Eliza Li Shan Fong

STEM CELL HOMING IN MUSCULOSKELETAL INJURY

The regenerative potential of injured adult tissue suggests the physiological existence of cells capable of participating in the reparative process. Recent studies indicate that stem-like cells residing in tissues contribute to tissue repair and are replenished by precursor bone marrow-derived cells. Mesenchymal stromal cells (MSC) are among the candidates for reparative cells. These cells can potentially be mobilized into the circulation in response to injury signals and exert their reparative effects at the site of injury. Current therapies for musculoskeletal injuries pose unavoidable risks which can impede full recovery. Trafficking of MSC to the injury site and their subsequent participation in the regenerative process is thought to be a natural healing response that can be imitated or augmented by enhancing the endogenous MSC pool with exogenously administered MSC. Therefore, a promising alternative to the existing strategies employed in the treatment of musculoskeletal injuries is to reinforce the inherent reparative capacity of the body by delivering MSC harvested from the patients own tissues to the site of injury. The aim of this review is to inform the reader of studies that have evaluated the intrinsic homing and regenerative abilities of MSC, with particular emphasis on the repair of musculoskeletal injuries. Research that supports the direct use of MSC (without in vitro differentiation into tissue-specific cells) will also be reported. Based on accruing evidence that the natural healing mechanism involves the recruitment of MSC and their subsequent reparative actions at the site of injury, as well as documented therapeutic response after the exogenous administration of MSC, the feasibility of the emerging strategy of instant stem-cell therapy will be proposed.

Modeling Ewing sarcoma tumors in vitro with 3D scaffolds

The pronounced biological influence of the tumor microenvironment on cancer progression and metastasis has gained increased recognition over the past decade, yet most preclinical antineoplastic drug testing is still reliant on conventional 2D cell culture systems. Although monolayer cultures recapitulate some of the phenotypic traits observed clinically, they are limited in their ability to model the full range of microenvironmental cues, such as ones elicited by 3D cell–cell and cell–extracellular matrix interactions. To address these shortcomings, we established an ex vivo 3D Ewing sarcoma model that closely mimics the morphology, growth kinetics, and protein expression profile of human tumors. We observed that Ewing sarcoma cells cultured in porous 3D electrospun poly(ε-caprolactone) scaffolds not only were more resistant to traditional cytotoxic drugs than were cells in 2D monolayer culture but also exhibited remarkable differences in the expression pattern of the insulin-like growth factor-1 receptor/mammalian target of rapamycin pathway. This 3D model of the bone microenvironment may have broad applicability for mechanistic studies of bone sarcomas and exhibits the potential to augment preclinical evaluation of antineoplastic drug candidates for these malignancies.

Building Bridges: Leveraging Interdisciplinary Collaborations in the Development of Biomaterials to Meet Clinical Needs

Our laboratory at Rice University has forged numerous collaborations with clinicians and basic scientists over the years to advance the development of novel biomaterials and the modification of existing materials to meet clinical needs. This review highlights collaborative advances in biomaterials research from our laboratory in the areas of scaffold development, drug delivery, and gene therapy, especially as related to applications in bone and cartilage tissue engineering.

Evaluation of cell-laden polyelectrolyte hydrogels incorporating poly(l-Lysine) for applications in cartilage tissue engineering

To address the lack of reliable long-term solutions for cartilage injuries, strategies in tissue engineering are beginning to leverage developmental processes to spur tissue regeneration. This study focuses on the use of poly(L-lysine) (PLL), previously shown to up-regulate mesenchymal condensation during developmental skeletogenesis in vitro, as an early chondrogenic stimulant of mesenchymal stem cells (MSCs). We characterized the effect of PLL incorporation on the swelling and degradation of oligo(poly(ethylene) glycol) fumarate) (OPF)-based hydrogels as functions of PLL molecular weight and dosage. Furthermore, we investigated the effect of PLL incorporation on the chondrogenic gene expression of hydrogel-encapsulated MSCs. The incorporation of PLL resulted in early enhancements of type II collagen and aggrecan gene expression and type II/type I collagen expression ratios when compared to blank controls. The presentation of PLL to MSCs encapsulated in OPF hydrogels also enhanced N-cadherin gene expression under certain culture conditions, suggesting that PLL may induce the expression of condensation markers in synthetic hydrogel systems. In summary, PLL can function as an inductive factor that primes the cellular microenvironment for early chondrogenic gene expression but may require additional biochemical factors for the generation of fully functional chondrocytes.

