Immunometabolism reprogramming is involved in progression, induction and therapy of several diseases such as cancer, infections, autoimmune disorders. Notably, modulation of immunometabolism can be performed by delivery of cell permeable metabolites, enzymatic inhibitors or through gene editing. For example, metabolites provided systemically may improve immune cell function. Importantly, these strategies can be targeted toward both the innate and adaptive branch of the immune system to generate effector functions. In this presentation I will introduce strategies for modifying immunometabolism of dendritic cells, macrophages, and T cells for supporting the induction of immune responses in melanoma (skin cancer). Specifically, these projects are geared toward developing immunotherapies by directly modulating the metabolism of immune cells to develop robust immune responses in mouse models. Overall, these projects will demonstrate the importance of metabolite modulation in reprogramming the immune responses.

Mitochondria transplantation (MT) presents a promising approach for treating diseases associated with mitochondrial dysfunction, including neurodegenerative diseases, metabolic disorders, and conditions requiring tissue regeneration like spinal cord injuries (SCI). The ability to augment or replace damaged mitochondria with functional ones offers a novel therapeutic pathway, potentially circumventing the limitations of traditional treatments that merely alleviate symptoms without addressing underlying cellular energetics and dysfunction. Nonetheless, challenges such as maintaining mitochondrial viability in adverse environments and ensuring efficient cellular uptake hinder its efficacy. Our research addresses these obstacles by investigating the potential of Poly(N-isopropylacrylamide) grafted onto Hyaluronic Acid (HA-PNIPAm) hydrogels as a novel delivery system for MT therapeutics. PNIPAm is known for its exceptional lower critical solution temperature (LCST) that is close to human body temperature. On the other hand, HA is recognized for its unique biocompatibility and anti-inflammatory properties, existing in many parts of biological systems across the human body including central nervous system. This combination of co-polymer constitutes an ideal pairing to facilitate sustained and controlled mitochondrial delivery. We synthesized various compositions of grafted hydrogels, evaluating its phase transition and gelation properties using ultraviolet-visible spectroscopy and dynamic scanning calorimetry. Hydrogel erosion and mitochondrial release over time were studied using a closed system drug dissolution module and a fluorescence microplate reader. Lastly, seahorse assay was used to study released mitochondria respiration and viability after incubation in HA-PNIPAm hydrogel.

Stroke is the fifth most common cause of the death apart from other diseases. About 87% of strokes are ischemic in nature which caused by blocked blood flow in the brain. Cell based therapies have a great potential as an alternative strategy to overcome different obstacles we face in current available treatments. Our lab previously demonstrated that the use of fibroblasts nanotransfected with factors Etv2, Fli1, and Foxc2 (EFF) can drive angiogenesis followed by increased perfusion, vascularity, and neuronal cellularity with decreased glial scarring at the infarct when injected intracranially. However, this therapy requires intracranial injection, which is highly invasive. To explore an alternative route of administration, we evaluated the use of myeloid-derived suppressor cells (MDSCs) in place of fibroblasts. MDSCs are a group of immature myeloid cells that infiltrate into diseased tissue from systemic circulation in chronic inflammatory conditions like ischemic stroke and can also induce angiogenesis. We performed an in vitro experiment to examine the role of extracellular vehicles (EVs) secreted from the EFF transfected MDSCs in transmitting the EFF cargo to the surrounding damaged tissues by exposing endothelial cell to conditioned media from MDSC transfection experiments which contained EVs and soluble proteins. The results showed that the conditioned media exposed group formed a more robust endothelial network compared to control. After promising in vitro results, we tested MDSCs transfected with EFF on in vivo middle cerebral artery occlusion (MCAO) mouse model by injecting into systemic circulation retro-orbitally and confirmed the migration of MDSCs to the infarcted hemisphere. Current studies are ongoing to determine angiogenesis, perfusion at the stroke area in both MCAO and focal ischemia mouse models followed by behavioral study.

