Bioengineering methods for vascularizing organoids

Background and Aims Induced pluripotent stem cell (iPSC)-derived human kidney organoids have enabled novel capacity to interrogate the mechanisms of kidney disease and screen for screen for new therapeutic strategies in vitro. However, current high-throughput (HT) protocols often rely on specialized equipment, are incompatible with imaging assays, or compromise on the cellular complexity of the organoid. The incompatibility with imaging in particular hinders robust analysis of how perturbations to the culture conditions alter organoid composition. This study aimed to develop a HT kidney organoid protocol that retains tissue complexity and compatibility with imaging to comprehensively assess cellular and molecular phenotypes. Methods iPSCs were differentiated to nephron and stromal precursor cell types for 7 days according to the Takasato protocol then dissociated into single cells and mixed with a hydrogel. Droplets of cell-hydrogel mix were deposited on the bottom of an imaging-compatible 96 or 384-well plate and cultured for an additional two weeks. Hydrogel composition, cell seeding density, medium additives, and differentiation timelines were optimized and evaluated by brightfield and high-content confocal imaging. Molecular and cell type markers were quantified within and between replicate organoids to define the area stained by a molecular marker, as a percentage of total organoid area. Hydrogel-embedded organoids were subjected to hypoxia (1% atmospheric O2 for 48 h) +/− experimental compounds to suppress hypoxia-induced acute kidney injury (HI-AKI), in comparison to vehicle and normoxic (21% O2) controls. Organoids were also augmented by integration of primary human endothelial cells and macrophages at the time of organoid formation and assessed by brightfield and confocal imaging. Results Hydrogel-embedded kidney precursor cell types successfully formed kidney organoids in 96 and 384-well imaging plates. Cell seeding density and hydrogel stiffness influenced the cellular composition of kidney organoids, with stiffer hydrogels producing more podocyte and vascular cell types and softer favouring proximal and distal tubule formation. Hydrogel composition was optimized to generate kidney organoids with balanced proportions of podocyte, proximal tubule, distal tubule, vasculature, and fibroblast cell types, with low inter-organoid variation. Importantly, hydrogel embedding enabled generation of a full 96-well plate of organoids in just 15 minutes, did not require specialized equipment, and enabled high resolution imaging on standard inverted microscopes. To evaluate drug screening in this protocol, hydrogel-embedded organoids were exposed to hypoxia +/− experimental compounds that targeted pathways associated with the occurrence of HI-AKI and vehicle controls. Hypoxic injury was characterized by loss of proximal tubule (9.3% decrease, P = 0.0199) and podocyte (29.9% decrease, P = 0.011) stainings in vehicle controls. Inhibiting the HIF-1α pathway was acutely toxic to the organoids, whereas the antioxidant N-acetyl cysteine (NAC) reduced proximal tubule loss. We next leveraged the ease of use and scalability of this protocol to improve the cellular composition and accuracy of kidney organoids by integrating primary human endothelial cells and macrophages, which are under-represented or absent in most kidney organoid models. Successful integration without an adverse effect on nephrogenesis was dependent on the ratio of these cell types relative to differentiated iPSCs and supplementations of key cytokines and growth factors. Conclusion We report a novel HT protocol to generate kidney organoids in hydrogel droplets with improved scale, imaging compatibility, and comparable cellular complexity to traditional methods. Our results identify NAC as a candidate agent to protect against ischemic AKI, while integration of vascular and immune cell types provides new possibilities to model human vascular and immune cell interactions in kidney disease. Ultimately, this new high-throughput method holds great promise to improve the biomedical and translational impact of human kidney organoids.

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

Lysophosphatidic acid and cardiovascular disease: seeing is believing

How well do brain organoids capture your brain?

