Abstract: Human pluripotent stem cells (hPSCs) provide a limitless source of human cell and tissues for modeling development and disease, evaluating the safety and efficacy of molecular therapeutics, and for developing cell therapies. For example, hPSC-derived cardiomyocytes are used in the drug development pipeline to identify cardiotoxicity and efficacy in a human model and are in clinical trials to restore cardiac function in patients with heart failure. Challenges associated with cardiomyocyte manufacturing, including difficulties in scaling processes to those needed for commercial and clinical applications and significant batch-to-batch variability, limit the application of these cells however. hPSCs transition through multiple progenitor states during cardiomyocyte differentiation. One of these states, termed the cardiac progenitor cell (CPC), is a multipotent cell type committed to the cardiac fate but capable of generating multiple cell types of the heart including cardiomyocytes, cardiac fibroblasts, smooth muscle cells, and epicardial cells. We identified CPC populations that give rise to high percentages of cardiomyocytes (high efficiency CPCs) and CPC populations that generate low purity cardiomyocytes (low efficiency CPCs), then performed an integrated transcriptomic (RNAseq) and epigenomic (ATACseq) analysis of these CPCs to identify features that predict CM generation. By comparing our datasets to those in the literature, we found a set of genes enriched in high efficiency CPCs.Our comparison of pathway enrichment in high and low efficiency CPCs also suggested MAP kinase and WNT signaling as significant drivers of off-target cell population generation, including skeletal myocytes, epicardial cells and a SLC7A11-expressing population of unknown cells. We found that stage-specific modulation of these pathways improves reliability of CM manufacturing. In conclusion, our study demonstrates how integrated multi-omics analysis of progenitor cells can identify quality attributes that enable prediction of cell differentiation potential, thereby improving differentiation protocols and increasing manufacturing robustness of cell therapies.

Abstract: In this talk, I will present our laboratory’s recent research efforts in developing rapid, digital light processing (DLP) based bioprinting methods to create 3D tissue constructs using a variety of biomaterials and cells. These 3D printed scaffolds are functionalized with precise control of micro-architecture, mechanical (e.g. stiffness), chemical, and biological properties. Such functional tissues allow us to investigate microscale cell-microenvironment interactions in response to integrated mechanical and chemical stimuli. From these fundamental studies we have been creating both in vitro and in vivo precision tissues for tissue regeneration, disease modeling, and drug discovery. Examples including 3D bioprinted liver and heart models will be discussed. I will also showcase 3D printed biomimetic scaffolds for nerve repair. Throughout the presentation, I will discuss engineer’s perspectives in terms of design innovation, biomaterials, mechanics, and scalable biomanufacturing. 1. You et al, “High Cell Density and High Resolution 3D Bioprinting for Fabricating Vascularized Tissues”, Science Advances, 9 (no. 8), eade7923, 2023. 2. Koffler et al., “Biomimetic 3D-Printed Scaffolds for Spinal Cord Injury”, Nature Medicine, 25, 263-269, 2019 3. Ma et al., Deterministically Patterned Biomimetic Human iPSC-derived Hepatic Model via Rapid 3D Bioprinting”, PNAS, 113 (no. 8), pp. 2206-2211, 2016.

Abstract: Over the last decades, the field of three-dimensional (3D) printing, or additive manufacturing, has witnessed tremendous progress. 3D printing enables precise control over the composition, spatial distribution, and architecture of the printed constructs facilitating the recapitulation of the delicate shapes and structures of target patterns. More recently it has been further combined with cells and cell-laden biomaterials to offer the versatility to fabricate biomimetic volumetric tissues that are both structurally and functionally relevant. Nevertheless, conventional 3D printing and bioprinting techniques are limited in certain aspects. This talk will thus discuss our recent efforts in developing a series of advanced additive (bio)manufacturing strategies that take unconventional approaches to tackle some of these problems and improve their capacities towards diverse applications in biomedicine with a focus on regenerative medicine. These platform technologies will likely provide new opportunities in areas from constructing functional tissues and microtissue models for promoting personalizable medicine, all the way to minimally invasive surgical implications.

