Jason A Wertheim

Interests

Teaching

My teaching activities center on instructing medical trainees in the surgical care of transplant patients or those in need of organ replacement. Within my laboratory, I mentor undergraduate and graduate students in dissertation-level research and post-doctoral scientists in transitioning their research toward independence. I am also dedicated to helping faculty develop their own research programs and mentoring junior faculty in their career progression.

Research

Our research is focused on studying the development of bio-artificial liver, kidney and vascular tissue. My laboratory utilizes novel bioreactors, 3D printing, biomaterial scaffolds and organoid tissue models to study disease and develop cells for therapy or transplantation.

Courses

Scholarly Contributions

Journals/Publications

• Jones, T. M., Espitia, C. M., Chipollini, J., Lee, B. R., Wertheim, J. A., Carew, J. S., & Nawrocki, S. T. (2023). Targeting NEDDylation is a Novel Strategy to Attenuate Cisplatin-induced Nephrotoxicity. Cancer research communications, 3(2), 245-257.
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  Although cisplatin remains a backbone of standard-of-care chemotherapy regimens for a variety of malignancies, its use is often associated with severe dose-limiting toxicities (DLT). Notably, 30%-40% of patients treated with cisplatin-based regimens are forced to discontinue treatment after experiencing nephrotoxicity as a DLT. New approaches that simultaneously prevent renal toxicity while improving therapeutic response have the potential to make a major clinical impact for patients with multiple forms of cancer. Here, we report that pevonedistat (MLN4924), a first-in-class NEDDylation inhibitor, alleviates nephrotoxicity and synergistically enhances the efficacy of cisplatin in head and neck squamous cell carcinoma (HNSCC) models. We demonstrate that pevonedistat protects normal kidney cells from injury while enhancing the anticancer activity of cisplatin through a thioredoxin-interacting protein (TXNIP)-mediated mechanism. Cotreatment with pevonedistat and cisplatin yielded dramatic HNSCC tumor regression and long-term animal survival in 100% of treated mice. Importantly, the combination decreased nephrotoxicity induced by cisplatin monotherapy as evidenced by the blockade of kidney injury molecule-1 (KIM-1) and TXNIP expression, a reduction in collapsed glomeruli and necrotic cast formation, and inhibition of cisplatin-mediated animal weight loss. Inhibition of NEDDylation represents a novel strategy to prevent cisplatin-induced nephrotoxicity while simultaneously enhancing its anticancer activity through a redox-mediated mechanism.
• Koss, K. M., Sereda, T. J., Kumirov, V. K., & Wertheim, J. A. (2023). A class of peptides designed to replicate and enhance the Receptor for Hyaluronic Acid Mediated Motility binding domain. Acta biomaterialia, 167, 293-308.
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  The extra-cellular matrix (ECM) is a complex and rich microenvironment that is exposed and over-expressed across several injury or disease pathologies. Biomaterial therapeutics are often enriched with peptide binders to target the ECM with greater specificity. Hyaluronic acid (HA) is a major component of the ECM, yet to date, few HA adherent peptides have been discovered. A class of HA binding peptides was designed using B(X)B hyaluronic acid binding domains inspired from the helical face of the Receptor for Hyaluronic Acid Mediated Motility (RHAMM). These peptides were bioengineered using a custom alpha helical net method, allowing for the enrichment of multiple B(X)B domains and the optimization of contiguous and non-contiguous domain orientations. Unexpectedly, the molecules also exhibited the behaviour of nanofiber forming self-assembling peptides and were investigated for this characteristic. Ten 23-27 amino acid residue peptides were assessed. Simple molecular modelling was used to depict helical secondary structures. Binding assays were performed with varying concentrations (1-10 mg/mL) and extra-cellular matrices (HA, collagens I-IV, elastin, and Geltrex). Concentration mediated secondary structures were assessed using circular dichroism (CD), and higher order nanostructures were visualized using transmission electron microscopy (TEM). All peptides formed the initial apparent 3/alpha-helices, yet peptides 17x-3, 4, BHP3 and BHP4 were HA specific and potent (i.e., a significant effect) binders at increasing concentrations. These peptides shifted from apparent 3/alpha-helical structures at low concentration to beta-sheets at increasing concentration and also formed nanofibers which are noted as self-assembling structures. Several of the HA binding peptides outperformed our positive control (mPEP35) at 3-4 times higher concentrations, and were enhanced by self-assembly as each of these groups had observable nanofibers. STATEMENT OF SIGNIFICANCE: Specific biomolecules or peptides have played a crucial role in developing materials or systems to deliver key drugs and therapeutics to a broad spectrum of diseases and disorders. In these diseased tissues, cells build protein/sugar networks, which are uniquely exposed and great targets to deliver drugs to. Hyaluronic acid (HA) is involved in every stage of injury and is abundant in cancer. To date, only two HA specific peptides have been discovered. In our work, we have designed a way to model and trace binding regions as they appear on the face of a helical peptide. Using this method we have created a family of peptides enriched with HA binding domains that stick with 3-4 higher affinity than those previously discovered.
• Koss, K. M., Son, T., Li, C., Hao, Y., Cao, J., Churchward, M. A., Zhang, Z. J., Wertheim, J. A., Derda, R., & Todd, K. G. (2023). Toward discovering a novel family of peptides targeting neuroinflammatory states of brain microglia and astrocytes. Journal of neurochemistry.