Purpose of ReviewHematopoietic stem cells (HSCs) sit at the top of the hierarchy that meets the daily burden of blood production. HSC maintenance relies on extrinsic cues from the bone marrow (BM) microenvironment to balance stem cell self-renewal and cell fate decisions. In this brief review, we will highlight the studies and model systems that define the centralized role of BM vascular endothelium in modulating HSC activity in health and stress.Recent FindingsThe BM microenvironment is composed of a diverse array of intimately associated vascular and perivascular cell types. Recent dynamic imaging studies, coupled with single-cell RNA sequencing (scRNA-seq) and functional readouts, have advanced our understanding of the HSC-supportive cell types and their cooperative mechanisms that govern stem cell fate during homeostasis, regeneration, and aging. These findings have established complex and discrete vascular microenvironments within the BM that express overlapping and unique paracrine signals that modulate HSC fate.SummaryUnderstanding the spatial and reciprocal HSC-niche interactions and the molecular mechanisms that govern HSC activity in the BM vascular microenvironment will be integral in developing therapies aimed at ameliorating hematological disease and supporting healthy hematopoietic output.

Engineering Stem and Stromal Cell Therapies for Musculoskeletal Tissue Repair.

The cytokines Interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF, also known as cachectin) exhibit multiple effects on circulating blood cells and cells of the blood vessel wall. For example, these mediators elicit a coordinated Drogram of functions of endothelial cells (EC) that promotes blood coagulation and thrombosis, and lead to clot stabilization. Furthermore, IL-1 and TNF promote adherence to vascular endothelium of leukocytes of many classes.Thus, these cytokines are likely to be involved in signaling the pathologic changes in blood vessels that characterize a number of inflammatory or infectious processes. These two cytokines were originally isolated frcm activated human mononuclear phagocytes, hence their comnon designation as monokines and the terminology "interleukin". However, recent findings have broadened this concept considerably. It is now clear that many cell types can produce IL-1-1ike activity.Several groups showed that human vascular EC can secrete material that stimulates proliferation of thymocytes incubated with suboptima1 doses of the mitogenic lectin phytohemagglutinin, a typical acitivty of IL-1 (thymocyte costimulation).Two related but distinct genes cloned frcm human peripheral blood monocytes encode IL-1 molecules. In human blood monocytes stimulated with bacterial lioopolysaccharide (LPS) IL-1 beta (pi ∼ 7) is the major form expressed while IL-1 alpha (pi ∼ 5) is the less abundant species secreted by human monocytes under these conditions. We found that EC and smooth muscle cells (SMC) isolated from adult human vessels can express these same IL-1 genes. LPS, a standard stimulus to IL-1 secretion in the monocyte, caused accumulation of IL-1 beta mRNA in both vascular cell types. Endothelial cells frcm adult human vessels also contained IL-1 alpha mRNA when treated with LPS in the presence of cycloheximide and LPS-stimulated smooth muscle cells contained RNA that hybridized with an IL-1 alpha cDNA probe as well. Although both vascular cell types can transcribe these IL-1 genes, the time course of this response differs. LPS induced IL-1 beta mRNA production by SMC maximally at 4-6 hr., whereas maximal IL-1 induction by LPS in EC occured 1 day after initiation of the exposure. Actinanycin D (1 ug/ml) blocked 3H-uridine incorporation into macromolecules by > 95% in both EC & SMC, and prevented the LPS-induced increases in IL-1 mRNA levels in these cells. This result suggests that this potentially injurious stimulus causes IL-1 mRNA accumulation by an increase in rates of transcription. These LPS-induced increases in IL-1 mRNA levels corresponded to production of biologically active IL-1 determined as thymocyte costimulation activity. Interestingly, gel filtration experiments revealed a molecular weight of around 22kD for both SMC and EC-derived IL-1 secreted into culture medium in response to LPS. This molecular weight contrasts with the 17 kD species which is the fully processed product secreted frcm activated human monocytes. A possible explanation for this disparity is that the vascular cells secrete a partially processed intermediate form of mature IL-1. Other stimuli for IL-1 mRNA accumulation and secretion of biological activity include TNF and IL-1 itself. Recombinant human INF (≥ 10 ng/ml) increased IL-1 beta mRNA levels in EC & SMC, and caused the EC & SMC to release IL-1-1 ike thymocyte costimulation activity. Of interest is the recent observation that IL-1 itself can stimulate expression of IL-rl genes in vascular wall cells. Both IL-1 aloha and beta can increase IL-1 beta mRNA content in EC & SMC. Hris observation was confirmed with homogenous IL-1 prepared by recombinant DNA technologies (rIL-1). These findings raise the possibility of a novel positive feedback loop in vascular pathophysiology. We also found that rIL-1 alpha or beta also induced the production of prostaglandin E2 (PGE2) by both vascular SMC & EC. This prostanoid, induced by IL-1, inhibits thymocyte _ proliferation. Thus, IL-1 not only induced its own expression but increased production of this immunosuppressive prostanoid. This mechanism provides a potential negative control loop in regulation of the local immune response in blood vessels. Vie conclude that these cells of the blood vessel wall are a source of the potent vasoregulatory and immune mediators IL-1 alpha and beta. Since IL-1 influences the thrombotic, hemostatic, and fibrinolytic functions of endothelium, as well as other responses to acute injury, our findings suggest novel local control mechanisms that may be important in a variety of pathologic states.

