A bioprinted sea-and-island multicellular model for dissecting human pancreatic tumor-stroma reciprocity and adaptive metabolism.

Pancreatic ductal adenocarcinoma (PDAC) presents a formidable clinical challenge due to its intricate microenvironment characterized by desmoplasia and complex tumor-stroma interactions. Conventional models hinder studying cellular crosstalk for therapeutic development. To recapitulate key features of PDAC masses, this study creates a novel sea-and-island PDAC tumor construct (s&i PTC). The s&i PTC consists of 3D-printed islands of human PDAC cells positioned within an interstitial extracellular matrix (ECM) populated by human cancer-associated fibroblasts (CAFs). This design closely mimics the in vivo desmoplastic architecture and nutrient-poor conditions. The model enables studying dynamic tumor-stroma crosstalk and signaling reciprocity, revealing both known and yet-to-be-discovered multicellular metabolic adaptations. Using the model, we discovered the orchestrated dynamic alterations of CAFs under nutrient stress, resembling critical in vivo human tumor niches, such as the secretion of pro-tumoral inflammatory factors. Additionally, nutrient scarcity induces dynamic alterations in the ECM composition and exacerbates poor cancer cell differentiation-features well-established in PDAC progression. Proteomic analysis unveiled the enrichment of proteins associated with aggressive tumor behavior and ECM remodeling in response to poor nutritional conditions, mimicking the metabolic stresses experienced by avascular pancreatic tumor cores. Importantly, the model's relevance to patient outcomes is evident through an inverse correlation between biomarker expression patterns in the s&i PTCs and PDAC patient survival rates. Key findings include upregulated MMPs and key ECM proteins (such as collagen 11 and TGFß) under nutrient-avid conditions, known to be regulated by CAFs, alongside the concomitant reduction in E-cadherin expression associated with a poorly differentiated PDAC state under nutrient deprivation. Furthermore, elevated levels of hyaluronic acid (HA) and integrins in response to nutrient deprivation underscore the model's fidelity to the PDAC microenvironment. We also observed increased IL-6 and reduced α-SMA expression under poor nutritional conditions, suggesting a transition of CAFs from myofibroblastic to inflammatory phenotypes under a nutrient stress akin to in vivo niches. In conclusion, the s&i PTC represents a significant advancement in engineering clinically relevant 3D models of PDAC masses. It offers a promising platform for elucidating tumor-stroma interactions and guiding future therapeutic strategies to improve patient outcomes.

Oxygenated Scaffolds for Pancreatic Endocrine Differentiation from Induced Pluripotent Stem Cells.

A 3D microenvironment is known to endorse pancreatic islet development from human induced pluripotent stem cells (iPSCs). However, oxygen supply becomes a limiting factor in a scaffold culture. In this study, oxygen-releasing biomaterials are fabricated and an oxygenated scaffold culture platform is developed to offer a better oxygen supply during 3D iPSC pancreatic differentiation. It is found that the oxygenation does not alter the scaffold's mechanical properties. The in situ oxygenation improves oxygen tension within the scaffolds. The unique 3D differentiation system enables the generation of islet organoids with enhanced expression of islet signature genes and proteins. Additionally, it is discovered that the oxygenation at the early stage of differentiation has more profound impacts on islet development from iPSCs. More C-peptide+ /MAFA+ ß and glucagon+ /MAFB+ α cells formed in the iPSC-derived islet organoids generated under oxygenated conditions, suggesting enhanced maturation of the organoids. Furthermore, the oxygenated 3D cultures improve islet organoids' sensitivity to glucose for insulin secretion. It is herein demonstrated that the oxygenated scaffold culture empowers iPSC islet differentiation to generate clinically relevant tissues for diabetes research and treatment.

Extracellular matrix-derived peptide stimulates the generation of endocrine progenitors and islet organoids from iPSCs.