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  Microglia are immune-derived cells critical to the development and healthy function of the brain and spinal cord, yet are implicated in the active pathology of many neuropsychiatric disorders. A range of functional phenotypes associated with the healthy brain or disease states has been suggested from in vivo work and were modeled in vitro as surveying, reactive, and primed sub-types of primary rat microglia and mixed microglia/astrocytes. It was hypothesized that the biomolecular profile of these cells undergoes a phenotypical change as well, and these functional phenotypes were explored for potential novel peptide binders using a custom 7 amino acid-presenting M13 phage library (SX7) to identify unique peptides that bind differentially to these respective cell types. Surveying glia were untreated, reactive were induced with a lipopolysaccharide treatment, recovery was modeled with a potent anti-inflammatory treatment dexamethasone, and priming was determined by subsequently challenging the cells with interferon gamma. Microglial function was profiled by determining the secretion of cytokines and nitric oxide, and expression of inducible nitric oxide synthase. After incubation with the SX7 phage library, populations of SX7-positive microglia and/or astrocytes were collected using fluorescence-activated cell sorting, SX7 phage was amplified in Escherichia coli culture, and phage DNA was sequenced via next-generation sequencing. Binding validation was done with synthesized peptides via in-cell westerns. Fifty-eight unique peptides were discovered, and their potential functions were assessed using a basic local alignment search tool. Peptides potentially originated from proteins ranging in function from a variety of supportive glial roles, including synapse support and pruning, to inflammatory incitement including cytokine and interleukin activation, and potential regulation in neurodegenerative and neuropsychiatric disorders.
• Nawrocki, S., Carew, J. S., Wertheim, J. A., Chipollini, J., Jones, T., Lee, B. R., Nawrocki, S., Carew, J. S., Wertheim, J. A., Chipollini, J., Jones, T., & Lee, B. R. (2023). Targeting NEDDylation is a novel strategy to attenuate cisplatin-induced nephrotoxicity. . American Association for Cancer Research.
• Sun, J., Ince, M. N., Abraham, C., Barrett, T., Brenner, L. A., Cong, Y., Dashti, R., Dudeja, P. K., Elliott, D., Griffith, T. S., Heeger, P. S., Hoisington, A., Irani, K., Kim, T. K., Kapur, N., Leventhal, J., Mohamadzadeh, M., Mutlu, E., Newberry, R., , Peled, J. U., et al. (2023). Modulating microbiome-immune axis in the deployment-related chronic diseases of Veterans: report of an expert meeting. Gut microbes, 15(2), 2267180.
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  The present report summarizes the United States Department of Veterans Affairs (VA) field-based meeting titled "Modulating microbiome-immune axis in the deployment-related chronic diseases of Veterans." Our Veteran patient population experiences a high incidence of service-related chronic physical and mental health problems, such as infection, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), various forms of hematological and non-hematological malignancies, neurologic conditions, end-stage organ failure, requiring transplantation, and posttraumatic stress disorder (PTSD). We report the views of a group of scientists who focus on the current state of scientific knowledge elucidating the mechanisms underlying the aforementioned disorders, novel therapeutic targets, and development of new approaches for clinical intervention. In conclusion, we dovetailed on four research areas of interest: 1) microbiome interaction with immune cells after hematopoietic cell and/or solid organ transplantation, graft-versus-host disease (GVHD) and graft rejection, 2) intestinal inflammation and its modification in IBD and cancer, 3) microbiome-neuron-immunity interplay in mental and physical health, and 4) microbiome-micronutrient-immune interactions during homeostasis and infectious diseases. At this VA field-based meeting, we proposed to explore a multi-disciplinary, multi-institutional, collaborative strategy to initiate a roadmap, specifically focusing on host microbiome-immune interactions among those with service-related chronic diseases to potentially identify novel and translatable therapeutic targets.
• Hekman, K. E., Koss, K. M., Ivancic, D. Z., He, C., & Wertheim, J. A. (2022). Autophagy Enhances Longevity of Induced Pluripotent Stem Cell-Derived Endothelium via mTOR-Independent ULK1 Kinase. Stem cells translational medicine, 11(11), 1151-1164.
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  Stem cells are enabling an improved understanding of the peripheral arterial disease, and patient-specific stem cell-derived endothelial cells (ECs) present major advantages as a therapeutic modality. However, applications of patient-specific induced pluripotent stem cell (iPSC)-derived ECs are limited by rapid loss of mature cellular function in culture. We hypothesized that changes in autophagy impact the phenotype and cellular proliferation of iPSC-ECs. Endothelial cells were differentiated from distinct induced pluripotent stem cell lines in 2D culture and purified for CD144 positive cells. Autophagy, mitochondrial morphology, and proliferation were characterized during differentiation and over serial passages in culture. We found that autophagy activity was stimulated during differentiation but stagnated in mature iPSC-ECs. Mitochondria remodeled through mitophagy during differentiation and demonstrated increasing membrane potential and mass through serial passages; however, these plateaued, coinciding with decreased proliferation. To evaluate for oxidative damage, iPSC-ECs were alternatively grown under hypoxic culture conditions; however, hypoxia only transiently improved the proliferation. Stimulating mTOR-independent ULK1-mediated autophagy with a plant derivative AMP kinase activator Rg2 significantly improved proliferative capacity of iPSC-ECs over multiple passages. Therefore, autophagy, a known mediator of longevity, played an active role in remodeling mitochondria during maturation from pluripotency to a terminally differentiated state. Autophagy failed to compensate for increasing mitochondrial mass over serial passages, which correlated with loss of proliferation in iPSC-ECs. Stimulating ULK1-kinase-driven autophagy conferred improved proliferation and longevity over multiple passages in culture. This represents a novel approach to overcoming a major barrier limiting the use of iPSC-ECs for clinical and research applications.
• Naved, B. A., Bonventre, J. V., Hubbell, J. A., Hukriede, N. A., Humphreys, B. D., Kesselman, C., Valerius, M. T., McMahon, A. P., Shankland, S. J., Wertheim, J. A., White, M. J., de Caestecker, M. P., & Drummond, I. A. (2022). Kidney repair and regeneration: perspectives of the NIDDK (Re)Building a Kidney consortium. Kidney international, 101(5), 845-853.
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  Acute kidney injury impacts ∼13.3 million individuals and causes ∼1.7 million deaths per year globally. Numerous injury pathways contribute to acute kidney injury, including cell cycle arrest, senescence, inflammation, mitochondrial dysfunction, and endothelial injury and dysfunction, and can lead to chronic inflammation and fibrosis. However, factors enabling productive repair versus nonproductive, persistent injury states remain less understood. The (Re)Building a Kidney (RBK) consortium is a National Institute of Diabetes and Digestive and Kidney Diseases consortium focused on both endogenous kidney repair mechanisms and the generation of new kidney tissue. This short review provides an update on RBK studies of endogenous nephron repair, addressing the following questions: (i) What is productive nephron repair? (ii) What are the cellular sources and drivers of repair? and (iii) How do RBK studies promote development of therapeutics? Also, we provide a guide to RBK's open access data hub for accessing, downloading, and further analyzing data sets.
