Journal of Biological Engineering

Apoptosis is an essential physiological process involved in embryonic development, immune responses, and tissue homeostasis. Despite many studies on pro-apoptotic genes, few reports have directly compared the lethality-inducing potential between them under identical conditions. In this study, we evaluated the lethality-inducing potential of three representative pro-apoptotic genes, Bax, Casp3, and Casp9, in mouse early embryos under defined conditions using the doxycycline (Dox)-inducible tetracycline-regulated gene expression system (Tet-On system) in combination with the PiggyBac transposon system. All genes were transcriptionally induced by Dox, and Bax showed the strongest lethal effect, followed by Casp9, while Casp3 did not show any effect. Notably, Bax expression severely impaired blastocyst formation and led to the intense accumulation of the DNA damage marker {gamma}H2AX. These findings suggest that introducing upstream apoptotic regulators leads to the more efficient and widespread activation of the apoptotic cascade. Additionally, an unexpected Dox-dependent increase in the expression of reverse tetracycline-controlled transactivator, which is typically driven by a constitutive promoter, was observed, raising the possibility of unanticipated regulatory mechanisms within the Tet-On system. Overall, this study is expected to contribute to a deeper understanding of apoptotic mechanisms and future advancements in regenerative medicine, reproductive engineering, and cancer research.

Cell plasticity, defined as the ability to undergo phenotypical transformation in a reversible manner, is a physiological processes that also exert important roles in disease progression Two forms of cellular plasticity are epithelial-mesenchymal transition (EMT) and its inverse process, mesenchymal-epithelial transition (MET). These processes have been correlated to the poor outcome of different types of neoplasias as well as drug resistance development. Since EMT/MET are transitional processes, we have generated and validated a reporter cell line. Specifically, a far-red fluorescent protein was knocked-in in-frame with the mesenchymal gene marker VIMENTIN (VIM) in H2170 lung cancer cells. The vimentin reporter cells (VRCs) are a reliable model for studying EMT and MET showing cellular plasticity upon a series of stimulations. These cells are a robust platform to dissect the molecular mechanisms of these processes, and for drug discovery in vitro and in the future in vivo.

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related deaths in the U.S., despite only being the 11th most common cancer. The high mortality rates of PDAC can be partially attributed to the tumor microenvironment. Unlike most carcinomas, PDAC is characterized by a strong desmoplastic reaction, or a fibrotic stiffening of the extracellular matrix (ECM) in response to chronic inflammation. The desmoplastic reaction is mediated by cancer-associated fibroblasts that deposit ECM proteins (collagens, laminins, fibronectin, etc.) and secrete matrix-remodeling proteins in the tumor parenchyma. Within the past decade, the enzyme quiescin sulfhydryl oxidase 1 (QSOX1) has gained recognition as a significant contributor to solid tumor pathogenesis, but its biological role remains uncertain. QSOX1 is a disulfide bond-generating catalyst that participates in oxidative protein folding in the mammalian cell. Current studies show that inhibiting or knocking down QSOX1 reduces pancreatic cancer cell migration and invasion, alters ECM deposition and organization, and decreases overall tumor growth in mice. However, it is unclear which features of the tumor microenvironment modulate QSOX1 and cause its overexpression in cancer. In this study, we explored potential regulators of QSOX1 expression and secretion by testing two major features of PDAC: hypoxia and mechanical stiffness. To induce hypoxia, we exposed pancreatic cancer cells to atmospheric (low O2) and chemical (CoCl2) hypoxia for up to 48 hours. QSOX1 gene and protein expression did not change in response to hypoxia. Substratum stiffness was modulated using polyacrylamide gels to represent the dynamic pathological range of elastic moduli found in PDAC tissue. We discovered that QSOX1 levels were decreased on softer surfaces compared to conventional tissue culture plastic. This paper presents new results and challenges prior findings on QSOX1 regulation in pancreatic tumor cells.