Journal of Hematotherapy & Stem Cell ResearchVol. 11, No. 1 State-of-the-Art ReviewsThe Search for the HemangioblastHanna K.A. Mikkola and Stuart H. OrkinHanna K.A. MikkolaSearch for more papers by this author and Stuart H. OrkinSearch for more papers by this authorPublished Online:9 Jul 2004https://doi.org/10.1089/152581602753448504AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited byMurine hematopoietic stem cell activity is derived from pre-circulation embryos but not yolk sacs20 December 2018 | Nature Communications, Vol. 9, No. 1Ontogeny of the Hematopoietic SystemThe role of cytomegalovirus in angiogenesisVirus Research, Vol. 157, No. 2Nkx2-5 Represses Gata1 Gene Expression and Modulates the Cellular Fate of Cardiac Progenitors During EmbryogenesisCirculation, Vol. 123, No. 15Human embryonic stem cells hemangioblast express HLA-antigens22 April 2009 | Journal of Translational Medicine, Vol. 7, No. 1Cerebral arteriovenous malformations. Part 1: cellular and molecular biologyNeurosurgical Focus, Vol. 26, No. 5Vascular and Neuronal Development: Intersecting Parallelisms and rossroadsSimilarities Between Angiogenesis and Neural Development: What Small Animal Models Can Tell UsBlood-Borne Stem Cells Differentiate into Vascular and Cardiac Lineages During Normal Development Ning Zhang, Deanna Mustin, Wade Reardon, Angela De Almeida, Paul Mozdziak, Michal Mrug, Leonard M. Eisenberg, and David Sedmera7 March 2006 | Stem Cells and Development, Vol. 15, No. 1Hedgehog signaling plays a conserved role in inhibiting fat formationCell Metabolism, Vol. 3, No. 1Patterning definitive hematopoietic stem cells from embryonic stem cellsExperimental Hematology, Vol. 33, No. 9The role of Smad signaling in hematopoiesis29 August 2005 | Oncogene, Vol. 24, No. 37Expression of the Hematopoietic Stem Cell Antigen Sca-1 (LY-6A/E) in Liver Sinusoidal Endothelial Cells: Possible Function of Sca-1 in Endothelial Cells Gonzalo Luna, Jesus Paez, and José E. Cardier2 November 2004 | Stem Cells and Development, Vol. 13, No. 5Manipulating angiogenesis in medicineJournal of Internal Medicine, Vol. 255, No. 5Commentary: The Role of Cell Migration in the Ontogeny of the Lymphoid System Malcolm A.S. Moore9 July 2004 | Stem Cells and Development, Vol. 13, No. 1Ontogeny of the Hematopoietic SystemVascular Endothelial Growth Factor and Its Receptors in Embryonic Zebrafish Blood Vessel DevelopmentConverging roads: evidence for an adult hemangioblastExperimental Hematology, Vol. 31, No. 11Blood vessels and nerves: common signals, pathways and diseasesNature Reviews Genetics, Vol. 4, No. 9Angiogenesis in health and diseaseNature Medicine, Vol. 9, No. 6Cell Therapy: Filling the Gap Between Basic Science and Clinical Trials October 15–17, 2001, Rome, Italy23 December 2008 | STEM CELLS, Vol. 21, No. 3Endothelial cell heterogeneityCritical Care Medicine, Vol. 31, No. SupplementAngiogenesis and Anti-Angiogenesis in Hematological Malignancies Domenico Ribatti, Angelo Vacca, Franco Dammacco, and Denis English9 July 2004 | Journal of Hematotherapy & Stem Cell Research, Vol. 12, No. 1Vascular Lineage Differentiation from Human Embryonic Stem CellsA developing picture of lymphopoiesis in bone marrow11 December 2002 | Immunological Reviews, Vol. 189, No. 1Coagulation Affects Vasculogenesis and Angiogenesis: Does It Affect the Development of the Heart? Charlotte Ferencz9 July 2004 | Journal of Hematotherapy & Stem Cell Research, Vol. 11, No. 4 Volume 11Issue 1Feb 2002 To cite this article:Hanna K.A. Mikkola and Stuart H. Orkin.The Search for the Hemangioblast.Journal of Hematotherapy & Stem Cell Research.Feb 2002.9-17.http://doi.org/10.1089/152581602753448504Published in Volume: 11 Issue 1: July 9, 2004PDF download