• Das, P., DiVito, M. D., Wertheim, J. A., & Tan, L. P. (2021). Bioengineered 3D electrospun nanofibrous scaffold with human liver cells to study alcoholic liver disease in vitro. Integrative biology : quantitative biosciences from nano to macro, 13(7), 184-195.
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  Alcohol injury induces hepatic fibrosis which gradually progresses to cirrhosis, sometimes may lead to liver cancer. Animal models are less efficient in mimicking responses of human liver cells, whereas in vitro models discussed so far are majorly based on rodent cells. In this work, a coculture of primary human hepatocytes (PHHs) with LX-2 cells was established on the unmodified (C:F_0:0), collagen-I modified (C:F_1:0), fibronectin modified (C:F_0:1) and 3:1 collagen-I to fibronectin modified (C:F_3:1) 3D electrospun fibrous scaffolds. The effect of alcohol injury was evaluated on this cell-scaffold model at 0-40 μl/ml alcohol concentrations over 14 days of culture period by using the gold standard sandwich culture as the control. Among all the culture groups, C:F_3:1 scaffold was able to maintain translational and transcriptional properties of human liver cells at all concentrations of alcohol treatment. The study reveals that, PHHs on C:F_3:1 were able to maintain ~4-fold and ~1.6-fold higher secretion of albumin than the gold standard sandwich culture on Day 3 and Day 7, respectively. When treated with alcohol, at concentrations of 20 and 40 μl/ml, albumin secretion was also observed to be higher (~2-fold) when compared to the gold standard sandwich culture. Again as expected, in C:F_3:1 culture group on 40 μl/ml alcohol treatment, albumin gene expression decreased by ~2-fold due to alcohol toxicity, whereas CYP2C9, CYP3A4, CYP2E1 and CYP1A2 gene expressions upregulated by ~3.5, ~~4, ~5 and ~15-fold, respectively in response to the alcohol injury. LX-2 cells also acquire more quiescent phenotype on C:F_3:1 scaffolds when compared to the gold standard sandwich culture upon alcohol treatment. Thus, C:F_3:1 scaffold with human liver cells was established as the potential platform to scan alcohol toxicity at varied alcohol concentrations. Thus, it can pave a promising path not only to support functional healthy human liver cells for liver tissue engineering but also to examine potential drugs to study the progression or inhibition of alcoholic liver fibrosis in vitro.
• Gupta, A. K., Ivancic, D. Z., Naved, B. A., Wertheim, J. A., & Oxburgh, L. (2021). An efficient method to generate kidney organoids at the air-liquid interface. Journal of biological methods, 8(2), e150.
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  The prevalence of kidney dysfunction continues to increase worldwide, driving the need to develop transplantable renal tissues. The kidney develops from four major renal progenitor populations: nephron epithelial, ureteric epithelial, interstitial and endothelial progenitors. Methods have been developed to generate kidney organoids but few or dispersed tubular clusters within the organoids hamper its use in regenerative applications. Here, we describe a detailed protocol of asynchronous mixing of kidney progenitors using organotypic culture conditions to generate kidney organoids tightly packed with tubular clusters and major renal structures including endothelial network and functional proximal tubules. This protocol provides guidance in the culture of human embryonic stem cells from a National Institute of Health-approved line and their directed differentiation into kidney organoids. Our 18-day protocol provides a rapid method to generate kidney organoids that facilitate the study of different nephrological events including tissue development, disease modeling and chemical screening. However, further studies are required to optimize the protocol to generate additional renal-specific cell types, interconnected nephron segments and physiologically functional renal tissues.
• Parsons, R. F., Baquerizo, A., Kirchner, V. A., Malek, S., Desai, C. S., Schenk, A., Finger, E. B., Brennan, T. V., Parekh, K. R., MacConmara, M., Brayman, K., Fair, J., Wertheim, J. A., & , A. C. (2021). Challenges, highlights, and opportunities in cellular transplantation: A white paper of the current landscape. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons.
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  Although cellular transplantation remains a relatively small field compared to solid organ transplantation, the prospects for advancement in basic science and clinical care remain bountiful. In this review, notable historical events and the current landscape of the field of cellular transplantation are reviewed with an emphasis on islets (allo- and xeno-), hepatocytes (including bioartificial liver), adoptive regulatory immunotherapy, and stem cells (SCs, specifically endogenous organ-specific and mesenchymal). Also, the nascent but rapidly evolving field of three-dimensional bioprinting is highlighted, including its major processing steps and latest achievements. To reach its full potential where cellular transplants are a more viable alternative than solid organ transplants, fundamental change in how the field is regulated and advanced is needed. Greater public and private investment in the development of cellular transplantation is required. Furthermore, consistent with the call of multiple national transplant societies for allo-islet transplants, the oversight of cellular transplants should mirror that of solid organ transplants and not be classified under the unsustainable, outdated model that requires licensing as a drug with the Food and Drug Administration. Cellular transplantation has the potential to bring profound benefit through progress in bioengineering and regenerative medicine, limiting immunosuppression-related toxicity, and providing markedly reduced surgical morbidity.
• Wysocki, J., Ye, M., Hassler, L., Gupta, A. K., Wang, Y., Nicoleascu, V., Randall, G., Wertheim, J. A., & Batlle, D. (2021). A Novel Soluble ACE2 Variant with Prolonged Duration of Action Neutralizes SARS-CoV-2 Infection in Human Kidney Organoids. Journal of the American Society of Nephrology : JASN.
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  There is an urgent need for approaches to prevent and treat SARS-CoV-2 infection. Administration of soluble ACE2 protein acting as a decoy to bind to SARS-CoV-2 should limit viral uptake mediated by binding to membrane-bound full-length ACE2, and further therapeutic benefit should result from ensuring enzymatic ACE2 activity to affected organs in patients with COVID-19.
