Stephanie M. Willerth

THE DIFFERENTIATION OF EMBRYONIC STEM CELLS SEEDED ON ELECTROSPUN NANOFIBERS INTO NEURAL LINEAGES

Due to advances in stem cell biology, embryonic stem (ES) cells can be induced to differentiate into a particular mature cell lineage when cultured as embryoid bodies. Although transplantation of ES cells-derived neural progenitor cells has been demonstrated with some success for either spinal cord injury repair in small animal model, control of ES cell differentiation into complex, viable, higher ordered tissues is still challenging. Mouse ES cells have been induced to become neural progenitors by adding retinoic acid to embryoid body cultures for 4 days. In this study, we examine the use of electrospun biodegradable polymers as scaffolds not only for enhancing the differentiation of mouse ES cells into neural lineages but also for promoting and guiding the neurite outgrowth. A combination of electrospun fiber scaffolds and ES cells-derived neural progenitor cells could lead to the development of a better strategy for nerve injury repair.

The Effects of Soluble Growth Factors on Embryonic Stem Cell Differentiation Inside of Fibrin Scaffolds

The goal of this research was to determine the effects of different growth factors on the survival and differentiation of murine embryonic stem cell‐derived neural progenitor cells (ESNPCs) seeded inside of fibrin scaffolds. Embryoid bodies were cultured for 8 days in suspension, retinoic acid was applied for the final 4 days to induce ESNPC formation, and then the EBs were seeded inside of three‐dimensional fibrin scaffolds. Scaffolds were cultured in the presence of media containing different doses of the following growth factors: neurotrophin‐3 (NT‐3), basic fibroblast growth factor (bFGF), platelet‐derived growth factor (PDGF)‐AA, ciliary neurotrophic factor, and sonic hedgehog (Shh). The cell phenotypes were characterized using fluorescence‐activated cell sorting and immunohistochemistry after 14 days of culture. Cell viability was also assessed at this time point. Shh (10 ng/ml) and NT‐3 (25 ng/ml) produced the largest fractions of neurons and oligodendrocytes, whereas PDGF (2 and 10 ng/ml) and bFGF (10 ng/ml) produced an increase in cell viability after 14 days of culture. Combinations of growth factors were tested based on the results of the individual growth factor studies to determine their effect on cell differentiation. The incorporation of ESNPCs and growth factors into fibrin scaffolds may serve as potential treatment for spinal cord injury.

The effect of controlled growth factor delivery on embryonic stem cell differentiation inside fibrin scaffolds.

The goal of this project was to develop 3-D biomaterial scaffolds that present cues to direct the differentiation of embryonic stem (ES) cell-derived neural progenitor cells, seeded inside the scaffolds, into mature neural phenotypes, specifically neurons and oligodendrocytes. Release studies were performed to determine the appropriate conditions for retention of neurotrophin-3 (NT-3), sonic hedgehog, and platelet-derived growth factor (PDGF) by an affinity-based delivery system incorporated into fibrin scaffolds. Embryoid bodies containing neural progenitors were formed from mouse ES cells, using a 4-/4+ retinoic acid treatment protocol, and then seeded inside fibrin scaffolds containing the drug delivery system. This delivery system was used to deliver various growth factor doses and combinations to the cells seeded inside the scaffolds. Controlled delivery of NT-3 and PDGF simultaneously increased the fraction of neural progenitors, neurons, and oligodendrocytes while decreasing the fraction of astrocytes obtained compared to control cultures seeded inside unmodified fibrin scaffolds with no growth factors present in the medium. These results demonstrate that such a strategy can be used to generate an engineered tissue for the potential treatment of spinal cord injury and could be extended to the study of differentiation in other tissues.

Emerging Biofabrication Strategies for Engineering Complex Tissue Constructs

The demand for organ transplantation and repair, coupled with a shortage of available donors, poses an urgent clinical need for the development of innovative treatment strategies for long-term repair and regeneration of injured or diseased tissues and organs. Bioengineering organs, by growing patient-derived cells in biomaterial scaffolds in the presence of pertinent physicochemical signals, provides a promising solution to meet this demand. However, recapitulating the structural and cytoarchitectural complexities of native tissues in vitro remains a significant challenge to be addressed. Through tremendous efforts over the past decade, several innovative biofabrication strategies have been developed to overcome these challenges. This review highlights recent work on emerging three-dimensional bioprinting and textile techniques, compares the advantages and shortcomings of these approaches, outlines the use of common biomaterials and advanced hybrid scaffolds, and describes several design considerations including the structural, physical, biological, and economical parameters that are crucial for the fabrication of functional, complex, engineered tissues. Finally, the applications of these biofabrication strategies in neural, skin, connective, and muscle tissue engineering are explored.