Simple SummaryThe ability to recapitulate stem cells’ self-organising potential, creating three-dimensional (3D) structures of stem cells, has revolutionised various fields. Recently, in vitro 3D organoid systems are now used as alternative research tools because they serve the same purpose as in vivo systems. In the present study, we developed bovine intestinal organoids based on a 3D culture system and evaluated their characteristics. These bovine intestinal organoids, used in an in vitro model as a potential alternative to an in vivo system, hold great promise for further potential use in determining disease-modelling-based host–pathogen interactions and nutritional research for feed efficiency measurements.Intestinal organoids offer great promise for disease-modelling-based host–pathogen interactions and nutritional research for feed efficiency measurement in livestock and regenerative medicine for therapeutic purposes. However, very limited studies are available on the functional characterisation and three-dimensional (3D) expansion of adult stem cells in livestock species compared to other species. Intestinal crypts derived from intestinal organoids under a 3D culture system from the small intestine in adult bovine were successfully established and characterised for functionality testing, including the cellular potentials and genetic properties based on immunohistochemistry, immunocytochemistry, epithelial barrier permeability assay, QuantSeq 3′ mRNA-Seq. data and quantitative reverse transcription-polymerase chain reaction. Intestinal organoids were long-term cultivated over several passages of culture without loss of the recapitulating capacity of crypts, and they had the specific expression of several specific markers involved in intestinal stem cells, intestinal epithelium, and nutrient absorption. In addition, they showed the key functionality with regard to a high permeability for compounds of up to FITC-dextran 4 kDa, while FITC-dextran 40 kDa failed to enter the organoid lumen and revealed that the genetic properties of bovine intestinal organoids were highly similar to those of in vivo. Collectively, these results provide a reliable method for efficient isolation of intestinal crypts from the small intestine and robust 3D expansion of intestinal organoids in adult bovine and demonstrate the in vitro 3D organoids mimics the in vivo tissue topology and functionality. Finally, intestinal organoids are potential alternatives to in vivo systems and will be facilitated as the practical model to replace animal experiments for various purposes in the fields of animal biotechnology.

In the field of regenerative medicine, stem cell therapeutic development has emerged as a key technological area. Since the establishment and characterization of human embryonic stem cell lines by James Thomson in 1998, stem cell research has expanded rapidly, accelerating industrialization through the strategic protection of intellectual property (IP) rights. As technological specialization and clinical trial expansion have progressed-particularly since 2015-the feasibility of commercialization has increased. However, IP analyses in the stem cell field have largely been limited to fragmented patent trend studies. This study analyzed the characteristics and limitations of stem cell-based IP and examined the correlations among scientific publications, patent activity, and clinical trials by stem cell type from 2000 to 2020. The findings revealed significant differences among stem cell categories. In particular, adult stem cells (ASCs) demonstrated strong positive correlations between patent filings and SCI-indexed publications (R2=0.947), as well as between patent activity and clinical trials (R2=0.945), suggesting a high level of technological maturity and translational potential. In contrast, embryonic and induced pluripotent stem cells showed relatively weaker correlations. The results indicate that ASC-based IP is closely associated with clinical development and commercialization potential. This study provides strategic insights into the potential for proactive commercialization based on the importance of patents in the stem cell therapeutic industry.

The lacrimal glands produce tears to support a healthy homeostatic environment on the ocular surface. The lacrimal gland dysfunction characteristic of dry eye disease causes ocular discomfort and visual disturbances and in severe cases can result in a loss of vision. The demand for adequate restoration of lacrimal gland function has been intensified due to advances in stem cell biology, developmental biology, and bioengineering technologies. In addition to conventional therapies, including artificial tears, tear alternatives (such as autologous serum eye drops) and salivary gland transplantation, a regenerative medicine approach has been identified as a novel strategy to restore the function of the lacrimal gland. Recent studies have demonstrated the potential of progenitor cell injection therapy to repair the tissue of the lacrimal glands. A current three-dimensional (3D) tissue engineering technique has been shown to regenerate a secretory gland structure by reproducing reciprocal epithelial-mesenchymal interactions during ontogenesis in vitro and in vivo. A novel direct reprogramming method has suggested a possibility to induce markers in the lacrimal gland developmental process from human pluripotent stem cells. The development of this method is supported by advances in our understanding of gene expression and regulatory networks involved in the development and differentiation of the lacrimal glands. Engineering science has proposed a medical device to stimulate tearing and a bio-hybrid scaffold to reconstruct the 3D lacrimal gland structure. In this review, we will summarize recent bioengineering advances in lacrimal gland regeneration toward the functional restoration of the lacrimal glands as a future dry eye therapy.

The increasing incidence of arteriosclerosis has become a significant global health burden. Arteriosclerosis is characterized by the thickening and hardening of arterial walls, which can lead to the narrowing or complete blockage of blood vessels. However, the pathogenesis of the disease remains incompletely understood. Recent research has shown that stem and progenitor cells found in the bone marrow and local vessel walls play a role in the development of arteriosclerosis by differentiating into various types of vascular cells, including endothelial cells, smooth muscle cells, fibroblasts, and inflammatory cells. This review aims to provide a comprehensive understanding of the role of stem and progenitor cells in the pathogenesis of arteriosclerosis, shedding light on the underlying mechanisms and potential therapeutic approaches for this disease.

Regeneration of complex oral organs using 3D cell organization technology

The complementary metal-oxide-semiconductor (CMOS) industry continues to push the boundaries of what is possible. Along with this, secondary ion mass spectrometry (SIMS) depth profiling continues to support CMOS R&D and high volume manufacturing (due to its unparalleled sensitivity and detection limits over predefined volumes and within acceptable time periods). New developments in SIMS are also being realized to support the move of the CMOS industry from planar structures to three-dimensional (3D) structures. This article presents a perspective of existing SIMS research areas for beyond one-dimensional structural analysis along with potential future SIMS implementation and data processing scenarios. The topics covered include: (1) existing SIMS depth profiling approaches for 3D structures, (2) in-fab SIMS deployment, (3) data analysis of in-fab SIMS derived depth profiles from 3D structures via pattern recognition, and (4) hybrid characterization approaches. With SIMS depth profiling of optimized (for SIMS) 3D structures already demonstrated, the remaining topics may serve to extend these and other capabilities and open new application areas for SIMS.

Living tissues have complex and well-organized microstructures. Although microtechnology has been used to create in vivo-like cell microstructures in vitro, most available microscale systems are two-dimensional, and few three-dimensional (3D) systems have been explored. This article demonstrates a 3D hierarchical biomimetic multilayer microsystem created by a generally applicable technique. The technique employs layer-by-layer microfluidics to build layers of cells and biopolymers in microchannels, allowing controlled patterning of cells and their microenvironments in the x, y, and z-dimension. As a prototype, a multilayer system was created using three vascular cell types within heterogeneous types of biopolymers to mimic the structure and composition of a blood vessel wall. The effects of matrix composition and multilayer configurations on 3D cell-cell interactions and cell biology were revealed. Cell migration in the z-dimension, matrix remodeling, intercellular adhesion molecule expression and actin organization were examined under different 3D coculture conditions. A more biomimetic coculture was found to reproduce a more stable structure and in vivo-like function. This approach provides a method to fabricate microscale hierarchical "neotissues" with 3D configurations of matrix materials and multiple cell types, and an in vitro cell coculture model to understand 3D processes of cell-cell and cell-matrix interactions.