• Das, P., DiVito, M. D., Wertheim, J. A., & Tan, L. P. (2020). Collagen-I and fibronectin modified three-dimensional electrospun PLGA scaffolds for long-term in vitro maintenance of functional hepatocytes. Materials science & engineering. C, Materials for biological applications, 111, 110723.
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  Extracellular matrix (ECM) proteins are important regulators of cellular behaviour in the native environment. It has been established that ECM proteins - collagen-I and fibronectin - are present in liver extracellular matrix and regulate specific functions of primary hepatocytes. While scaffolds grafted with the individual ECM protein have shown support for hepatocyte functional properties in vitro, the synergistic effects of both ECM proteins remain to be explored. Such studies are even more limited when three-dimensional (3D) scaffolds are involved. In the current work, the fabrication of a series of highly porous poly(lactic-co-glycolic acid) (PLGA) 3D electrospun scaffolds, simultaneously modified with both collagen-I and fibronectin, has been demonstrated. Different ratios of collagen-I to fibronectin were optimized to study the synergistic effects of the proteins in supporting the viability and functional properties of Huh-7.5 cells. The ratio of collagen-I to fibronectin at 3:1 was found to provide the most efficient chemisorption on the 3D scaffolds. At this ratio, the total protein content that can be grafted on the scaffolds was the highest and the most homogeous. This led to remarkable enhancement of cell seeding efficiency as well as proliferation. Most importantly, liver specific genes such as albumin and cytochrome P450 enzymes i.e. CYP3A4 and CYP3A7 were significantly upregulated by ~12.5, 7 and 4.5 fold respectively, as compared to unmodified PLGA scaffolds after 28 days of culture. Compared to single-protein modified scaffolds, scaffolds modified with 3:1 collagen to fibronectin result in a rise of the albumin gene expression of cultured cells by ~8 to 10 fold, whereas CYP3A4 gene expression improved by ~5 to 7 fold and CYP3A7 gene expression improved by ~4 to 4.5 fold after a long culture period of 28 days. Albumin secretion was improved by ~4 fold compared to unmodified PLGA scaffolds, ~3 fold compared to collagen-I modified culture groups and ~2 fold compared to fibronectin modified culture groups. The multi-protein modified scaffolds, at the optimum ratio, were able to significantly enhance functional properties of the liver cells. This simple yet highly functioning platform would be useful for in vitro culture of liver cells for both drug screening as well as translational purposes.
• Ferrer, J. R., Sinegra, A. J., Ivancic, D., Yeap, X. Y., Qiu, L., Wang, J. J., Zhang, Z. J., Wertheim, J. A., & Mirkin, C. A. (2020). Structure-Dependent Biodistribution of Liposomal Spherical Nucleic Acids. ACS nano, 14(2), 1682-1693.
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  Spherical nucleic acids (SNAs) are a class of nanomaterials with a structure defined by a radial distribution of densely packed, short DNA or RNA sequences around a nanoparticle core. This structure allows SNAs to rapidly enter mammalian cells, protects the displayed oligonucleotides from nuclease degradation, and enables co-delivery of other drug cargoes. Here, we investigate the biodistribution of liposomal spherical nucleic acid (LSNA) conjugates, SNA architectures formed from liposome templates and DNA modified with hydrophobic end groups (tails). We compared linear DNA with two types of LSNAs that differ only by the affinity of the modified DNA sequence for the liposome template. We use single-stranded DNA (ssDNA) terminated with either a low-affinity cholesterol tail (CHOL-LSNA) or a high-affinity diacylglycerol lipid tail (DPPE-LSNA). Both LSNA formulations, independent of DNA conjugation, reduce the inflammatory cytokine response to intravenously administered DNA. The difference in the affinity for the liposome template significantly affects DNA biodistribution. DNA from CHOL-LSNAs accumulates in greater amounts in the lungs than DNA from DPPE-LSNAs. In contrast, DNA from DPPE-LSNAs exhibits greater accumulation in the kidneys. Flow cytometry and fluorescence microscopy of tissue sections indicate that different cell populations-immune and nonimmune-sequester the DNA depending upon the chemical makeup of the LSNA. Taken together, these data suggest that the chemical structure of the LSNAs represents an opportunity to direct the biodistribution of nucleic acids to major tissues outside of the liver.
• Kumar Gupta, A., Sarkar, P., Wertheim, J. A., Pan, X., Carroll, T. J., & Oxburgh, L. (2020). Asynchronous mixing of kidney progenitor cells potentiates nephrogenesis in organoids. Communications biology, 3(1), 231.
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  A fundamental challenge in emulating kidney tissue formation through directed differentiation of human pluripotent stem cells is that kidney development is iterative, and to reproduce the asynchronous mix of differentiation states found in the fetal kidney we combined cells differentiated at different times in the same organoid. Asynchronous mixing promoted nephrogenesis, and heterochronic organoids were well vascularized when engrafted under the kidney capsule. Micro-CT and injection of a circulating vascular marker demonstrated that engrafted kidney tissue was connected to the systemic circulation by 2 weeks after engraftment. Proximal tubule glucose uptake was confirmed, but despite these promising measures of graft function, overgrowth of stromal cells prevented long-term study. We propose that this is a technical feature of the engraftment procedure rather than a specific shortcoming of the directed differentiation because kidney organoids derived from primary cells and whole embryonic kidneys develop similar stromal overgrowth when engrafted under the kidney capsule.
• Zheng, F., Tully, A., Koss, K. M., Zhang, X., Qiu, L., Wang, J. J., Naved, B. A., Ivancic, D. Z., Mathew, J. M., Wertheim, J. A., & Zhang, Z. J. (2020). Taking the Next Step: a Neural Coaptation Orthotopic Hind Limb Transplant Model to Maximize Functional Recovery in Rat. Journal of visualized experiments : JoVE.