Induced pluripotent stem cells (iPSCs) have enormous potential in producing human tissues endlessly. We previously reported that type V collagen (COL5), a pancreatic extracellular matrix protein, promotes islet development and maturation from iPSCs. In this study, we identified a bioactive peptide domain of COL5, WWASKS, through bioinformatic analysis of decellularized pancreatic ECM (dpECM)-derived collagens. RNA-sequencing suggests that WWASKS induces the formation of pancreatic endocrine progenitors while suppressing the development of other types of organs. The expressions of hypoxic genes were significantly downregulated in the endocrine progenitors formed under peptide stimulation. Furthermore, we unveiled an enhancement of iPSC-derived islets' (i-islets) glucose sensitivity under peptide stimulation. These i-islets secrete insulin in a glucose responsive manner. They were comprised of α, ß, δ, and γ cells and were assembled into a tissue architecture similar to that of human islets. Mechanistically, the peptide is able to activate the canonical Wnt signaling pathway, permitting the translocation of ß-catenin from the cytoplasm to the nucleus for pancreatic progenitor development. Collectively, for the first time, we demonstrated that an ECM-derived peptide dictates iPSC fate toward the generation of endocrine progenitors and subsequent islet organoids.

Bioink Formulation and Machine Learning-Empowered Bioprinting Optimization.

Bioprinting enables the fabrication of complex, heterogeneous tissues through robotically-controlled placement of cells and biomaterials. It has been rapidly developing into a powerful and versatile tool for tissue engineering. Recent advances in bioprinting modalities and biofabrication strategies as well as new materials and chemistries have led to improved mimicry and development of physiologically relevant tissue architectures constituted with multiple cell types and heterogeneous spatial material properties. Machine learning (ML) has been applied to accelerate these processes. It is a new paradigm for bioprinting. In this review, we explore current trends in bioink formulation and how ML has been used to accelerate optimization and enable real-time error detection as well as to reduce the iterative steps necessary for bioink formulation. We examined how rheometric properties, including shear storage, loss moduli, viscosity, shear-thinning property of biomaterials affect the printability of a bioink. Furthermore, we scrutinized the interplays between yield shear stress and the printability of a bioink. Moreover, we systematically surveyed the application of ML in precision in situ surgical site bioprinting, closed-loop AI printing, and post-printing optimization.

Decellularization for the retention of tissue niches.

Decellularization of natural tissues to produce extracellular matrix is a promising method for three-dimensional scaffolding and for understanding microenvironment of the tissue of interest. Due to the lack of a universal standard protocol for tissue decellularization, recent investigations seek to develop novel methods for whole or partial organ decellularization capable of supporting cell differentiation and implantation towards appropriate tissue regeneration. This review provides a comprehensive and updated perspective on the most recent advances in decellularization strategies for a variety of organs and tissues, highlighting techniques of chemical, physical, biological, enzymatic, or combinative-based methods to remove cellular contents from tissues. In addition, the review presents modernized approaches for improving standard decellularization protocols for numerous organ types.

Systematic evaluating and modeling of SARS-CoV-2 UVC disinfection.

The ongoing COVID-19 global pandemic has necessitated evaluating various disinfection technologies for reducing viral transmission in public settings. Ultraviolet (UV) radiation can inactivate pathogens and viruses but more insight is needed into the performance of different UV wavelengths and their applications. We observed greater than a 3-log reduction of SARS-CoV-2 infectivity with a dose of 12.5 mJ/cm2 of 254 nm UV light when the viruses were suspended in PBS, while a dose of 25 mJ/cm2 was necessary to achieve a similar reduction when they were in an EMEM culture medium containing 2%(v/v) FBS, highlighting the critical effect of media in which the virus is suspended, given that SARS-CoV-2 is always aerosolized when airborne or deposited on a surface. It was found that SARS-CoV-2 susceptibility (a measure of the effectiveness of the UV light) in a buffer such as PBS was 4.4-fold greater than that in a cell culture medium. Furthermore, we discovered the attenuation of UVC disinfection by amino acids, vitamins, and niacinamide, highlighting the importance of determining UVC dosages under a condition close to aerosols that wrap the viruses. We developed a disinfection model to determine the effect of the environment on UVC effectiveness with three different wavelengths, 222 nm, 254 nm, and 265 nm. An inverse correlation between the liquid absorbance and the viral susceptibility was observed. We found that 222 nm light was most effective at reducing viral infectivity in low absorbing liquids such as PBS, whereas 265 nm light was most effective in high absorbing liquids such as cell culture medium. Viral susceptibility was further decreased in N95 masks with 222 nm light being the most effective. The safety of 222 nm was also studied. We detected changes to the mechanical properties of the stratum corneum of human skins when the 222 nm accumulative exposure exceeded 50 J/cm2.The findings highlight the need to evaluate each UV for a given application, as well as limiting the dose to the lowest dose necessary to avoid unnecessary exposure to the public.

Epithelial-to-Mesenchymal Transition Signaling Pathways Responsible for Breast Cancer Metastasis.

Breast carcinoma is highly metastatic and invasive. Tumor metastasis is a convoluted and multistep process involving tumor cell disseminating from their primary site and migrating to the secondary organ. Epithelial-mesenchymal transition (EMT) is one of the crucial steps that initiate cell progression, invasion, and metastasis. During EMT, epithelial cells alter their molecular features and acquire a mesenchymal phenotype. The regulation of EMT is centered by several signaling pathways, including primary mediators TGF-ß, Notch, Wnt, TNF-α, Hedgehog, and RTKs. It is also affected by hypoxia and microRNAs (miRNAs). All these pathways are the convergence on the transcriptional factors such as Snail, Slug, Twist, and ZEB1/2. In addition, a line of evidence suggested that EMT and cancer stem like cells (CSCs) are associated. EMT associated cancer stem cells display mesenchymal phenotypes and resist to chemotherapy or targeted therapy. In this review, we highlighted recent discoveries in these signaling pathways and their regulation in breast cancer metastasis and invasion. While the clinical relevance of EMT and breast cancers remains controversial, we speculated a convergent signaling network pivotal to elucidating the transition of epithelial to mesenchymal phenotypes and onset of metastasis of breast cancer cells.

Prevalence and related factors of peripheral arterial disease in diabetes mellitus inpatients: a cross-sectional study in China.

Peripheral arterialdisease (PAD) can result in diabetic foot ulcers, gangrene, and even amputation. Since most cases of PAD in diabetic patients are associated with peripheral neuropathy, the symptoms of vascular disease are easily concealed by the symptoms of neuropathy and are ignored by people, so it is critical for health care providers to screen PAD for the diabetes patients. This study was carried out to identify theprevalence and related factors of PAD in diabetes mellitus inpatients. This was a cross-sectional observational study. A total of 855 patients were enrolled in the study from December 2018 to December 2019. The patients were divided into a non-PAD group (ABI = 0.9-1.3) and a PAD group (ABI <0.9). Logistic multivariate regression analysis showed that age, LDL-C, dorsalis pedis artery pulsation (left foot), and sensory-current threshold (right foot) were related factors for peripheral arterial disease. Patients who are older and have a higher LDL-C level, abnormal dorsal foot pulse, and abnormal sensory-current threshold must be vigilant, and receive early screening for PAD diagnosis and treatment to avoid a malignant outcome. In clinical work, medical staff should actively apply PAD screening to diabetic patients, identify risk factors as early as possible, conduct early interventions, reduce the risk of PAD in patients, and avoid the occurrence of adverse outcomes.

Proteomic and Bioinformatic Analysis of Decellularized Pancreatic Extracellular Matrices.