Controlling basic fibroblast growth factor (bFGF) signaling is important for both tissue-engineering purposes, controlling proliferation and differentiation potential, and for cancer biology, influencing tumor progression and metastasis. Here, we observed that human mesenchymal stromal cells (hMSCs) no longer responded to soluble or covalently bound bFGF when cultured on microfibrillar substrates, while fibroblasts did. This correlated with a downregulation of FGF receptor 1 (FGFR1) expression of hMSCs on microfibrillar substrates, compared to hMSCs on conventional tissue culture plastic (TCP). hMSCs also expressed less SRF on ESP scaffolds, compared to TCP, while fibroblasts maintained high FGFR1 and SRF expression. Inhibition of actin-myosin tension or the MRTF/SRF pathway decreased FGFR1 expression in hMSCs, fibroblasts and MG63 osteosarcoma cells. This downregulation was functional, as hMSCs became irresponsive to bFGF in the presence of MRTF/SRF inhibitor. Together, our data show that hMSCs, but not fibroblasts, are irresponsive to bFGF when cultured on microfibrillar susbtrates by downregulation of FGFR1 through the MRTF/SRF pathway. This is the first time FGFR1 expression has been shown to be mechanosensitive and adds to the sparse literature on FGFR1 regulation. These results could open up new targets for cancer treatments and could aid designing tissue engineering constructs that better control cell proliferation.

Adipose tissue is an active endocrine organ that can signal bidirectionally to many tissues and organ systems in the body. With obesity, adipose tissue is a source of low-level inflammation that contributes to various co-morbidities and damage to downstream effector tissues. The ability to synthesize genetically engineered adipose tissue could have critical applications in studying adipokine signaling and the use of adipose tissue for novel therapeutic strategies. This study aimed to develop a method for non-viral adipogenic differentiation of genome-edited murine induced pluripotent stem cells (iPSCs) and to test the ability of such cells to engraft in mice in vivo. Designer adipocytes were created from iPSCs, which can be readily genetically engineered using CRISPR-Cas9 to knock out or insert individual genes of interest. As a model system for adipocyte-based drug delivery, an existing iPSC cell line that transcribes interleukin 1 receptor antagonist under the endogenous macrophage chemoattractant protein-1 promoter was tested for adipogenic capabilities under these same differentiation conditions. To understand the role of various adipocyte subtypes and their impact on health and disease, an efficient method was devised for inducing browning and whitening of IPSC-derived adipocytes in culture. Finally, to study the downstream effects of designer adipocytes in vivo, we transplanted the designer adipocytes into fat-free lipodystrophic mice as a model system for studying adipose signaling in different models of disease or repair. This novel translational tissue engineering and regenerative medicine platform provides an innovative approach to studying the role of adipose interorgan communication in various conditions.

The development of regenerative strategies to repair the heart is of high importance. Our lab has shown that extracellular matrix derived from decellularized fetal myocardium promotes neonatal cardiomyocyte proliferation in vitro. The goal of this study was to identify specific peptide(s)/protein(s) in solubilized cardiac ECM responsible for this proliferative effect. We hypothesized that isolation and then treatment with one or more small synthetic peptide derived from this source could replicate the cellular response to whole solubilized ECM. Decellularized fetal and adult rat hearts were fractionated by molecular weight using SDS-PAGE and transferred to PVDF membranes. Analysis of cardiomyocytes cultured on the membranes revealed regions of enhanced cardiomyocyte proliferation. Subsequent isolation and proteomic analysis of the protein bands that that correlated with proliferative regions identified fibrillin-1 as the predominant ECM protein associated with these regions of cardiomyocyte proliferation. One region (residues 55-86) of fibrillin-1 was synthesized as a peptide and tested for a direct effect on cardiomyocyte proliferation. Compared to positive and negative controls, as well as scrambled and alkylated versions, this peptide led to 3-4 fold increase in cardiomyocyte proliferation. Analysis of the amino acid sequence demonstrated high homology with laent-TGF-{beta} binding proteins and subsequent experiments showed that the matrikine could also reduce TGF-{beta} induced activation of cardiac fibroblasts. These data suggest that individual peptides derived from soluble ECM could have utility as a novel therapeutic for cardiac tissue engineering and regeneration.

Adipocyte differentiation plays an important role in bone remodeling due to secretory factors that can directly modulate osteoblast and osteoclast, thus affecting overall bone mass and skeletal integrity. Excessive adipocyte differentiation within bone marrow microenvironment can lead to decreased bone mass, eventually causing osteoporosis. Mechanical microenvironment of bone marrow, including fluid shear, maintains the balance of adipocyte and osteoblast differentiation during bone remodeling. However, how mechanical cues interact with long noncoding RNA (lncRNA) and regulating adipocyte differentiation remains unexplored. In this study, we investigated the mechanosensitive role of lncRNA MALAT1 during mesenchymal stem cells (MSCs) adipocyte differentiation. By applying physiologically relevant shear stress, MSCs experienced morphological changes and adipocyte differentiation differences. Shear stress inhibits MSCs adipocyte differentiation with reduced oil-red-o-stained lipid droplets. Silencing MALATs results in reduced adipocyte differentiation. By leveraging a novel gapmer double stranded locked nuclei acid (dsLNA) nanobiosensor, we showed that shear stress inhibits MALAT1 expression with significantly reduced fluorescence intensity. Our findings suggest that shear stress influences adipocyte differentiation primarily through the downregulation of MALAT1, highlighting a significant interplay between biophysical cues and lncRNAs. This interaction is crucial for understanding the complexities of bone remodeling and the potential therapeutic targeting of lncRNAs to treat bone-related disorders.

Estrogens are global regulators of cellular signaling pathways, impacting fundamental processes and phenotypes that are essential for tissue remodeling and homeostasis. Traditional cell culture media contains estrogen-mimetic compounds, including phenol red and endogenous estrogen in fetal bovine serum (FBS). However, the potential of these compounds to bias in vitro studies, particularly when considering sex as a biological variable, remains unclear. This gap in understanding critically impacts the culture of human mesenchymal stromal cells (hMSCs), whose basic functions and differentiation potential, central to cell therapy and tissue engineering, are sensitive to perturbations in the culture conditions. Despite this, the effect of estrogens from cell culture media on male and female hMSCs is not currently considered in cell processing for clinical trials. As such, a baseline understanding of these estrogen-mimetic media influences on hMSCs is critical for clinical efficacy and adequate study design in research. To this end, we investigated the effects of phenol red and fetal bovine serum on the proliferation, metabolism, senescence, and differentiation capacity of male and female hMSCs. Phenol red, FBS, donor sex, and 17{beta}-estradiol (E2) supplementation all had significant impacts on hMSC health and differentiation potential in culture. Notably, dosing with estrogen at the levels found in FBS did not recover most of the hMSC metrics tested. The only outcomes that were not significantly different based on donor sex were senescence and mRNA transcripts for RUNX2 and PPARG, transcriptional regulators for osteogenesis and adipogenesis. Overall, these findings reveal the sex-biased effects of estrogen and estrogen-mimetic compounds in traditional culture media, underscoring a current gap in considering sex as a biological variable in cell therapy and tissue engineering research and manufacturing.

In prokaryotes, the Rho protein mediates Rho-dependent termination (RDT) by identifying a non-specific cytosine-rich Rho utilization site on the newly synthesized RNA. As a result of RDT, downstream RNA transcription is reduced. Due to the bias in reverse transcription and PCR amplification, we were unable to identify the RDT site by directly measuring the amount of mRNA upstream and downstream of RDT sites. To overcome this difficulty, we employed a 77 bp reporter gene argX, coding transfer RNA that binds L-arginine, tRNAarg from Brevibacterium albidum, and transcriptionally fused it to the sequences to be assayed. We constructed a series of plasmids by combining a segment of the galactose (gal) operon sequences, both with and without the RDT regions at the ends of cistrons (galE, galT, and galM) upstream of argX. The RNA polymerase will transcribe the gal operon sequence and argX unless it encounters the RDT encoded by the inserted sequence. We observed similar tRNAarg half-lives expressed in these transcriptional fusion plasmids. Therefore, the amount of tRNAarg directly represents the number of transcripts transcribed. Using this approach, we were able to effectively assay the RDTs in the gal operon by quantifying the relative amount of tRNAarg using quantitative real-time PCR (qRT-PCR) analyses. The resultant RDT% for galET, galTK, and at the end of galM were 36, 26, and 63, individually. Our findings demonstrate that combining tRNAarg, with qRT-PCR can directly measure RDT efficiencies in vivo, making it a useful tool for gene expression research.

Progress toward the development of sex-specific tissue engineered systems has been hampered by the lack of research efforts to define the effects of sex-specific hormone concentrations on relevant human cell types. Here, we investigated the effects of defined concentrations of estradiol (E2) and dihydrotestosterone (DHT) on primary human dermal and lung fibroblasts (HDF and HLF), and human umbilical vein endothelial cells (HUVEC) from female (XX) and male (XY) donors in both 2D expansion cultures and 3D stromal vascular tissues. Sex-matched E2 and DHT stimulation in 2D expansion cultures significantly increased the proliferation index, mitochondrial membrane potential, and the expression of genes associated with bioenergetics (Na+/K+ ATPase, somatic cytochrome C) and beneficial stress responses (chaperonin) in all cell types tested. Notably, cross sex hormone stimulation, i.e., DHT treatment of XX cells in the absence of E2 and E2 stimulation of XY cells in the absence of DHT, decreased bioenergetic capacity and inhibited cell proliferation. We used a microengineered 3D vasculogenesis assay to assess hormone effects on tissue scale morphogenesis. E2 increased metrics of vascular network complexity compared to vehicle in XX tissues. Conversely, and in line with results from 2D expansion cultures, E2 potently inhibited vasculogenesis compared to vehicle in XY tissues. DHT did not significantly alter vasculogenesis in XX or XY tissues but increased the number of non-participating endothelial cells in both sexes. This study establishes a scientific rationale and adaptable methods for using sex hormone stimulation to develop sex-specific culture systems.

Mesenchymal stem cells (MSC) maintain the musculoskeletal system by differentiating into multiple cell types including osteocytes and adipocytes. Mechanical signals, including strain and low intensity vibration (LIV), are important regulators of MSC differentiation. Lamin A/C is a vital protein for nuclear architecture that supports chromatin organization, as well as mechanical integrity and mechano-sensitivity of the nucleus in MSCs. Here, we investigated whether Lamin A/C and mechano-responsiveness are functionally coupled during adipogenesis. Lamin depletion in MSCs using siRNA increased nuclear area, height and volume and decreased circularity and stiffness, while phosphorylation of focal adhesions and dynamic substrate strain in response to LIV remained intact. Lamin A/C depletion decelerates adipogenesis as reflected by delayed appearance of key biomarkers (e.g., adiponectin/ADIPOQ). Based on RNA-seq data, reduced Lamin A/C levels decrease the activation of the adipocyte transcriptome that is normally observed in response to adipogenic cues mediating differentiation of MSCs. Mechanical stimulation via daily LIV application reduced the expression levels of ADIPOQ in both control and Lamin A/C depleted cells. Yet, treatment with LIV did not induce major transcriptome changes in either control or Lamin A/C depleted MSCs, suggesting that the biological effects of LIV on adipogenesis may not occur at the transcriptional level. We conclude that while Lamin A/C activation is essential for normal adipogenesis, it is dispensible for activation of focal adhesions by dynamic vibration induced mechanical signals.

Mechanotransduction is a biological phenomenon where mechanical stimuli are converted to biochemical responses. A model system for studying mechanotransduction are the chondrocytes of articular cartilage. Breakdown of this tissue results in decreased mobility, increased pain, and reduced quality of life. Either disuse or overloading can disrupt cartilage homeostasis, but physiological cyclical loading promotes cartilage homeostasis. To model this, we exposed SW1353 cells to cyclical mechanical stimuli, shear and compression, for different durations of time (15 and 30 min). By utilizing liquid chromatography-mass spectroscopy (LC-MS), metabolomic profiles were generated detailing metabolite features and biological pathways that are altered in response to mechanical stimulation. In total, 1,457 metabolite features were detected. Statistical analyses identified several pathways of interest. Taken together, differences between experimental groups were associated with inflammatory pathways, lipid metabolism, beta-oxidation, central energy metabolism, and amino acid production. These findings expand our understanding of chondrocyte mechanotransduction under varying loading conditions and time periods.

Blood vessel formation is an important initial step for bone formation during development as well as during remodelling and repair in the adult skeleton. This results in a heavily vascularized tissue where endothelial cells and skeletal cells are constantly in crosstalk to facilitate homeostasis, a process that is mediated by numerous environment signals, including mechanical loading. Breakdown in this communication can lead to disease and/or poor fracture repair. Therefore, this study aimed to determine the role of mature bone cells in regulating angiogenesis, how this is influenced by a dynamic mechanical environment, and understand the mechanism by which this could occur. Herein, we demonstrate that both osteoblasts and osteocytes coordinate endothelial cell proliferation, migration, and blood vessel formation via a mechanically dependent paracrine mechanism. Moreover, we identified that this process is mediated via the secretion of extracellular vesicles (EVs), as isolated EVs from mechanically stimulated bone cells elicited the same response as seen with the full secretome, while the EV depleted secretome did not elicit any effect. Despite mechanically activated bone cell derived EVs (MA-EVs) driving a similar response to VEGF treatment, MA-EVs contain minimal quantities of this angiogenic factor. Lastly, a miRNA screen identified mechanoresponsive miRNAs packaged within MA-EVs which are linked with angiogenesis. Taken together, this study has highlighted an important mechanism in osteogenic-angiogenic coupling in bone and has identified the mechanically activated bone cell derived EVs as a therapeutic to promote angiogenesis and potentially bone repair.

Adeno-associated viruses (AAVs) have emerged as powerful tools for delivery of genes to a variety of cell types including pancreatic endocrine cells. Currently, AAV serotype 8 (AAV8) is the main AAV vector employed for infecting pancreatic cells for transgene transfer. We aimed to address whether alternative serotypes (AAV2, AAV6, and AAV9) commonly used for gene transfer can be effective in transducing pancreatic cells efficiently. We also screened the additives heparin and neuraminidase to further understand the interaction between the individual AAV types included in this work and the cells for optimal infection. Murine pancreatic {beta}-cells and -cells as well as fibroblasts were infected with AAV serotypes 2, 6, and 9 carrying the transgene for enhanced green fluorescent protein (eGFP). AAV2 outperformed AAV9 in transducing pancreatic cells, while AAV6 induced cytotoxicity. Both AAV2 and AAV9 displayed slightly higher tropism for -cells than for {beta}-cells. Compared to the pancreatic cells, the fraction of GFP-expressing cells at various multiplicities of infection was consistently lower for fibroblasts. Incubation of AAV2 with heparin prior to transduction failed to induce any GFP expression in {beta}-cells, indicating that the primary site used for initial interaction with pancreatic cells are heparan sulfate proteoglycans. Treatment of {beta}-cells with neuraminidase prior to AAV9 infection appeared to improve the number of GFP-positive cells, but the increase was not statistically significant. These findings expand the repertoire of available serotypes for AAV-mediated delivery of transgenes to pancreatic endocrine cells and may contribute to gene therapy strategies for pancreas pathologies.

Transcription factor HAND2 has a significant role in vascularization, angiogenesis, and cardiac neural crest development. Also, it is one of the key cardiac factors crucial for the enhanced derivation of functional and mature myocytes from non-myocyte cells. Here, we report the generation of the recombinant human HAND2 fusion protein from the heterologous system. First, we cloned the full-length human HAND2 gene (only protein-coding sequence) after codon optimization along with the fusion tags (for cell penetration, nuclear translocation, and affinity purification) into the expression vector. We then transformed and expressed it in Escherichia coli (E. coli) strain, BL21(DE3). Next, the effect (in terms of expression) of tagging of fusion tags with this recombinant protein at two different terminals was also investigated. Notably, using affinity chromatography, we established the one-step homogeneous purification of human recombinant HAND2 protein; and through circular dichroism spectroscopy, we established that this purified protein had retained its secondary structure. Furthermore, we show that this purified human protein could transduce the human cells and translocate to its nucleus. Prospectively, the purified recombinant HAND2 protein can potentially be a safe and effective molecular tool in the direct cardiac reprogramming process and other biological applications.