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  Limb transplant in particular and vascularized composite allotransplant (VCA) in general have wide therapeutic promise that have been stymied by current limitations in immunosuppression and functional neuromotor recovery. Many animal models have been developed for studying unique features of VCA, but here we present a robust reproducible model of orthotopic hind limb transplant in rats designed to simultaneously investigate both aspects of current VCA limitation: immunosuppression strategies and functional neuromotor recovery. At the core of the model rests a commitment to meticulous, time-tested microsurgical techniques such as hand sewn vascular anastomoses and hand sewn neural coaptation of the femoral nerve and the sciatic nerve. This approach yields durable limb reconstructions that allow for longer lived animals capable of rehabilitation, resumption of daily activities, and functional testing. With short-term treatment of conventional immunosuppressive agents, allotransplanted animals survived up to 70 days post-transplant, and isotransplanted animals provide long lived controls beyond 200 days post-operatively. Evidence of neurologic functional recovery is present by 30 days post operatively. This model not only provides a useful platform for interrogating immunological questions unique to VCA and nerve regeneration, but also allows for in vivo testing of new therapeutic strategies specifically tailored for VCA.
• Ferrer, J. R., Wertheim, J. A., & Mirkin, C. A. (2019). Dual Toll-Like Receptor Targeting Liposomal Spherical Nucleic Acids. Bioconjugate chemistry, 30(3), 944-951.
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  Liposomal spherical nucleic acids (LSNAs) are a class of nanomaterial used broadly for biomedical applications. Their intrinsic capacity to rapidly enter cells and engage cell surface and intracellular ligands stems from their unique three-dimensional architecture, which consists of densely packed and uniformly oriented oligonucleotides on the surface of a liposomal core. Such structures are promising for therapeutics because they can carry chemical cargo within the lipid core in addition to the nucleic acids that define them, in principle enabling delivery of multiple signals to a single cell. On the basis of these traits, we have designed novel dual-targeting LSNAs that deliver a nucleic acid specific for TLR9 inhibition and a small molecule (TAK-242) that inhibits TLR4. Toll-like receptors (TLRs) play a large role in pathogen recognition and disease initiation, and TLR subtypes are differentially located within the lipid membranes of the cell surface and within intracellular endosomes. Oftentimes, in acute or chronic inflammatory conditions, multiple TLRs are activated, leading to stimulation of distinct, and sometimes overlapping, downstream pathways. As such, these inflammatory conditions may respond to attenuation of more than one initiating receptor. We show that dual targeting LSNAs, comprised of unilamellar liposomal cores, the INH-18 oligonucleotide sequence, and TAK-242 robustly inhibit TLR-9 and TLR-4 respectively, in engineered TLR reporter cells and primary mouse peritoneal macrophages. Importantly, the LSNAs exhibit up to a 10- and a 1000-fold increase, respectively, in TLR inhibition compared to the linear sequence and TAK-242 alone. Moreover, the timing of delivery is shown to be a critical factor in effecting TLR-inhibition, with near-complete TLR-4 inhibition occurring when cells were pretreated with SNAs for 4 h prior to stimulation. The most pronounced effect observed from this approach is the benefit of delivering the small molecule within the SNA via the receptor-mediated internalization pathway common to SNAs.
• Su, J., Satchell, S. C., Wertheim, J. A., & Shah, R. N. (2019). Poly(ethylene glycol)-crosslinked gelatin hydrogel substrates with conjugated bioactive peptides influence endothelial cell behavior. Biomaterials, 201, 99-112.
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  The basement membrane is a specialized extracellular matrix substrate responsible for support and maintenance of epithelial and endothelial structures. Engineered basement membrane-like hydrogel systems have the potential to advance understanding of cell-cell and cell-matrix interactions by allowing precise tuning of the substrate or matrix biochemical and biophysical properties. In this investigation, we developed tunable hydrogel substrates with conjugated bioactive peptides to modulate cell binding and growth factor signaling by endothelial cells. Hydrogels were formed by employing a poly(ethylene glycol) crosslinker to covalently crosslink gelatin polymers and simultaneously conjugate laminin-derived YIGSR peptides or vascular endothelial growth factor (VEGF)-mimetic QK peptides to the gelatin. Rheological characterization revealed rapid formation of hydrogels with similar stiffnesses across tested formulations, and swelling analysis demonstrated dependency on peptide and crosslinker concentrations in hydrogels. Levels of phosphorylated VEGF Receptor 2 in cells cultured on hydrogel substrates revealed that while human umbilical vein endothelial cells (HUVECs) responded to both soluble and conjugated forms of the QK peptide, conditionally-immortalized human glomerular endothelial cells (GEnCs) only responded to the conjugated presentation of the peptide. Furthermore, whereas HUVECs exhibited greatest upregulation in gene expression when cultured on YIGSR- and QK-conjugated hydrogel substrates after 5 days, GEnCs exhibited greatest upregulation when cultured on Matrigel control substrates at the same time point. These results indicate that conjugation of bioactive peptides to these hydrogel substrates significantly influenced endothelial cell behavior in cultures but with differential responses between HUVECs and GEnCs.
• Brown, J. H., Das, P., DiVito, M. D., Ivancic, D., Tan, L. P., & Wertheim, J. A. (2018). Nanofibrous PLGA electrospun scaffolds modified with type I collagen influence hepatocyte function and support viability in vitro. Acta biomaterialia, 73, 217-227.
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  A major challenge of maintaining primary hepatocytes in vitro is progressive loss of hepatocyte-specific functions, such as protein synthesis and cytochrome P450 (CYP450) catalytic activity. We developed a three-dimensional (3D) nanofibrous scaffold made from poly(l-lactide-co-glycolide) (PLGA) polymer using a newly optimized wet electrospinning technique that resulted in a highly porous structure that accommodated inclusion of primary human hepatocytes. Extracellular matrix (ECM) proteins (type I collagen or fibronectin) at varying concentrations were chemically linked to electrospun PLGA using amine coupling to develop an in vitro culture system containing the minimal essential ECM components of the liver micro-environment that preserve hepatocyte function in vitro. Cell-laden nanofiber scaffolds were tested in vitro to maintain hepatocyte function over a two-week period. Incorporation of type I collagen onto PLGA scaffolds (PLGA-C: 100 µg/mL) led to 10-fold greater albumin secretion, 4-fold higher urea synthesis, and elevated transcription of hepatocyte-specific CYP450 genes (CYP3A4, 3.5-fold increase and CYP2C9, 3-fold increase) in primary human hepatocytes compared to the same cells grown within unmodified PLGA scaffolds over two weeks. These indices, measured using collagen-bonded scaffolds, were also higher than scaffolds coupled to fibronectin or an ECM control sandwich culture composed of type I collagen and Matrigel. Induction of CYP2C9 activity was also higher in these same type I collagen PLGA scaffolds compared to other ECM-modified or unmodified PLGA constructs and was equivalent to the ECM control at 7 days. Together, we demonstrate a minimalist ECM-based 3D synthetic scaffold that accommodates primary human hepatocyte inclusion into the matrix, maintains long-term in vitro survival and stimulates function, which can be attributed to coupling of type I collagen.
