Reciprocal interactions between alveolar fibroblasts and epithelial cells are crucial for lung homeostasis, injury repair, and fibrogenesis, but underlying mechanisms remain unclear. To investigate, we administered the fibroblast-selective TGFβ1 signaling inhibitor, epigallocatechin gallate (EGCG), to Interstitial Lung Disease (ILD) patients undergoing diagnostic lung biopsy and conducted single-cell RNA sequencing on spare tissue. Biopsies from untreated patients showed higher fibroblast TGFβ1 signaling compared to non-disease donor or end-stage ILD tissues. In vivo, EGCG downregulated TGFβ1 signaling and several pro-inflammatory and stress pathways in biopsy samples. Notably, EGCG reduced fibroblast secreted frizzle-like receptor protein 2 (sFRP2), an unrecognized TGFβ1 fibroblast target gene induced near type II alveolar epithelial cells (AEC2s) in situ. Using AEC2-fibroblast coculture organoids and precision cut lung slices (PCLS) from non-diseased donors, we found TGFβ1 signaling promotes a spread AEC2 KRT17+ basaloid state, whereupon sFRP2 then activates a mature Krt5+ basal cell program. Wnt-receptor Frizzled 5 (Fzd5) expression and downstream calcineurin signaling were required for sFRP2-induced nuclear NFATc3 accumulation and KRT5 expression. These findings highlight stage-specific TGFβ1 signaling in ILD, the therapeutic potential of EGCG in reducing IPF-related transcriptional changes, and identify TGFβ1-non-canonical Wnt pathway crosstalk via sFRP2 as a novel mechanism for dysfunctional epithelial signaling in Idiopathic Pulmonary Fibrosis/ILD.

Within this interdisciplinary study, we demonstrate the applicability of a 6D printer for soft tissue engineering models. For this purpose, a special plant was constructed, combining the technical requirements for 6D printing with the biological necessities, especially for soft tissue. Therefore, a commercial 6D robot arm was combined with a sterilizable housing (including a high-efficiency particulate air (HEPA) filter and ultraviolet radiation (UVC) lamps) and a custom-made printhead and printbed. Both components allow cooling and heating, which is desirable for working with viable cells. In addition, a spraying unit was installed that allows the distribution of fine droplets of a liquid. Advanced geometries on uneven or angled surfaces can be created with the use of all six axes. Based on often used bioinks in the field of soft tissue engineering (gellan gum, collagen, and gelatin methacryloyl) with very different material properties, we could demonstrate the flexibility of the printing system. Furthermore, cell-containing constructs using primary human adipose-derived stem cells (ASCs) could be produced in an automated manner. In addition to cell survival, the ability to differentiate along the adipogenic lineage could also be demonstrated as a representative of soft tissue engineering.

Galactic cosmic radiation (GCR) is one of the most serious risks posed to astronauts during missions to the Moon and Mars. Experimental models capable of recapitulating human physiology are critical to understanding the effects of radiation on human organs and developing radioprotective measures against space travel exposures. The effects of systemic radiation are studied using a multi-organ-on-a-chip (multi-OoC) platform containing engineered tissue models of human bone marrow (site of hematopoiesis and acute radiation damage), cardiac muscle (site of chronic radiation damage) and liver (site of metabolism), linked by vascular circulation with an endothelial barrier separating individual tissue chambers from the vascular perfusate. Following protracted neutron radiation, the most damaging radiation component in deep space, a greater deviation of tissue function is observed as compared to the same cumulative dose delivered acutely. Further, by characterizing engineered bone marrow (eBM)-derived immune cells in circulation, 58 unique genes specific to the effects of protracted neutron dosing are identified, as compared to acutely irradiated and healthy tissues. It propose that this bioengineered platform allows studies of human responses to extended radiation exposure in an \"astronaut-on-a-chip\" model that can inform measures for mitigating cosmic radiation injury.

BACKGROUND: Tuberous sclerosis complex (TSC) is a multi-system genetic disease that causes benign tumors in the brain and other vital organs. The most debilitating symptoms result from involvement of the central nervous system and lead to a multitude of severe symptoms including seizures, intellectual disability, autism, and behavioral problems. TSC is caused by heterozygous mutations of either the TSC1 or TSC2 gene and dysregulation of mTOR kinase with its multifaceted downstream signaling alterations is central to disease pathogenesis. Although the neurological sequelae of the disease are well established, little is known about how these mutations might affect cellular components and the function of the blood-brain barrier (BBB).
METHODS: We generated TSC disease-specific cell models of the BBB by leveraging human induced pluripotent stem cell and microfluidic cell culture technologies.
RESULTS: Using microphysiological systems, we demonstrate that a BBB generated from TSC2 heterozygous mutant cells shows increased permeability. This can be rescued by wild type astrocytes or by treatment with rapamycin, an mTOR kinase inhibitor.
CONCLUSIONS: Our results demonstrate the utility of microphysiological systems to study human neurological disorders and advance our knowledge of cell lineages contributing to TSC pathogenesis and informs future therapeutics.

We previously reported that human muscle-derived stem cells (hMuStem cells) contribute to tissue repair after local administration into injured skeletal muscle or infarcted heart in immunodeficient rodent models. However, extrapolation of these findings to a clinical context is problematic owing to the considerable differences often seen between in vivo findings in humans versus rodents. Therefore, we investigated whether the muscle regenerative behavior of hMuStem cells is maintained in a clinically relevant transplantation context. Human MuStem cells were intramuscularly administered by high-density microinjection matrices into nonhuman primates receiving tacrolimus-based immunosuppression thereby reproducing the protocol that has so far produced the best results in clinical trials of cell therapy in myopathies. Four and 9 weeks after administration, histological analysis of cell injection sites revealed large numbers of hMuStem cell-derived nuclei in all cases. Most graft-derived nuclei were distributed in small myofiber groups in which no signs of a specific immune response were observed. Importantly, hMuStem cells contributed to simian tissue repair by fusing mainly with host myofibers, demonstrating their capacity for myofiber regeneration in this model. Together, these findings obtained in a valid preclinical model provide new insights supporting the potential of hMuStem cells in future cell therapies for muscle diseases.

Utilizing tissue-specific extracellular matrices (ECMs) is vital for replicating the composition of native tissues and developing biologically relevant biomaterials. Human- or animal-derived donor tissues and organs are the current gold standard for the source of these ECMs. To overcome the several limitations related to these ECM sources, including the highly limited availability of donor tissues, cell-derived ECM offers an alternative approach for engineering tissue-specific biomaterials, such as bioinks for three-dimensional (3D) bioprinting. 3D bioprinting is a state-of-the-art biofabrication technology that addresses the global need for donor tissues and organs. In fact, there is a vast global demand for human donor corneas that are used for treating corneal blindness, often resulting from damage in the corneal stromal microstructure. Human adipose tissue is one of the most abundant tissues and easy to access, and adipose tissue-derived stem cells (hASCs) are a highly advantageous cell type for tissue engineering. Furthermore, hASCs have already been studied in clinical trials for treating corneal stromal pathologies. In this study, a corneal stroma-specific ECM was engineered without the need for donor corneas by differentiating hASCs toward corneal stromal keratocytes (hASC-CSKs). Furthermore, this ECM was utilized as a component for corneal stroma-specific bioink where hASC-CSKs were printed to produce corneal stroma structures. This cost-effective approach combined with a clinically relevant cell type provides valuable information on developing more sustainable tissue-specific solutions and advances the field of corneal tissue engineering.

Mutations in ATP-binding cassette A3 (ABCA3), a phospholipid transporter critical for surfactant homeostasis in pulmonary alveolar type II epithelial cells (AEC2s), are the most common genetic causes of childhood interstitial lung disease (chILD). Treatments for patients with pathological variants of ABCA3 mutations are limited, in part due to a lack of understanding of disease pathogenesis resulting from an inability to access primary AEC2s from affected children. Here, we report the generation of AEC2s from affected patient induced pluripotent stem cells (iPSCs) carrying homozygous versions of multiple ABCA3 mutations. We generated syngeneic CRISPR/Cas9 gene-corrected and uncorrected iPSCs and ABCA3-mutant knockin ABCA3:GFP fusion reporter lines for in vitro disease modeling. We observed an expected decreased capacity for surfactant secretion in ABCA3-mutant iPSC-derived AEC2s (iAEC2s), but we also found an unexpected epithelial-intrinsic aberrant phenotype in mutant iAEC2s, presenting as diminished progenitor potential, increased NFκB signaling, and the production of pro-inflammatory cytokines. The ABCA3:GFP fusion reporter permitted mutant-specific, quantifiable characterization of lamellar body size and ABCA3 protein trafficking, functional features that are perturbed depending on ABCA3 mutation type. Our disease model provides a platform for understanding ABCA3 mutation-mediated mechanisms of alveolar epithelial cell dysfunction that may trigger chILD pathogenesis.

Three-dimensional (3D) bioprinting offers an automated, customizable solution to manufacture highly detailed 3D tissue constructs and holds great promise for regenerative medicine to solve the severe global shortage of donor tissues and organs. However, uni-material 3D bioprinting is not sufficient for manufacturing heterogenous 3D constructs with native-like microstructures and thus, innovative multi-material solutions are required. Here, we developed a novel multi-material 3D bioprinting strategy for bioprinting human corneal stroma. The human cornea is the transparent outer layer of your eye, and vision loss due to corneal blindness has serious effects on the quality of life of individuals. One of the main reasons for corneal blindness is the damage in the detailed organization of the corneal stroma where collagen fibrils are arranged in layers perpendicular to each other and the corneal stromal cells grow along the fibrils. Donor corneas for treating corneal blindness are scarce, and the current tissue engineering (TE) technologies cannot produce artificial corneas with the complex microstructure of native corneal stroma. To address this, we developed a novel multi-material 3D bioprinting strategy to mimic detailed organization of corneal stroma. These multi-material 3D structures with heterogenous design were bioprinted by using human adipose tissue -derived stem cells (hASCs) and hyaluronic acid (HA) -based bioinks with varying stiffnesses. In our novel design of 3D models, acellular stiffer HA-bioink and cell-laden softer HA-bioink were printed in alternating filaments, and the filaments were printed perpendicularly in alternating layers. The multi-material bioprinting strategy was applied for the first time in corneal stroma 3D bioprinting to mimic the native microstructure. As a result, the soft bioink promoted cellular growth and tissue formation of hASCs in the multi-material 3D bioprinted composites, whereas the stiff bioink provided mechanical support as well as guidance of cellular organization upon culture. Interestingly, cellular growth and tissue formation altered the mechanical properties of the bioprinted composite constructs significantly. Importantly, the bioprinted composite structures showed good integration to the host tissue in ex vivo cornea organ culture model. As a conclusion, the developed multi-material bioprinting strategy provides great potential as a biofabrication solution for manufacturing organized, heterogenous microstructures of native tissues. To the best of our knowledge, this multi-material bioprinting strategy has never been applied in corneal bioprinting. Therefore, our work advances the technological achievements in additive manufacturing and brings the field of corneal TE to a new level.

Tuberous sclerosis complex (TSC) is a multi-system genetic disease that causes benign tumors in the brain and other vital organs. The most debilitating symptoms result from involvement of the central nervous system and lead to a multitude of severe symptoms including seizures, intellectual disability, autism, and behavioral problems. TSC is caused by heterozygous mutations of either the TSC1 or TSC2 gene. Dysregulation of mTOR kinase with its multifaceted downstream signaling alterations is central to disease pathogenesis. Although the neurological sequelae of the disease are well established, little is known about how these mutations might affect cellular components and the function of the blood-brain barrier (BBB). We generated disease-specific cell models of the BBB by leveraging human induced pluripotent stem cell and microfluidic cell culture technologies. Using these microphysiological systems, we demonstrate that the BBB generated from TSC2 heterozygous mutant cells shows increased permeability which can be rescued by wild type astrocytes and with treatment with rapamycin, an mTOR kinase inhibitor. Our results further demonstrate the utility of microphysiological systems to study human neurological disorders and advance our knowledge of the cell lineages contributing to TSC pathogenesis.

UNASSIGNED: To explore the advances and future research directions in image analysis and computational modelling of human stem cells (hSCs) for ophthalmological applications.
UNASSIGNED: hSCs hold great potential in ocular regenerative medicine due to their application in cell-based therapies and in disease modelling and drug discovery using state-of-the-art 2D and 3D organoid models. However, a deeper characterisation of their complex, multi-scale properties is required to optimise their translation to clinical practice. Image analysis combined with computational modelling is a powerful tool to explore mechanisms of hSC behaviour and aid clinical diagnosis and therapy.
UNASSIGNED: Many computational models draw on a variety of techniques, often blending continuum and discrete approaches, and have been used to describe cell differentiation and self-organisation. Machine learning tools are having a significant impact in model development and improving image classification processes for clinical diagnosis and treatment and will be the focus of much future research.