Three-dimensional (3D) culture of bone-derived human 786-O renal cell carcinoma retains relevant clinical characteristics of bone metastases

Bone metastases from renal cell carcinoma (RCC) are typically lytic, destructive, and resistant to treatment regimens. Current in vitro models for studying metastasis introduce artifacts that limit their usefulness. Many features of tumors growing in bone are lost when human RCC cells are cultured in two-dimensional (2D) plastic substrata. In this study, we established that RCC spheroids, consisting of aggregates of cells, can be grown in a three-dimensional (3D) hyaluronate hydrogel-based culture system. The bone-derived human 786-O RCC subline proliferated and survived long term in these hydrogels. Additionally, RCC spheroids in 3D hydrogels demonstrated lower proliferation rates than their counterparts grown in 2D. Overall, gene expression patterns of RCC spheroids in 3D more closely mimicked those observed in vivo than did those of cells grown in 2D. Of particular importance, selected adhesion molecules, angiogenesis factors, and osteolytic factors that have been shown to be involved in RCC bone metastasis were found to be expressed at higher levels in 3D than in 2D cultures. We propose that the 3D culture system provides an improved platform for RCC bone metastasis studies compared with 2D systems.

IL-1β induces p62/SQSTM1 and represses androgen receptor expression in prostate cancer cells.

Chronic inflammation is associated with advanced prostate cancer (PCa), although the mechanisms governing inflammation‐mediated PCa progression are not fully understood. PCa progresses to an androgen independent phenotype that is incurable. We previously showed that androgen independent, androgen receptor negative (AR−) PCa cell lines have high p62/SQSTM1 levels required for cell survival. We also showed that factors in the HS‐5 bone marrow stromal cell (BMSC) conditioned medium can upregulate p62 in AR+ PCa cell lines, leading us to investigate AR expression under those growth conditions. In this paper, mRNA, protein, and subcellular analyses reveal that HS‐5 BMSC conditioned medium represses AR mRNA, protein, and nuclear accumulation in the C4‐2 PCa cell line. Using published gene expression data, we identify the inflammatory cytokine, IL‐1β, as a candidate BMSC paracrine factor to regulate AR expression and find that IL‐1β is sufficient to both repress AR and upregulate p62 in multiple PCa cell lines. Immunostaining demonstrates that, while the C4‐2 population shows a primarily homogeneous response to factors in HS‐5 BMSC conditioned medium, IL‐1β elicits a strikingly heterogeneous response; suggesting that there are other regulatory factors in the conditioned medium. Finally, while we observe concomitant AR loss and p62 upregulation in IL‐1β‐treated C4‐2 cells, silencing of AR or p62 suggests that IL‐1β regulates their protein accumulation through independent pathways. Taken together, these in vitro results suggest that IL‐1β can drive PCa progression in an inflammatory microenvironment through AR repression and p62 induction to promote the development and survival of androgen independent PCa. J. Cell. Biochem. 115: 2188–2197, 2014.

A vertical-flow bioreactor array compacts hepatocytes for enhanced polarity and functions

Although hepatocytes in vivo experience intra-abdominal pressure (IAP), pressure is typically not incorporated in hepatocyte culture systems. The cuboidal cell shape and extent of intercellular contact between cultured hepatocytes are critical parameters that influence the differentiated hepatic phenotype. Using a microfluidic device, the application of pressure to artificially compact cells and forge cell-cell interactions was previously demonstrated to be effective in accelerating hepatic repolarization. In seeking to implement this approach to higher throughput culture platforms for potential drug screening applications, we specifically designed a vertical-flow compaction bioreactor array (VCBA) that compacts hepatocytes within the range of IAP and portal pressure in vivo in a multi-well setup. As a result of vertical perfusion-generated forces, hepatocytes not only exhibited accelerated repolarization, an in vivo-like cuboidal morphology, but also better maintained hepatic functions in long-term culture as compared to the same cells cultured under static conditions. As a novel engineering tool to modulate cell compaction and intercellular interactions, this platform is a promising approach to confer tight control over hepatocyte repolarization for in vitro culture.