• Friedrich, E. E., Lanier, S. T., Niknam-Bienia, S., Arenas, G. A., Rajendran, D., Wertheim, J. A., & Galiano, R. D. (2018). Residual sodium dodecyl sulfate in decellularized muscle matrices leads to fibroblast activation in vitro and foreign body response in vivo. Journal of tissue engineering and regenerative medicine, 12(3), e1704-e1715.
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  Detergents such as sodium dodecyl sulfate (SDS) are commonly used to extract cells from tissues in a process called "decellularization". Residual SDS is difficult to completely remove and may lead to an undesirable host response towards an implanted biomaterial. In this study, we developed a modification for SDS cell extraction from muscle equally efficient to previous methods but leading to significantly less residual SDS remnants in the matrices. Muscle-derived matrices were prepared via 2 SDS-based decellularization methods, which led to removal of either 81.4% or 98.4% of the SDS. In vitro, matrices were seeded with thp1 macrophages and primary human foreskin fibroblasts. By Day 2, both matrices demonstrated similar macrophage polarization; however, fibroblasts cultured on matrices with greater residual SDS expressed higher levels of mRNA associated with fibroblast activation: α-smooth muscle actin and connective tissue growth factor. In vivo, Collagen I gels spiked with increasing concentrations of SDS displayed a corresponding decrease in cell infiltration when implanted subcutaneously in rats after 4 days. Finally, as a model for muscle regeneration, matrices produced by each method were implanted in rat latissimus dorsi defects. At POD 30 greater levels of IL-1β mRNA were present in defects treated with matrices containing higher levels of SDS, indicating a more severe inflammatory response. Although matrices containing higher levels of residual SDS became encapsulated by POD 30 and showed evidence of a foreign body response, matrices with the lower levels of SDS integrated into the defect area with lower levels of inflammatory and fibrosis-related gene expression.
• Sawicki, K. T., Chang, H. C., Shapiro, J. S., Bayeva, M., De Jesus, A., Finck, B. N., Wertheim, J. A., Blackshear, P. J., & Ardehali, H. (2018). Hepatic tristetraprolin promotes insulin resistance through RNA destabilization of FGF21. JCI insight, 3(13).
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  The role of posttranscriptional metabolic gene regulatory programs in diabetes is not well understood. Here, we show that the RNA-binding protein tristetraprolin (TTP) is reduced in the livers of diabetic mice and humans and is transcriptionally induced in response to insulin treatment in murine livers in vitro and in vivo. Liver-specific Ttp-KO (lsTtp-KO) mice challenged with high-fat diet (HFD) have improved glucose tolerance and peripheral insulin sensitivity compared with littermate controls. Analysis of secreted hepatic factors demonstrated that fibroblast growth factor 21 (FGF21) is posttranscriptionally repressed by TTP. Consistent with increased FGF21, lsTtp-KO mice fed HFD have increased brown fat activation, peripheral tissue glucose uptake, and adiponectin production compared with littermate controls. Downregulation of hepatic Fgf21 via an adeno-associated virus-driven shRNA in mice fed HFD reverses the insulin-sensitizing effects of hepatic Ttp deletion. Thus, hepatic TTP posttranscriptionally regulates systemic insulin sensitivity in diabetes through liver-derived FGF21.
• Su, J., Satchell, S. C., Shah, R. N., & Wertheim, J. A. (2018). Kidney decellularized extracellular matrix hydrogels: Rheological characterization and human glomerular endothelial cell response to encapsulation. Journal of biomedical materials research. Part A, 106(9), 2448-2462.
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  Hydrogels, highly-hydrated crosslinked polymer networks, closely mimic the microenvironment of native extracellular matrix (ECM) and thus present as ideal platforms for three-dimensional cell culture. Hydrogels derived from tissue- and organ-specific decellularized ECM (dECM) may retain bioactive signaling cues from the native tissue or organ that could in turn modulate cell-material interactions and response. In this study, we demonstrate that porcine kidney dECM can be processed to form hydrogels suitable for cell culture and encapsulation studies. Scanning electron micrographs of hydrogels demonstrated a fibrous ultrastructure with interconnected pores, and rheological analysis revealed rapid gelation times with shear moduli dependent upon the protein concentration of the hydrogels. Conditionally-immortalized human glomerular endothelial cells (GEnCs) cultured on top of or encapsulated within hydrogels exhibited high cell viability and proliferation over a one-week culture period. However, gene expression analysis of GEnCs encapsulated within kidney dECM hydrogels revealed significantly lower expression of several relevant genes of interest compared to those encapsulated within hydrogels composed of only purified collagen I. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2448-2462, 2018.
• Giwa, S., Lewis, J. K., Alvarez, L., Langer, R., Roth, A. E., Church, G. M., Markmann, J. F., Sachs, D. H., Chandraker, A., Wertheim, J. A., Rothblatt, M., Boyden, E. S., Eidbo, E., Lee, W. P., Pomahac, B., Brandacher, G., Weinstock, D. M., Elliott, G., Nelson, D., , Acker, J. P., et al. (2017). The promise of organ and tissue preservation to transform medicine. Nature biotechnology, 35(6), 530-542.