Biomaterial-based drug delivery systems for the controlled release of neurotrophic factors

This review highlights recent work on the use of biomaterial-based drug delivery systems to control the release of neurotrophic factors as a potential strategy for the treatment of neurological disorders. Examples of neurotrophic factors include the nerve growth factor, the glial cell line-derived neurotrophic factor, the brain-derived neurotrophic factor and neurotrophin-3. In particular, this review focuses on two methods of drug delivery: affinity-based and reservoir-based systems. We review the advantages and challenges associated with both types of drug delivery system and how these systems can be applied to neurological diseases and disorders. While a limited number of affinity-based delivery systems have been developed for the delivery of neurotrophic factors, we also examine the broad spectrum of reservoir-based delivery systems, including microspheres, electrospun nanofibers, hydrogels and combinations of these systems. Finally, conclusions are drawn about the current state of such drug delivery systems as applied to neural tissue engineering along with some thoughts on the future direction of the field.

Neural tissue engineering using embryonic and induced pluripotent stem cells

With the recent start of the first clinical trial evaluating a human embryonic stem cell-derived therapy for the treatment of acute spinal cord injury, it is important to review the current literature examining the use of embryonic stem cells for neural tissue engineering applications with a focus on diseases and disorders that affect the central nervous system. Embryonic stem cells exhibit pluripotency and thus can differentiate into any cell type found in the body, including those found in the nervous system. A range of studies have investigated how to direct the differentiation of embryonic cells into specific neural phenotypes using a variety of cues to achieve the goal of replacing diseased or damaged neural tissue. Additionally, the recent development of induced pluripotent stem cells provides an intriguing alternative to the use of human embryonic stem cell lines for these applications. This review will discuss relevant studies that have used embryonic stem cells to replicate the tissue found in the central nervous system as well as evaluate the potential of induced pluripotent stem cells for the aforementioned applications.

Development of a low bias method for characterizing viral populations using next generation sequencing technology.

Background With an estimated 38 million people worldwide currently infected with human immunodeficiency virus (HIV), and an additional 4.1 million people becoming infected each year, it is important to understand how this virus mutates and develops resistance in order to design successful therapies. Methodology/Principal Findings We report a novel experimental method for amplifying full-length HIV genomes without the use of sequence-specific primers for high throughput DNA sequencing, followed by assembly of full length viral genome sequences from the resulting large dataset. Illumina was chosen for sequencing due to its ability to provide greater coverage of the HIV genome compared to prior methods, allowing for more comprehensive characterization of the heterogeneity present in the HIV samples analyzed. Our novel amplification method in combination with Illumina sequencing was used to analyze two HIV populations: a homogenous HIV population based on the canonical NL4-3 strain and a heterogeneous viral population obtained from a HIV patients infected T cells. In addition, the resulting sequence was analyzed using a new computational approach to obtain a consensus sequence and several metrics of diversity. Significance This study demonstrates how a lower bias amplification method in combination with next generation DNA sequencing provides in-depth, complete coverage of the HIV genome, enabling a stronger characterization of the quasispecies present in a clinically relevant HIV population as well as future study of how HIV mutates in response to a selective pressure.

Fabrication of poly (ϵ-caprolactone) microfiber scaffolds with varying topography and mechanical properties for stem cell-based tissue engineering applications

Highly porous poly (ϵ-caprolactone) microfiber scaffolds can be fabricated using electrospinning for tissue engineering applications. Melt electrospinning produces such scaffolds by direct deposition of a polymer melt instead of dissolving the polymer in a solvent as performed during solution electrospinning. The objective of this study was to investigate the significant parameters associated with the melt electrospinning process that influence fiber diameter and scaffold morphology, including processing temperature, collection distance, applied, voltage and nozzle size. The mechanical properties of these microfiber scaffolds varied with microfiber diameter. Additionally, the porosity of scaffolds was determined by combining experimental data with mathematical modeling. To test the cytocompatability of these fibrous scaffolds, we seeded neural progenitors derived from murine R1 embryonic stem cell lines onto these scaffolds, where they could survive, migrate, and differentiate into neurons; demonstrating the potential of these melt electrospun scaffolds for tissue engineering applications.

Electrospun biomaterial scaffolds with varied topographies for neuronal differentiation of human-induced pluripotent stem cells

In this study, we investigated the effect of micro and nanoscale scaffold topography on promoting neuronal differentiation of human induced pluripotent stem cells (iPSCs) and directing the resulting neuronal outgrowth in an organized manner. We used melt electrospinning to fabricate poly (ε-caprolactone) (PCL) scaffolds with loop mesh and biaxial aligned microscale topographies. Biaxial aligned microscale scaffolds were further functionalized with retinoic acid releasing PCL nanofibers using solution electrospinning. These scaffolds were then seeded with neural progenitors derived from human iPSCs. We found that smaller diameter loop mesh scaffolds (43.7 ± 3.9 µm) induced higher expression of the neural markers Nestin and Pax6 compared to thicker diameter loop mesh scaffolds (85 ± 4 µm). The loop mesh and biaxial aligned scaffolds guided the neurite outgrowth of human iPSCs along the topographical features with the maximum neurite length of these cells being longer on the biaxial aligned scaffolds. Finally, our novel bimodal scaffolds also supported the neuronal differentiation of human iPSCs as they presented both physical and chemical cues to these cells, encouraging their differentiation. These results give insight into how physical and chemical cues can be used to engineer neural tissue.

Preparation of 3D fibrin scaffolds for stem cell culture applications.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue. A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside. This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach.