Waseem K. Raja

Self-Organizing 3D Human Neural Tissue Derived from Induced Pluripotent Stem Cells Recapitulate Alzheimer’s Disease Phenotypes

The dismal success rate of clinical trials for Alzheimer’s disease (AD) motivates us to develop model systems of AD pathology that have higher predictive validity. The advent of induced pluripotent stem cells (iPSCs) allows us to model pathology and study disease mechanisms directly in human neural cells from healthy individual as well as AD patients. However, two-dimensional culture systems do not recapitulate the complexity of neural tissue, and phenotypes such as extracellular protein aggregation are difficult to observe. We report brain organoids that use pluripotent stem cells derived from AD patients and recapitulate AD-like pathologies such as amyloid aggregation, hyperphosphorylated tau protein, and endosome abnormalities. These pathologies are observed in an age-dependent manner in organoids derived from multiple familial AD (fAD) patients harboring amyloid precursor protein (APP) duplication or presenilin1 (PSEN1) mutation, compared to controls. The incidence of AD pathology was consistent amongst several fAD lines, which carried different mutations. Although these are complex assemblies of neural tissue, they are also highly amenable to experimental manipulation. We find that treatment of patient-derived organoids with β- and γ-secretase inhibitors significantly reduces amyloid and tau pathology. Moreover, these results show the potential of this model system to greatly increase the translatability of pre-clinical drug discovery in AD.

Bioengineering embryonic stem cell microenvironments for exploring inhibitory effects on metastatic breast cancer cells.

The recreation of an in vitro microenvironment to understand and manipulate the proliferation and migration of invasive breast cancer cells may allow one to put a halt to their metastasis capacity. Invasive cancer cells have been linked to embryonic stem (ES) cells as they possess certain similar characteristics and gene signatures. Embryonic microenvironments have the potential to reprogram cancer cells into a less invasive phenotype and help elucidate tumorigenesis and metastasis. In this study, we explored the feasibility of reconstructing embryonic microenvironments using mouse ES cells cultured in alginate hydrogel and investigated the interactions of ES cells and highly invasive breast cancer cells in 2D, 2&1/2D, and 3D cultures. Results showed that mouse ES cells inhibited the growth and tumor spheroid formation of breast cancer cells. The mouse ES cell microenvironment was further constructed and optimized in 3D alginate hydrogel microbeads, and co-cultured with breast cancer cells. Migration analysis displayed a significant reduction in the average velocity and trajectory of breast cancer cell locomotion compared to control, suggesting that bioengineered mouse ES cell microenvironments inhibited the proliferation and migration of breast cancer cells. This study may act as a platform to open up new options to understand and harness tumor cell plasticity and develop therapeutics for metastatic breast cancer.

A new chemotaxis device for cell migration studies

This study presents the design and optimization for in vitro use of a new versatile chemotaxis device called the NANIVID (NANo IntraVital Imaging Device), developed using advanced nano/micro fabrication techniques. The device is fabricated using microphotolithographic techniques and two substrates are bonded together using a thin polymer layer creating a sealed device with one outlet. The main structure of the device consists of two Pyrex substrates: an etched chemoattractant reservoir and a top cover, with a final size of 0.2 × 2 × 3 mm. This reservoir contains a hydrogel blend with EGF which diffuses out through a small (∼9.10(3)μm(2)) outlet. This reservoir sustains a steady release of growth factor into the surrounding environment for several hours establishing a consistent concentration gradient from the device. The focus of this study was to design and optimize the new device for cell chemotaxis studies in breast cancer cells in cell culture. Our results show that we have created a flexible, cheap, miniature and autonomous chemotaxis device and demonstrate its usefulness in 2D and 3D cell culture. We also provide preliminary data for use of the device in vivo.

Transdermal Delivery Devices: Fabrication, Mechanics and Drug Release from Silk

Microneedles are a relatively simple, minimally invasive and painless approach to deliver drugs across the skin. However, there remain limitations with this approach because of the materials most commonly utilized for such systems. Silk protein, with tunable and biocompatibility properties, is a useful biomaterial to overcome the current limitations with microneedles. Silk devices preserve drug activity, offer superior mechanical properties and biocompatibility, can be tuned for biodegradability, and can be processed under aqueous, benign conditions. In the present work, the fabrication of dense microneedle arrays from silk with different drug release kinetics is reported. The mechanical properties of the microneedle patches are tuned by post-fabrication treatments or by loading the needles with silk microparticles, to increase capacity and mechanical strength. Drug release is further enhanced by the encapsulation of the drugs in the silk matrix and coating with a thin dissolvable drug layer. The microneedles are used on human cadaver skin and drugs are delivered successfully. The various attributes demonstrated suggest that silk-based microneedle devices can provide significant benefit as a platform material for transdermal drug delivery.

Ion electrodiffusion governs silk electrogelation

Silk electrogelation involves the transition of an aqueous silk fibroin solution to a gel state (E-gel) in the presence of an electric current. The process is based on local pH changes as a result of water electrolysis - generating H(+) and OH(-) ions at the (+) and (-) electrodes, respectively. Silk fibroin has a pI=4.2 and when local pH<pI, E-gel forms. An experimental system was constructed that allowed the measurement of E-gel growth and pH distribution for an applied current. To explain the observed rectangular pH profile of pH(gel)~4 surrounded by pH(silk-solution)~10, a finite-element ion electrodiffusion model was developed. The model relies on electrodiffusion of the generated H(+) and OH(-) ions. Initially, inputs into the model were the measured E-gel and voltage curves. The governing ion electrodiffusion equations were solved and the calculated pH matched the experimental pH profile, indicating that ion electrodiffusion dictates local pH changes and E-gel growth. Furthermore, the model predicted the constant currents (2mA and 3mA) necessary for two hypothetical E-gel growth curves and these results were then validated experimentally. The model thus shows how ion electrodiffusion governs the electrogelation process and also provides predictable outcomes for fundamental and practical E-gel applications.

Neural responses to electrical stimulation on patterned silk films

Peripheral nerve injury is a critical issue for patients with trauma. Following injury, incomplete axon regeneration or misguided axon innervation into tissue will result in loss of sensory and motor functions. The objective of this study was to examine axon outgrowth and axon alignment in response to surface patterning and electrical stimulation. To accomplish our objective, metal electrodes with dimensions of 1.5 mm × 4 cm, were sputter coated onto micropatterned silk protein films, with surface grooves 3.5 μm wide × 500 nm deep. P19 neurons were seeded on the patterned electronic silk films and stimulated at 120 mV, 1 kHz, for 45 min each day for 7 days. Responses were compared with neurons on flat electronic silk films, patterned silk films without stimulation, and flat silk films without stimulation. Significant alignment was found on the patterned film groups compared with the flat film groups. Axon outgrowth was greater (p < 0.05) on electronic films on days 5 and 7 compared with the unstimulated groups. In conclusion, electrical stimulation, at 120 mV, 1 kHz, for 45 min daily, in addition to surface patterning, of 3.5 μm wide × 500 nm deep grooves, offered control of nerve axon outgrowth and alignment.

Long term perfusion system supporting adipogenesis

Adipose tissue engineered models are needed to enhance our understanding of disease mechanisms and for soft tissue regenerative strategies. Perfusion systems generate more physiologically relevant and sustainable adipose tissue models, however adipocytes have unique properties that make culturing them in a perfusion environment challenging. In this paper we describe the methods involved in the development of two perfusion culture systems (2D and 3D) to test their applicability for long term in vitro adipogenic cultures. It was hypothesized that a silk protein biomaterial scaffold would provide a 3D framework, in combination with perfusion flow, to generate a more physiologically relevant sustainable adipose tissue engineered model than 2D cell culture. Consistent with other studies evaluating 2D and 3D culture systems for adipogenesis we found that both systems successfully model adipogenesis, however 3D culture systems were more robust, providing the mechanical structure required to contain the large, fragile adipocytes that were lost in 2D perfused culture systems. 3D perfusion also stimulated greater lipogenesis and lipolysis and resulted in decreased secretion of LDH compared to 2D perfusion. Regardless of culture configuration (2D or 3D) greater glycerol was secreted with the increased nutritional supply provided by perfusion of fresh media. These results are promising for adipose tissue engineering applications including long term cultures for studying disease mechanisms and regenerative approaches, where both acute (days to weeks) and chronic (weeks to months) cultivation are critical for useful insight.