Tissue microenvironments are rich in signaling molecules. However, factors in the tissue matrix that can serve as tissue-specific cues for engineering pancreatic tissues have not been thoroughly identified. In this study, we performed a comprehensive proteomic analysis of porcine decellularized pancreatic extracellular matrix (dpECM). By profiling dpECM collected from subjects of different ages and genders, we showed that the detergent-free decellularization method developed in this study permits the preservation of approximately 62.4% more proteins than a detergent-based method. In addition, we demonstrated that dpECM prepared from young pigs contained approximately 68.5% more extracellular matrix proteins than those prepared from adult pigs. Furthermore, we categorized dpECM proteins by biological process, molecular function, and cellular component through gene ontology analysis. Our study results also suggested that the protein composition of dpECM is significantly different between male and female animals while a KEGG enrichment pathway analysis revealed that dpECM protein profiling varies significantly depending on age. This study provides the proteome of pancreatic decellularized ECM in different animal ages and genders, which will help identify the bioactive molecules that are pivotal in creating tissue-specific cues for engineering tissues in vitro.

Angiopoietins stimulate pancreatic islet development from stem cells.

In vitro differentiation of human induced pluripotent stem cells (iPSCs) into functional islets holds immense potential to create an unlimited source of islets for diabetes research and treatment. A continuous challenge in this field is to generate glucose-responsive mature islets. We herein report a previously undiscovered angiopoietin signal for in vitro islet development. We revealed, for the first time, that angiopoietins, including angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) permit the generation of islets from iPSCs with elevated glucose responsiveness, a hallmark of mature islets. Angiopoietin-stimulated islets exhibited glucose synchronized calcium ion influx in repetitive glucose challenges. Moreover, Ang2 augmented the expression of all islet hormones, including insulin, glucagon, somatostatin, and pancreatic polypeptide; and ß cell transcription factors, including NKX6.1, MAFA, UCN3, and PDX1. Furthermore, we showed that the Ang2 stimulated islets were able to regulate insulin exocytosis through actin-filament polymerization and depolymerization upon glucose challenge, presumably through the CDC42-RAC1-gelsolin mediated insulin secretion signaling pathway. We also discovered the formation of endothelium within the islets under Ang2 stimulation. These results strongly suggest that angiopoietin acts as a signaling molecule to endorse in vitro islet development from iPSCs.

Decellularized Tissue Matrix Enhances Self-Assembly of Islet Organoids from Pluripotent Stem Cell Differentiation.

Regenerating human islet organoids from stem cells remains a significant challenge because of our limited knowledge on cues essential for developing the endocrine organoids in vitro. In this study, we discovered that a natural material prepared from a decellularized rat pancreatic extracellular matrix (dpECM) induces the self-assembly of human islet organoids during induced pluripotent stem cell (iPSC) pancreatic differentiation. For the first time, we demonstrated that the iPSC-derived islet organoids formed in the presence of the dpECM are capable of glucose-responsive secretion of both insulin and glucagon, two major hormones that maintain blood glucose homeostasis. The characterization of the organoids revealed that the organoids consisted of all major endocrine cell types, including α, ß, δ, and pancreatic polypeptide cells, that were assembled into a tissue architecture similar to that of human islets. The exposure of iPSCs to the dpECM during differentiation resulted in considerably elevated expression of key pancreatic transcription factors such as PDX-1, MAFA, and NKX6.1 and the production of all major hormones, including insulin, glucagon, somatostatin, and pancreatic polypeptide from stem cell-derived organoids. This study highlights the importance of natural, bioactive biomaterials for building microenvironments crucial to regenerating islet organoids from stem cells.

Proteomic analysis of decellularized pancreatic matrix identifies collagen V as a critical regulator for islet organogenesis from human pluripotent stem cells.