3D Culture as a Clinically Relevant Model for Personalized Medicine

Advances in understanding many of the fundamental mechanisms of cancer progression have led to the development of molecular targeted therapies. While molecular targeted therapeutics continue to improve the outcome for cancer patients, tumor heterogeneity among patients, as well as intratumoral heterogeneity, limits the efficacy of these drugs to specific patient subtypes, as well as contributes to relapse. Thus, there is a need for a more personalized approach toward drug development and diagnosis that takes into account the diversity of cancer patients, as well as the complex milieu of tumor cells within a single patient. Three-dimensional (3D) culture systems paired with patient-derived xenografts or patient-derived organoids may provide a more clinically relevant system to address issues presented by personalized or precision medical approaches. In this review, we cover the current methods available for applying 3D culture systems toward personalized cancer research and drug development, as well as key challenges that must be addressed in order to fully realize the potential of 3D patient-derived culture systems for cancer drug development. Greater implementation of 3D patient-derived culture systems in the cancer research field should accelerate the development of truly personalized medical therapies for cancer patients.

Actomyosin contractility drives bile regurgitation as an early response during obstructive cholestasis

BACKGROUND & AIMS A wide range of liver diseases manifest as biliary obstruction, or cholestasis. However, the sequence of molecular events triggered as part of the early hepatocellular homeostatic response in obstructive cholestasis is poorly elucidated. Pericanalicular actin is known to accumulate during obstructive cholestasis. Therefore, we hypothesized that the pericanalicular actin cortex undergoes significant remodeling as a regulatory response to obstructive cholestasis. METHODS In vivo investigations were performed in a bile duct-ligated mouse model. Actomyosin contractility was assessed using sandwich-cultured rat hepatocytes transfected with various fluorescently labeled proteins and pharmacological inhibitors of actomyosin contractility. RESULTS Actomyosin contractility induces transient deformations along the canalicular membrane, a process we have termed inward blebbing. We show that these membrane intrusions are initiated by local ruptures in the pericanalicular actin cortex; and they typically retract following repair by actin polymerization and actomyosin contraction. However, above a certain osmotic pressure threshold, these inward blebs pinch away from the canalicular membrane into the hepatocyte cytoplasm as large vesicles (2-8μm). Importantly, we show that these vesicles aid in the regurgitation of bile from the bile canaliculi. CONCLUSION Actomyosin contractility induces the formation of bile-regurgitative vesicles, thus serving as an early homeostatic mechanism against increased biliary pressure during cholestasis. LAY SUMMARY Bile canaliculi expand and contract in response to the amount of secreted bile, and resistance from the surrounding actin bundles. Further expansion due to bile duct blockade leads to the formation of inward blebs, which carry away excess bile to prevent bile build up in the canaliculi.

Data describing the swelling behavior and cytocompatibility of biodegradable polyelectrolyte hydrogels incorporating poly(L-lysine) for applications in cartilage tissue engineering

This data article presents data associated with the research article entitled “Evaluation of cell-laden polyelectrolyte hydrogels incorporating poly(L-lysine) for applications in cartilage tissue engineering” (Lam et al., 2016) [1]. Synthetic hydrogel composites fabricated using oligo(poly(ethylene glycol) fumarate) (OPF) macromers were utilized as vehicles for the incorporation of poly(L-lysine) (PLL) as well as the encapsulation of mesenchymal stem cells (MSCs). PLL-laden and PLL-free hydrogels were fabricated to characterize the main and interaction effects of OPF molecular weight, PLL molecular weight, and PLL loading density on the swelling and degradation of synthetic OPF hydrogels. Cells were then encapsulated within such hydrogels for in vitro culture and examined for viability, biochemical activity, and chondrogenic gene expression. These data, which are supplementary to the associated research article (Lam et al., 2016) [1], are presented here.