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  The ability to replace organs and tissues on demand could save or improve millions of lives each year globally and create public health benefits on par with curing cancer. Unmet needs for organ and tissue preservation place enormous logistical limitations on transplantation, regenerative medicine, drug discovery, and a variety of rapidly advancing areas spanning biomedicine. A growing coalition of researchers, clinicians, advocacy organizations, academic institutions, and other stakeholders has assembled to address the unmet need for preservation advances, outlining remaining challenges and identifying areas of underinvestment and untapped opportunities. Meanwhile, recent discoveries provide proofs of principle for breakthroughs in a family of research areas surrounding biopreservation. These developments indicate that a new paradigm, integrating multiple existing preservation approaches and new technologies that have flourished in the past 10 years, could transform preservation research. Capitalizing on these opportunities will require engagement across many research areas and stakeholder groups. A coordinated effort is needed to expedite preservation advances that can transform several areas of medicine and medical science.
• Jiang, B., Suen, R., Wang, J. J., Zhang, Z. J., Wertheim, J. A., & Ameer, G. A. (2017). Vascular scaffolds with enhanced antioxidant activity inhibit graft calcification. Biomaterials, 144, 166-175.
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  There is a need for off-the-shelf, small-diameter vascular grafts that are safe and exhibit high long-term patency. Decellularized tissues can potentially be used as vascular grafts; however, thrombogenic and unpredictable remodeling properties such as intimal hyperplasia and calcification are concerns that hinder their clinical use. The objective of this study was to investigate the long-term function and remodeling of extracellular matrix (ECM)-based vascular grafts composited with antioxidant poly(1, 8-octamethylene-citrate-co-cysteine) (POCC) with or without immobilized heparin. Rat aortas were decellularized to create the following vascular grafts: 1) ECM hybridized with POCC (Poly-ECM), 2) Poly-ECM subsequently functionalized with heparin (Poly-ECM-Hep), and 3) non-modified vascular ECM. Grafts were evaluated as interposition grafts in the abdominal aorta of adult rats at three months. All grafts displayed antioxidant activity, were patent, and exhibited minimal intramural cell infiltration with varying degrees of calcification. Areas of calcification co-localized with osteochondrogenic differentiation of vascular smooth muscle cells, lipid peroxidation, oxidized DNA damage, and cell apoptosis, suggesting an important role for oxidative stress in the calcification of grafts. The extent of calcification within grafts was inversely proportional to their antioxidant activity: Poly-ECM-Hep > ECM > Poly-ECM. The incorporation of antioxidants into vascular grafts may be a viable strategy to inhibit degenerative changes.
• Oxburgh, L., Carroll, T. J., Cleaver, O., Gossett, D. R., Hoshizaki, D. K., Hubbell, J. A., Humphreys, B. D., Jain, S., Jensen, J., Kaplan, D. L., Kesselman, C., Ketchum, C. J., Little, M. H., McMahon, A. P., Shankland, S. J., Spence, J. R., Valerius, M. T., Wertheim, J. A., Wessely, O., , Zheng, Y., et al. (2017). (Re)Building a Kidney. Journal of the American Society of Nephrology : JASN, 28(5), 1370-1378.
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  (Re)Building a Kidney is a National Institute of Diabetes and Digestive and Kidney Diseases-led consortium to optimize approaches for the isolation, expansion, and differentiation of appropriate kidney cell types and the integration of these cells into complex structures that replicate human kidney function. The ultimate goals of the consortium are two-fold: to develop and implement strategies for engineering of replacement kidney tissue, and to devise strategies to stimulate regeneration of nephrons to restore failing kidney function. Projects within the consortium will answer fundamental questions regarding human gene expression in the developing kidney, essential signaling crosstalk between distinct cell types of the developing kidney, how to derive the many cell types of the kidney through directed differentiation of human pluripotent stem cells, which bioengineering or scaffolding strategies have the most potential for kidney tissue formation, and basic parameters of the regenerative response to injury. As these projects progress, the consortium will incorporate systematic investigations in physiologic function of and differentiated kidney tissue, strategies for engraftment in experimental animals, and development of therapeutic approaches to activate innate reparative responses.
• Uzarski, J. S., DiVito, M. D., Wertheim, J. A., & Miller, W. M. (2017). Essential design considerations for the resazurin reduction assay to noninvasively quantify cell expansion within perfused extracellular matrix scaffolds. Biomaterials, 129, 163-175.
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  Precise measurement of cellularity within bioartificial tissues and extracellular matrix (ECM) scaffolds is necessary to augment rigorous characterization of cellular behavior, as accurate benchmarking of tissue function to cell number allows for comparison of data across experiments and between laboratories. Resazurin, a soluble dye that is reduced to highly fluorescent resorufin in proportion to the metabolic activity of a cell population, is a valuable, noninvasive tool to measure cell number. We investigated experimental conditions in which resazurin reduction is a reliable indicator of cellularity within three-dimensional (3D) ECM scaffolds. Using three renal cell populations, we demonstrate that correlation of viable cell numbers with the rate of resorufin generation may deviate from linearity at higher cell densities, lower resazurin working volumes, or longer incubation times that all contribute to depleting the pool of resazurin. In conclusion, while the resazurin reduction assay provides a powerful, noninvasive readout of metrics enumerating cellularity and growth within ECM scaffolds, assay conditions may strongly influence its applicability for accurate quantification of cell number. The approach and methodological recommendations presented herein may be used as a guide for application-specific optimization of this assay to obtain rigorous and accurate measurement of cellular content in bioengineered tissues.
• Jiang, B., Suen, R., Wang, J. J., Zhang, Z. J., Wertheim, J. A., & Ameer, G. A. (2016). Mechanocompatible Polymer-Extracellular-Matrix Composites for Vascular Tissue Engineering. Advanced healthcare materials, 5(13), 1594-605.