In pancreatic tissue engineering, generating human pancreatic islet organoids from stem cells has been challenging due mainly to a poor understanding of niches required for multicellular tissue self-assembly in vitro. In this study, we aimed to identify bioactive, chemically defined niches from natural, biological materials for islet development in vitro. We investigated the proteomics of decellularized rat pancreatic extracellular matrix (dpECM) hydrogel using advanced bioinformatics analysis, and identified that type V collagen (ColV) is constantly and abundantly present in dpECM hydrogel. Niches provided to human pluripotent stem cells (iPSCs) by presenting ColV in matrix coating substrates permitted stem cells progression into islet-like organoids that consist of all major pancreatic endocrine cell types, i.e. α, ß, δ, and pancreatic polypeptide cells. In the presence of ColV niches, gene expressions of all key pancreatic transcription factors and major hormone genes significantly increased in iPSC-derived organoids. Most importantly, ColV-containing microenvironment resulted in enhanced glucose responsive secretions of both insulin and glucagon hormone from organoids. The study demonstrates that ColV is a critical regulator that augments islet self-assembly from iPSCs, and it is feasible to utilize natural biomaterials to build tissue cues essential for multicellular tissue production in vitro.

Inducing Tumor Suppressive Microenvironments through Genome Edited CD47-/- Syngeneic Cell Vaccination.

Tumors can escape from the immune system by overexpressing CD47 and other checkpoint blockades. CD47 is expressed ubiquitously by all cells in the body, posing an obstacle for CD47 blocking treatments due to their systemic toxicity. We performed a study to determine how the tumor microenvironment changes after vaccination with genome edited CD47-/- syngeneic tumor cells. We discovered that inactivated CD47-depleted mouse melanoma cells can protect mice from melanoma. Our animal study indicated that 33% of vaccinated mice remained tumor-free, and 100% of mice had 5-fold reduced growth rates. The characterization of immunomodulatory effects of the vaccine revealed a highly anti-tumorigenic and homogenous microenvironment after vaccination. We observed consistently that in the tumors that failed to respond to vaccines, there were reduced natural killer cells, elevated regulatory T cells, M2-type macrophages, and high PD-L1 expression in these cells. These observations suggested that the tumor microenvironments became more suppressive to tumor growth after vaccination, suggesting a potential new immunotherapy for solid tumors.

A bioink blend for rotary 3D bioprinting tissue engineered small-diameter vascular constructs.

3D bioprinted vascular constructs have gained increased interest due to their significant potential for creating customizable alternatives to autologous vessel grafts. In this study, we developed a new approach for biofabricating fibrin-based vascular constructs using a novel rotary 3D bioprinter developed in our lab. We formulated a new bioink by incorporating fibrinogen with gelatin to achieve a desired shear-thinning property for rotary bioprinting. The blending of heat-treated gelatin with fibrinogen turned unprintable fibrinogen into a printable biomaterial for vessel bioprinting by leveraging the favorable rheological properties of gelatin. We discovered that the heat-treatment of gelatin remarkably affects the rheological properties of a gelatin-fibrinogen blended bioink, which in turn influences the printability of the ink. Further characterizations revealed that not only concentration of the gelatin but the heat treatment also affects cell viability during printing. Notably, the density of cells included in the bioinks also influenced printability and tissue volumetric changes of the printed vessel constructs during cultures. We observed increased collagen deposition and construct mechanical strength during two months of the cultures. The burst pressure of the vessel constructs reached 1110 mmHg, which is about 52% of the value of the human saphenous vein. An analysis of the tensile mechanical properties of the printed vessel constructs unveiled an increase in both the circumferential and axial elastic moduli during cultures. This study highlights important considerations for bioink formulation when bioprinting vessel constructs. STATEMENT OF SIGNIFICANCE: There has been an increased demand for small-diameter tissue-engineered vascular grafts. Vascular 3D bioprinting holds the potential to create equivalent vascular grafts but with the ability to tailor them to meet patient's needs. Here, we presented a new and innovative 3D rotary bioprinter and a new bioink formulation for printing vascular constructs using fibrinogen, a favorable biomaterial for vascular tissue engineering. The bioink was formulated by blending fibrinogen with a more printable biomaterial, gelatin. The systematic characterization of the effects of heat treatment and gelatin concentration as well as bioink cell concentration on the printability of the bioink offers new insight into the development of printable biomaterials for tissue biofabrication.