3D bioprinting is a versatile approach for fabricating tissues with complex geometries and features ranging from the cellular to organ length scales. However, there are disadvantages with current approaches, and the need to improve the structure and function of the engineered tissue constructs. To address this, we have developed approaches to build extracellular matrix (ECM) scaffolds that mimic the structure and composition of the heart and other tissues and organs. Termed Freeform Reversible Embedding of Suspended Hydrogels (FRESH), we can 3D bioprint collagen, fibrin, decellularized ECM, growth factors, and multiple cell types into complex 3D architectures. This includes organ-scale constructs based on patient-specific MRI scans that incorporate functional blood vessels and valves. Ongoing work is focused on cellularizing these constructs with human iPSC-derived cardiomyocytes to create beating cardiac tissues and extending these approaches to additional tissue and organ systems for in vitro disease modeling and in vivo regeneration.

Granular gels, comprising microgel subunits, present compelling prospects in biomedical applications, as they mimic the extracellular matrix and foster a conducive microenvironment for tissue regeneration. Their significance in regenerative medicine lies in enabling enhanced cell invasion crucial for promoting tissue growth. This study investigates the influence of diverse composite granular gel morphologies on early-stage of lymphatic tube formation. Using norbornene-modified hyaluronic acid (NorHA), microgels were fabricated through pipetting and vortexing techniques, subsequently forming granular gels via centrifugation. Evaluation of microgel generation methods showcased distinct granular hydrogel morphologies. Vortexing-produced gels exhibited higher porosity but wider microgel size distribution, leading to tighter packing compared to pipetting, resulting in smaller pores. This morphology yielded vortexing gels with a higher storage modulus and wider linear viscoelastic region (LVR) ranges due to increased interparticle contact. Morphological variances significantly affected lymphatic sprout formation. Pipetting gels facilitated linear-like sprouts, potentially forming lumen-like tubes. Conversely, vortexing gel's tight packing enhanced microparticle coverage. Quantitative RT-PCR data supported these observations, indicating a four-fold upregulation of LYVE-1 and PDPN markers in pipetting gels during early-stage lymphatic sprout formation. Furthermore, altering interstitial matrix composition affected sprout morphology, mirrored in gene expression levels. These findings emphasize the need for synchronized morphological and compositional tuning in granular hydrogel design to enhance lymphatic sprouting in composite granular hydrogels. Overall, this study provides new insights into 3D in vitro lymphatic tube formation, which is beneficial for basic lymphatic biology, as well as various approaches in lymphatic regeneration.

Peripheral nerve injuries (PNIs) can result in sensory/motor deficits and muscle loss if not treated immediately.  In this study, we describe the use of a novel technology, Tissue Nano-Transfection (TNT), that can deliver gene therapies to nerve/muscle aimed at improving the rate of peripheral nerve regeneration and healthy maintenance of injured nerve tissue and denervated muscle through neurogenic and vasculogenic reprogramming. Vasculogenic reprogramming factors Etv2, Fli1, and Foxc2 (EFF) were delivered into an injured mouse (C57BL/6) sciatic nerve using TNT to reprogram native fibroblast/Schwann cells into functioning endothelial cells; with the intent of increasing vascularity which should lead to axonal preservation.  Varying concentrations of these three EFF factors were assessed in their effectiveness of increasing vascularity in the sciatic nerve by comparing laser speckle imaging (LSI) perfusion in the nerves between all concentration groups and a control (pCMV6) 7 and 14 days after treatment.  Histology was also used for comparing the amount of blood vessels (CD31) and axons (NF) formed in the nerves between each group.  Another set of experiments were done using TNT delivery of pro-neuronal factors Ascl1, Brn2, and Myt1l (ABM) to help reinnervate leg muscle in cases of sciatic nerve transection.  Again, optimization of the concentration ratios is being done currently to determine the most efficient combination of factors for conversion of native myoblasts to induced neurons. At days 7 and 14 post-treatment using TNT of the EFF vasculogenic factors in crushed nerve tissue, there was a significant increase in blood perfusion in the sciatic nerve measured by LSI.  Histology also showed an increase in the number of sciatic nerve blood vessels in the EFF cocktail group where Foxc2 was doubled in concentration (1:1:2) at both 7 and 14 days post treatment. The conversion of muscle to induced neurons using ABM is still an ongoing study.