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  Small-diameter vascular grafts developed from vascular extracellular matrix (ECM) can potentially be used for bypass surgeries and other vascular reconstruction and repair procedures. The addition of heparin to the ECM improves graft hemocompatibility but often involves chemical cross-linking, which increases ECM mechanical stiffness compared to native arteries. Herein, the importance of maintaining ECM mechanocompatibility is demonstrated, and a mechanocompatible strategy to immobilize heparin onto the ECM via a biodegradable elastomer is described. Specifically, poly(1,8-octamethylene citrate)-co-cysteine is hybridized to the ECM, forming a polymer-ECM composite that allows for heparin immobilization via maleimide-thiol "click" chemistry. Heparinized composites reduce platelet adhesion by >60% in vitro, without altering the elastic modulus of the ECM. In a rat abdominal aortic interposition model, intimal hyperplasia in heparinized mechanocompatible grafts is 65% lower when compared to ECM-only control grafts at four weeks. In contrast, grafts that are heparinized with carbodiimide chemistry exhibit increased intimal hyperplasia (4.2-fold) and increased macrophage infiltration (3.5-fold) compared to ECM-only control grafts. All grafts show similar, partial endothelial cell coverage and little to no ECM remodeling. Overall, a mechanocompatible strategy to improve ECM thromboresistance is described and the importance of ECM mechanical properties for proper in vivo graft performance is highlighted.
• Jiang, B., Suen, R., Wertheim, J. A., & Ameer, G. A. (2016). Targeting Heparin to Collagen within Extracellular Matrix Significantly Reduces Thrombogenicity and Improves Endothelialization of Decellularized Tissues. Biomacromolecules, 17(12), 3940-3948.
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  Thrombosis within small-diameter vascular grafts limits the development of bioartificial, engineered vascular conduits, especially those derived from extracellular matrix (ECM). Here we describe an easy-to-implement strategy to chemically modify vascular ECM by covalently linking a collagen binding peptide (CBP) to heparin to form a heparin derivative (CBP-heparin) that selectively binds a subset of collagens. Modification of ECM with CBP-heparin leads to increased deposition of functional heparin (by ∼7.2-fold measured by glycosaminoglycan composition) and a corresponding reduction in platelet binding (>70%) and whole blood clotting (>80%) onto the ECM. Furthermore, addition of CBP-heparin to the ECM stabilizes long-term endothelial cell attachment to the lumen of ECM-derived vascular conduits, potentially through recruitment of heparin-binding growth factors that ultimately improve the durability of endothelialization in vitro. Overall, our findings provide a simple yet effective method to increase deposition of functional heparin on the surface of ECM-based vascular grafts and thereby minimize thrombogenicity of decellularized tissue, overcoming a significant challenge in tissue engineering of bioartificial vessels and vascularized organs.
• Tsukamoto, A., Abbot, S. E., Kadyk, L. C., DeWitt, N. D., Schaffer, D. V., Wertheim, J. A., Whittlesey, K. J., & Werner, M. J. (2016). Challenging Regeneration to Transform Medicine. Stem cells translational medicine, 5(1), 1-7.
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  The aging population in the U.S. and other developed countries has led to a large increase in the number of patients suffering from degenerative diseases. Transplantation surgery has been a successful therapeutic option for certain patients; however, the availability of suitable donor organs and tissues significantly limits the number of patients who can benefit from this approach. Regenerative medicine has witnessed numerous recent and spectacular advances, making the repair or replacement of dysfunctional organs and tissues an achievable goal. Public-private partnerships and government policies and incentives would further catalyze the development of universally available donor tissues, resulting in broad medical and economic benefits. This article describes a Regenerative Medicine Grand Challenge that the Alliance for Regenerative Medicine recently shared with the White House's Office of Science and Technology Policy in response to a White House call to action in scientific disciplines suggesting that the development of "universal donor tissues" should be designated as a Regenerative Medicine Grand Challenge. Such a designation would raise national awareness of the potential of regenerative medicine to address the unmet needs of many diseases and would stimulate the scientific partnerships and investments in technology needed to expedite this goal. Here we outline key policy changes and technological challenges that must be addressed to achieve the promise of a major breakthrough in the treatment of degenerative disease. A nationalized effort and commitment to develop universal donor tissues could realize this goal within 10 years and along the way result in significant innovation in manufacturing technologies.
• Wang, B., Jakus, A. E., Baptista, P. M., Soker, S., Soto-Gutierrez, A., Abecassis, M. M., Shah, R. N., & Wertheim, J. A. (2016). Functional Maturation of Induced Pluripotent Stem Cell Hepatocytes in Extracellular Matrix-A Comparative Analysis of Bioartificial Liver Microenvironments. Stem cells translational medicine, 5(9), 1257-67.
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  : Induced pluripotent stem cells (iPSCs) are new diagnostic and potentially therapeutic tools to model disease and assess the toxicity of pharmaceutical medications. A common limitation of cell lineages derived from iPSCs is a blunted phenotype compared with fully developed, endogenous cells. We examined the influence of novel three-dimensional bioartificial microenvironments on function and maturation of hepatocyte-like cells differentiated from iPSCs and grown within an acellular, liver-derived extracellular matrix (ECM) scaffold. In parallel, we also compared a bioplotted poly-l-lactic acid (PLLA) scaffold that allows for cell growth in three dimensions and formation of cell-cell contacts but is infused with type I collagen (PLLA-collagen scaffold) alone as a "deconstructed" control scaffold with narrowed biological diversity. iPSC-derived hepatocytes cultured within both scaffolds remained viable, became polarized, and formed bile canaliculi-like structures; however, cells grown within ECM scaffolds had significantly higher P450 (CYP2C9, CYP3A4, CYP1A2) mRNA levels and metabolic enzyme activity compared with iPSC hepatocytes grown in either bioplotted PLLA collagen or Matrigel sandwich control culture. Additionally, the rate of albumin synthesis approached the level of primary cryopreserved hepatocytes with lower transcription of fetal-specific genes, α-fetoprotein and CYP3A7, compared with either PLLA-collagen scaffolds or sandwich culture. These studies show that two acellular, three-dimensional culture systems increase the function of iPSC-derived hepatocytes. However, scaffolds derived from ECM alone induced further hepatocyte maturation compared with bioplotted PLLA-collagen scaffolds. This effect is likely mediated by the complex composition of ECM scaffolds in contrast to bioplotted scaffolds, suggesting their utility for in vitro hepatocyte assays or drug discovery.
• Wertheim, J. A. (2016). Novel technology for liver regeneration and replacement. Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society, 22(S1), 41-46.