Mechanistic Analysis of Physicochemical Cues in Promoting Human Pluripotent Stem Cell Self-Renewal and Metabolism.

We have previously reported that a porous membrane of polyethylene terephthalate (PET) enables significant augmentation of human pluripotent stem cell (hPSC) proliferation and differentiation. The interaction between hPSCs and the PET surface induces ß-catenin-mediated wingless/integrated (Wnt) signaling, leading to upregulation of the expression of adhesion molecules in hPSCs. In this study, we sought to unveil mechanisms underlying the role of the PET membrane in hPSC self-renewal and metabolism. We discovered that physicochemical cues of the PET membrane considerably alter hPSC metabolism by increasing the cell yield and suppressing the generation of toxic byproduct, indicating an effective cell self-renewal and a less apoptotic culture environment in the membrane culture system. Furthermore, we discovered that a caspase-8 medicated apoptotic pathway plays a profound role in obstructing hPSCs grown on a traditional tissue culture plate (TCP). Treating hPSCs seeded on a TCP surface with a caspase-8 inhibitor significantly suppressed cellular apoptotic pathway and improved cell proliferation and metabolism. Our experimental results provided valuable insights into signal pathways influencing hPSC self-renewal during routine maintenance and expansion, which would shed light on large-scale preparation of hPSCs for clinical applications.

Tissue and Organ 3D Bioprinting.

Three-dimensional (3D) bioprinting enables the creation of tissue constructs with heterogeneous compositions and complex architectures. It was initially used for preparing scaffolds for bone tissue engineering. It has recently been adopted to create living tissues, such as cartilage, skin, and heart valve. To facilitate vascularization, hollow channels have been created in the hydrogels by 3D bioprinting. This review discusses the state of the art of the technology, along with a broad range of biomaterials used for 3D bioprinting. It provides an update on recent developments in bioprinting and its applications. 3D bioprinting has profound impacts on biomedical research and industry. It offers a new way to industrialize tissue biofabrication. It has great potential for regenerating tissues and organs to overcome the shortage of organ transplantation.

Perspective: The promise of multi-cellular engineered living systems.

Recent technological breakthroughs in our ability to derive and differentiate induced pluripotent stem cells, organoid biology, organ-on-chip assays, and 3-D bioprinting have all contributed to a heightened interest in the design, assembly, and manufacture of living systems with a broad range of potential uses. This white paper summarizes the state of the emerging field of "multi-cellular engineered living systems," which are composed of interacting cell populations. Recent accomplishments are described, focusing on current and potential applications, as well as barriers to future advances, and the outlook for longer term benefits and potential ethical issues that need to be considered.

Advanced Cell and Tissue Biomanufacturing.

This position paper assesses state-of-the-art advanced biomanufacturing and identifies paths forward to advance this emerging field in biotechnology and biomedical engineering, including new research opportunities and translational and corporate activities. The vision for the field is to see advanced biomanufacturing emerge as a discipline in academic and industrial communities as well as a technological opportunity to spur research and industry growth. To navigate this vision, the paths to move forward and to identify major barriers were a focal point of discussions at a National Science Foundation-sponsored workshop focused on the topic. Some of the major needs include but are not limited to the integration of specific scientific and engineering disciplines and guidance from regulatory agencies, infrastructure requirements, and strategies for reliable systems integration. Some of the recommendations, major targets, and opportunities were also outlined, including some "grand challenges" to spur interest and progress in the field based on the participants at the workshop. Many of these recommendations have been expanded, materialized, and adopted by the field. For instance, the formation of an initial collaboration network in the community was established. This report provides suggestions for the opportunities and challenges to help move the field of advanced biomanufacturing forward. The field is in the early stages of effecting science and technology in biomanufacturing with a bright and important future impact evident based on the rapid scientific advances in recent years and industry progress.