Breast cancer often results in the removal of the breast, creating a need for replacement tissue. Tissue engineering offers the promise of generating such replacements by combining cells with biomaterial scaffolds and serves as an attractive potential alternative to current surgical repair methods. Such engineered tissues can also serve as important tools for drug screening and provide in vitro models for analysis. 3D bioprinting serves as an exciting technology with significant implications and applications in the field of tissue engineering. Here we review the work that has been undertaken in hopes of generating the recognized in-demand replacement breast tissue using different types of bioprinting. We then offer suggestions for future work needed to advance this field for both in vitro and in vivo applications.

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Recent work demonstrated that 3D fibrin scaffolds function as an effective substrate for engineering tissues from pluripotent stem cells. However, the rapid degradation rate of fibrin remains a major limitation when differentiating human pluripotent stem cells for tissue engineering applications. The addition of crosslinking agents, such as genipin, during the polymerization process increases scaffold stability while decreasing the degradation rate of fibrin. Genipin crosslinking alters the physical characteristics of the fibrin scaffolds, which influences the behaviour of the differentiating cells seeded inside. It also possesses neuritogenic and neuroprotective properties, making it particularly attractive for engineering neural tissue from pluripotent stem cells. Here we show that genipin enhances neuronal differentiation of neural progenitors derived from human induced pluripotent stem cells (hiPSCs) in 2D culture and genipin concentration influences the morphological and mechanical properties of 3D fibrin scaffolds. These mechanically stable genipin-crosslinked fibrin scaffolds support hiPSC-derived neural aggregates and induce neurite outgrowth while remaining intact for 2 weeks as opposed to 5 days for unmodified fibrin scaffolds.

Parkinson's disease (PD) is a neurodegenerative disorder that results when the dopaminergic neurons (DNs) present in the substantia nigra necessary for voluntary motor control are depleted, making patients with this disorder ideal candidates for cell replacement therapy. Human induced pluripotent stem cells (hiPSCs), obtained by reprogramming adult cells, possess the properties of pluripotency and immortality while enabling the possibility of patient-specific therapies. An effective cell therapy for PD requires an efficient, defined method of DN generation, as well as protection from the neuroinflammatory environment upon engraftment. Although similar in pluripotency to human embryonic stem cells (hESCs), hiPSCs differentiate less efficiently into neuronal subtypes. Previous work has shown that treatment with guggulsterone can efficiently differentiate hESCs into DNs. Our work shows that guggulsterone is able to derive DNs from hiPSCs with comparable efficiency, and furthermore, this differentiation can be achieved inside three-dimensional fibrin scaffolds that could enhance cell survival upon engraftment.

We have designed and validated a set of robust and non-toxic protocols for directly evaluating the properties of engineered neural tissue. These protocols characterize the mechanical properties of engineered neural tissues and measure their electrophysical activity. The protocols obtain elastic moduli of very soft fibrin hydrogel scaffolds and voltage readings from motor neuron cultures. Neurons require soft substrates to differentiate and mature, however measuring the elastic moduli of soft substrates remains difficult to accurately measure using standard protocols such as atomic force microscopy or shear rheology. Here we validate a direct method for acquiring elastic modulus of fibrin using a modified Hertz model for thin films. In this method, spherical indenters are positioned on top of the fibrin samples, generating an indentation depth that is then correlated with elastic modulus. Neurons function by transmitting electrical signals to one another and being able to assess the development of electrical signaling serves is an important verification step when engineering neural tissues. We then validated a protocol wherein the electrical activity of motor neural cultures is measured directly by a voltage sensitive dye and a microplate reader without causing damage to the cells. These protocols provide a non-destructive method for characterizing the mechanical and electrical properties of living spinal cord tissues using novel biosensing methods.

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Glioblastoma multiforme, a type of deadly brain cancer, originates most commonly from astrocytes found in the brain. Current multimodal treatments for glioblastoma minimally increase life expectancy, but significant advancements in prognosis have not been made in the past few decades. Here we investigate cellular reprogramming for inhibiting the aggressive proliferation of glioblastoma cells. Cellular reprogramming converts one differentiated cell type into another type based on the principles of regenerative medicine. In this study, we used cellular reprogramming to investigate whether small molecule mediated reprogramming could convert glioblastoma cells into neurons. We investigated a novel method for reprogramming U87MG human glioblastoma cells into terminally differentiated neurons using a small molecule cocktail consisting of forskolin, ISX9, CHIR99021 I-BET 151, and DAPT. Treating U87MG glioblastoma cells with this cocktail successfully reprogrammed the malignant cells into early neurons over 13 days. The reprogrammed cells displayed morphological and immunofluorescent characteristics associated with neuronal phenotypes. Genetic analysis revealed that the chemical cocktail upregulates the Ngn2, Ascl1, Brn2, and MAP2 genes, resulting in neuronal reprogramming. Furthermore, these cells displayed decreased viability and lacked the ability to form high numbers of tumor-like spheroids. Overall, this study validates the use of a novel small molecule cocktail for reprogramming glioblastoma into nonproliferating neurons.

Ongoing clinical trials are evaluating the use of stem cells as a way to treat traumatic spinal cord injury (SCI). However, the inhibitory environment present in the injured spinal cord makes it challenging to achieve the survival of these cells along with desired differentiation into the appropriate phenotypes necessary to regain function. Transplanting stem cells along with an instructive biomaterial scaffold can increase cell survival and improve differentiation efficiency. This study reviews the literature discussing different types of instructive biomaterial scaffolds developed for transplanting stem cells into the injured spinal cord. We have chosen to focus specifically on biomaterial scaffolds that direct the differentiation of neural stem cells and pluripotent stem cells since they offer the most promise for producing the cell phenotypes that could restore function after SCI. In terms of biomaterial scaffolds, this article reviews the literature associated with using hydrogels made from natural biomaterials and electrospun scaffolds for differentiating stem cells into neural phenotypes. It then presents new data showing how these different types of scaffolds can be combined for neural tissue engineering applications and provides directions for future studies.