Inhibition of p25/Cdk5 Attenuates Tauopathy in Mouse and iPSC Models of Frontotemporal Dementia

Increased p25, a proteolytic fragment of the regulatory subunit p35, is known to induce aberrant activity of cyclin-dependent kinase 5 (Cdk5), which is associated with neurodegenerative disorders, including Alzheimers disease. Previously, we showed that replacing endogenous p35 with the noncleavable mutant p35 (Δp35) attenuated amyloidosis and improved cognitive function in a familial Alzheimers disease mouse model. Here, to address the role of p25/Cdk5 in tauopathy, we generated double-transgenic mice by crossing mice overexpressing mutant human tau (P301S) with Δp35KI mice. We observed significant reduction of phosphorylated tau and its seeding activity in the brain of double transgenic mice compared with the P301S mice. Furthermore, synaptic loss and impaired LTP at hippocampal CA3 region of P301S mice were attenuated by blocking p25 generation. To further validate the role of p25/Cdk5 in tauopathy, we used frontotemporal dementia patient-derived induced pluripotent stem cells (iPSCs) carrying the Tau P301L mutation and generated P301L:Δp35KI isogenic iPSC lines using CRISPR/Cas9 genome editing. We created cerebral organoids from the isogenic iPSCs and found that blockade of p25 generation reduced levels of phosphorylated tau and increased expression of synaptophysin. Together, these data demonstrate a crucial role for p25/Cdk5 in mediating tau-associated pathology and suggest that inhibition of this kinase can remedy neurodegenerative processes in the presence of pathogenic tau mutation. SIGNIFICANCE STATEMENT Accumulation of p25 results in aberrant Cdk5 activation and induction of numerous pathological phenotypes, such as neuroinflammation, synaptic loss, Aβ accumulation, and tau hyperphosphorylation. However, it was not clear whether p25/Cdk5 activity is necessary for the progression of these pathological changes. We recently developed the Δp35KI transgenic mouse that is deficient in p25 generation and Cdk5 hyperactivation. In this study, we used this mouse model to elucidate the role of p25/Cdk5 in FTD mutant tau-mediated pathology. We also used a frontotemporal dementia patient-derived induced pluripotent stem cell carrying the Tau P301L mutation and generated isogenic lines in which p35 is replaced with noncleavable mutant Δp35. Our data suggest that p25/Cdk5 plays an important role in tauopathy in both mouse and human model systems.

Stability of Silk and Collagen Protein Materials in Space

Collagen and silk materials, in neat forms and as silica composites, were flown for 18 months on the International Space Station [Materials International Space Station Experiment (MISSE)-6] to assess the impact of space radiation on structure and function. As natural biomaterials, the impact of the space environment on films of these proteins was investigated to understand fundamental changes in structure and function related to the future utility in materials and medicine in space environments. About 15% of the film surfaces were etched by heavy ionizing particles such as atomic oxygen, the major component of the low-Earth orbit space environment. Unexpectedly, more than 80% of the silk and collagen materials were chemically crosslinked by space radiation. These findings are critical for designing next-generation biocompatible materials for contact with living systems in space environments, where the effects of heavy ionizing particles and other cosmic radiation need to be considered.

APOE4 Causes Widespread Molecular and Cellular Alterations Associated with Alzheimer’s Disease Phenotypes in Human iPSC-Derived Brain Cell Types

The apolipoprotein E4 (APOE4) variant is the single greatest genetic risk factor for sporadic Alzheimers disease (sAD). However, the cell-type-specific functions of APOE4 in relation to AD pathology remain understudied. Here, we utilize CRISPR/Cas9 and induced pluripotent stem cells (iPSCs) to examine APOE4 effects on human brain cell types. Transcriptional profiling identified hundreds of differentially expressed genes in each cell type, with the most affected involving synaptic function (neurons), lipid metabolism (astrocytes), and immune response (microglia-like cells). APOE4 neurons exhibited increased synapse number and elevated Aβ42 secretion relative to isogenic APOE3 cells while APOE4 astrocytes displayed impaired Aβ uptake and cholesterol accumulation. Notably, APOE4 microglia-like cells exhibited altered morphologies, which correlated with reduced Aβ phagocytosis. Consistently, converting APOE4 to APOE3 in brain cell types from sAD iPSCs was sufficient to attenuate multiple AD-related pathologies. Our study establishes a reference for human cell-type-specific changes associated with the APOE4 variant. VIDEO ABSTRACT.