Abstract: Gene therapy has experienced an increasing number of successful human clinical trials, leading to 6 FDA approved products using delivery vectors based on adeno-associated viruses (AAV). These clear successes have been made possible by the identification of disease targets that are suitable for the delivery properties of natural variants of AAV. However, vectors in general face a number of barriers and challenges that preclude their extension to most disease targets, including pre-existing antibodies against AAVs, suboptimal biodistribution, limited spread within tissues, an inability to target delivery to specific cells, and/or limited delivery efficiency to target cells. These barriers are not surprising, since the parent viruses upon which vectors are based were not evolved by nature for our convenience to use as human therapeutics. Unfortunately, for most applications, there is insufficient mechanistic knowledge of underlying virus structure-function relationships to empower rational design improvements. As an alternative, for two decades we have been implementing directed evolution – the iterative genetic diversification of the viral genome and functional selection for desired properties – to engineer highly optimized, next generation AAV variants for delivery to any cell or tissue target. We have genetically diversified AAV using a broad range of approaches from fully random (e.g. error prone PCR) to computationally guided (e.g. by machine learning). The resulting large (~109) libraries are then functionally selected for substantially enhanced delivery, yielding AAVs capable of highly efficient therapeutic gene delivery in animal models and in several human clinical trials.

Abstract: Glioblastoma (GBM), the most common type of primary brain tumor, is characterized by high mortality rate, short lifespan, and poor prognosis with a high tendency of recurrence. Functional therapeutics, including PRMT5 inhibitors, radiosensitizers, and emerging chimeric antigen receptor (CAR)-T immunotherapy, have been developed to treat GBM. However, the existence of physiological blood-brain barrier (BBB) or blood-brain-tumor barrier has impeded the efficient delivery of such promising therapeutics into the brain and limited their therapeutic efficacy. Given the native ability of neutrophils to cross BBB and penetrate the brain parenchyma, here we tested the therapeutic concept that neutrophils could be engineered with synthetic CARs to specifically target GBM and effectively deliver chemo-drugs to brain tumor as a novel dual chemoimmunotherapy for the first time. Primary neutrophils are short-lived and resistant to genetic modification. Therefore, we genetically engineered human pluripotent stem cells with different chlorotoxin (CLTX) CARs and differentiated them into functional CAR-neutrophils. As compared to CAR-natural killer (NK) cells, systemically administered hPSC-derived CLTX CAR-neutrophils significantly reduced tumor burden in xenograft mouse models and extended their lifespan, suggesting superior abilities of neutrophils in crossing BBB and penetrating GBM xenograft in mice. We also loaded hypoxia-activated prodrug tirapazamine (TPZ) into CAR-neutrophils using silica nanoparticles with rough surfaces (R-SiO2-TPZ) and demonstrated their enhanced antitumor activities in xenograft mouse models, serving as a novel dual chemoimmunotherapy against GBM. Our results established that CAR neutrophil-mediated drug delivery may provide an effective and universal strategy for specific targeting of solid tumors.

Abstract: Synthetic biology has transformed how cells can be reprogrammed, providing a means to reliably and predictably control cell behavior with the assembly of genetic parts into more complex synthetic gene circuits. Using these approaches, we are programming stem cells with novel genetic tools to control genes and pathways that result in changes in their native function for desired outcomes. We are particularly interested in building genetic tools to define the molecular rules governing hematopoietic cell fate transitions and the dynamic processes that guide stem cell differentiation. We have also shown that megakaryocytes, the progenitor cells for platelets, can be reprogrammed and loaded with non-native proteins to produce engineered platelets that function as local and systemic delivery devices.

