundance). Both culture models were treated with ITS+, 10mM ß-glycerophosphate (ßGP), or ITS+/10mM ßGP, which resulted in three levels of Pi abundance in cultures: basal (Pi/DNA <10ng/µg), moderate (Pi/DNA=25.3 - 32.3ng/µg), and high abundance (Pi/DNA >60ng/µg). In chondrogenic medium alone, the abundance levels were at the basal level in 2D culture and moderate in 3D cultures. The addition of 10mM ßGP resulted in moderate abundance in 2D and high abundance in 3D cultures. Moderate Pi abundance enhanced early chondrogenesis and production of aggrecan and type II collagen whereas high Pi abundance inhibited chondrogenic differentiation and induced rapid mineralization. Inhibition of sodium phosphate transporters reduced phosphate-induced expression of chondrogenic markers. When 3D ITS+/ßGP cultures were treated with levamisole to reduce ALP activity, Pi abundance was decreased to moderate levels, which resulted in significant upregulation of chondrogenic markers, similar to the response in 2D cultures. Delay of phosphate delivery until after early chondrogenesis occurs (7 days) no longer enhanced chondrogenesis, but instead accelerated hypertrophy and mineralization. Together, our data highlights the dependence of chondroprogenitor cell response to Pi on its availability to individual cells and the chondrogenic maturation stage of these cells and suggest that appropriate temporal delivery of phosphate to ATDC5 cells in 3D cultures represents a rapid model for mechanistic studies into the effects of exogenous cues on chondrogenic differentiation, chondrocyte maturation, and matrix mineralization.

Nanowire lasers as intracellular probes.

We investigate a cadmium sulfide (CdS) nanowire (NW) laser that is spontaneously internalized into a single cell to serve as a stand-alone intracellular probe. By pumping with nano-joule light pulses, green laser emission (500-520 nm) can be observed inside cells with a peak linewidth as narrow as 0.5 nm. Due to the sub-micron diameter (∼200 nm), the NW has an appreciable fraction of the evanescent field outside, facilitating a sensitive detection of cellular environmental changes. By monitoring the lasing peak wavelength shift in response to the intracellular refractive index change, our NW laser probe shows a sensitivity of 55 nm per RIU (refractive index units) and a figure of merit of approximately 98.

An integrated microwell array platform for cell lasing analysis.

Biological cell lasers are emerging as a novel technology in biological studies and biomedical engineering. The heterogeneity of cells, however, can result in various lasing behaviors from cell to cell. Thus, the capability to track individual cells during laser investigation is highly desired. In this work, a microwell array was integrated with high-quality Fabry-Pérot cavities for addressable and automated cell laser studies. Cells were captured in the microwells and the corresponding cell lasing was achieved and analyzed using SYTO9-stained Sf9 cells as a model system. It is found that the presence of the microwells does not affect the lasing performance, but the cell lasers exhibit strong heterogeneity due to different cell sizes, cycle stages and polyploidy. Time series laser measurements were also performed automatically with the integrated microarray, which not only enables the tracking and multiplexed detection of individual cells, but also helps identify "abnormal" cells that deviate from a large normal cell population in their lasing performance. The microarrayed cell laser platform developed here could provide a powerful tool in single cell analysis using lasing emission that complements conventional fluorescence-based cell analysis.

Characterizing natural hydrogel for reconstruction of three-dimensional lymphoid stromal network to model T-cell interactions.

Hydrogels have been used in regenerative medicine because they provide a three-dimensional environment similar to soft tissues, allow diffusion of nutrients, present critical biological signals, and degrade via endogenous enzymatic mechanisms. Herein, we developed in vitro system mimicking cell-cell and cell-matrix interactions in secondary lymphoid organs (SLOs). Existing in vitro culture systems cannot accurately represent the complex interactions happening between T-cells and stromal cells in immune response. To model T-cell interaction in SLOs in vitro, we encapsulated stromal cells in fibrin, collagen, or fibrin-collagen hydrogels and studied how different mechanical and biological properties affect stromal network formation. Overall, fibrin supplemented with aprotinin was superior to collagen and fibrin-collagen in terms of network formation and promotion of T-cell penetration. After 8 days of culture, stromal networks formed through branching and joining with other adjacent cell populations. T-cells added to the newly formed stromal networks migrated and attached to stromal cells, similar to the T-cell zones of the lymph nodes in vivo. Our results suggest that the constructed three-dimensional lymphoid stromal network can mimic the in vivo environment and allow the modeling of T-cell interaction in SLOs.