Abstract: The seamless integration of synthetic materials with biological systems long remains a grand challenge, often curtailed by the sheer complexity of the cell-material interface. For decades, biomaterial scientists and engineers have designed around this complexity by rationally designing new materials one experiment at a time. However, recent advances in laboratory automation, high throughput analytics, and artificial intelligence / machine learning (AI/ML) now provide a unique opportunity to fully automate the design process. In this seminar, we put forth our efforts to develop a biomaterials acceleration platform (BioMAP) (i.e., self-driving biomaterials lab) that can rapidly iterate through design spaces and identify unique material properties that perfectly synergize with biological complexity.

Abstract The human placenta is a transient organ, key for proper fetal growth and development. Abnormal development and function of this organ is associated with adverse pregnancy outcomes, including pre-eclampsia, fetal growth restriction, and stillbirth. The human placenta is also unique in its structure, cellular composition, and signaling pathways, particularly in the early embryonic period, thus limiting the utility of animal models for its study. Specification and early development of trophoblast, the epithelial cells of the placenta, have been the subject of intense study over the past decade, with the advent of single cell and spatial ‘omics technology and their convergence on the fields of human embryology, pluripotent stem cell, and placental biology. In particular, establishment of culture conditions for derivation of trophoblast stem cells (TSC) and trophoblast organoids (TOrg) from both placental tissues and pluripotent stem cells has significantly advanced our ability to study trophoblast differentiation. Nevertheless, many questions remain, including whether these models reflect trophoblast differentiation in vivo. In addition, significant barriers exist regarding access to these model systems and funding for such research. It is therefore most important to identify the strengths and limitations of each of these models, while simultaneously educating the public about this research and advocating for reasonable regulation and increased funding. Over the past decade, our group has optimized pluripotent stem cell-derived models for trophoblast differentiation, in particular, asking whether they recapitulate diseased trophoblast, while also comparing them to primary (placenta-derived) cells from placentas of different gestational ages. Overall, our data reveal significant differences between established TSC and the vast majority of primary cytotrophoblast progenitor cells in early gestation placenta. At the same time, we see a high level of similarity between primary and pluripotent stem cell-derived TSC, confirming the latter as a potentially robust, easily-accessible model for studying human trophoblast differentiation.

Abstract: Cardiovascular disease is the leading cause of death in both men and women, yet our mechanistic knowledge of the sex-specific molecular and cellular mechanisms that guide cardiovascular disease progression, particularly in women, remain poorly characterized. Studies evaluating disease mechanisms rarely state the sex of cells used for in vitro studies or are performed primarily in male animal models, causing our gap in knowledge. My laboratory uses precision biomaterials as in vitro and in vivo tools to dissect mechanisms that contribute to sexual dimorphisms in cardiovascular diseases, specifically aortic valve stenosis. In my talk, I will discuss how we have used hydrogel biomaterials as engineered valve matrix mimics to explore sex dimorphisms in valvular interstitial cell phenotypes in vitro and describe sex-specific molecular mechanisms that may drive dimorphisms in aortic valve stenosis. Our work seeks to leverage biomaterial technologies to understand sex dimorphisms in health and disease, with the long-term goal of achieving sex and gender equity in cardiovascular disease treatments and outcomes.

Abstract: Our understanding of human pre-, peri- and early post-implantation development at the cellular, molecular, and genetic levels is limited, posing a significant obstacle to comprehending the basis of implantation and developmental defects. Studying early human development is challenging due to restricted access to human embryos for research. While rodent models have been instrumental, they are not ideal because many aspects of human development differ molecularly and temporally from rodents. To reduce dependency on human embryos for studying human embryogenesis, we have developed several integrated models of early human development using pluripotent stem cells and/or their derivatives. These models provide a well-controlled cellular substrate for testing biological hypotheses and will enhance our fundamental understanding of the key signaling events and cellular interactions in pre- and peri-implantation human development. This, in turn, will help us understand the molecular and cellular contributors to implantation and developmental failures in humans.