Diabetes is a chronic metabolic disorder that affects 422 million people worldwide and can lead to diabetic myopathy and bone diseases. The etiology of musculoskeletal complications in diabetes and the interplay between the muscular and osseous systems are poorly understood. Exercise training promises to prevent diabetic myopathy and diabetic bone disease and offer protective effects on muscle and bone. Although the muscle-bone interaction is largely biomechanical, the muscle secretome, specifically the myokines, has significant implications for bone biology. Here, we have developed an in vitro model to elucidate the effects of mechanical strain on myokine secretion and its impact on bone metabolism decoupled from physical stimuli. We developed modular bone constructs using crosslinked gelatin hydrogels which facilitated osteogenic differentiation of osteoprogenitor cells. Then muscle constructs were made from fibrin hydrogel, which enabled myoblast differentiation and formed mature myotubes. We investigated the myokine expression by the muscle constructs under strain regimens replicating endurance (END) and high-intensity interval training (HIIT) in hyperglycemic conditions. In monocultures, both regimens induced higher expression of Il15 and Igf1, while END supported more myoblasts differentiation and myotube maturation than HIIT. When cocultured with bone constructs, the HIIT regimen increased Glut4 expression in muscle contructs that END supporting higher glucose uptake. Likewise, the muscle constructs under the HIIT regimen promoted a healthier and matured bone phenotype than END. Interestingly, under static conditions, myostatin (Mstn) expression was significantly downregulated in muscle constructs cocultured with bone constructs compared to monocultures. Our in vivo analysis of the role of myostatin on bone structure and function also showed that myostatin knockout (GDF8-/-) enhanced muscle mass and moderately influenced bone phenotype in adult mice. Together, our in vitro coculture system allowed orthogonal manipulation of mechanical strain on muscle constructs while facilitating biochemical crosstalk between bone and muscle constructs. Such systems can provide an individualized microenvironment and allow decoupled biomechanical manipulation, which is unachievable using traditional models. In the long-term, these in-vitro systems will help identify molecular targets and develop engineered therapies for diabetic bone disease.

Nephropathic cystinosis is a rare monogenetic kidney disease caused by mutations in the lysosomal transporter cystinosin (encoded by CTNS) that, to date, has no cure. The hallmark of this disease is lysosomal accumulation of cystine and decline in proximal tubular function leading to kidney failure early in life. In this project, we developed a novel gene repair strategy using CRISPR/Cas9 Homology-Independent Targeted Integration (HITI) to restore CTNS. A novel, non-viral peptide-mediated approach was used to deliver the Cas9-guideRNA ribonucleoprotein (RNP) complex and repair templates to conditionally immortalized proximal tubule epithelial cell (ciPTEC) lines. The repair constructs contained either mCherry (1.7 kb), the CTNS Superexon (1.7 Kb) or both (3.2 Kb). The results demonstrated that the smaller mCherry construct achieved a higher repair efficiency (63%) compared to the CTNS-mCherry construct (16%). Clonal expansion of repaired cells showed restoration of lysosomal cystine levels in 70-75% of the clones, which was accompanied by improved mitochondrial bioenergetics. In conclusion, CRISPR/Cas9 HITI can be used to precisely insert repair templates into the genome, resulting in a functional cystinosin restoration, and a reversal of the cystinotic disease phenotype.

Osteoarthritis is a debilitating disease of the joint that affects over 230 million people worldwide. Currently there are no treatments that slow the progression of this disease. For these reasons, new biological treatment options are currently being explored. Inorganic polyphosphates are naturally occurring biological molecules that have an anabolic effect on chondrocytes grown in vitro in the presence of Ca2+. We hypothesized that when examining significant changes in protein phosphorylation, key candidates would emerge that could help to elucidate the anabolic effects of polyphosphate on chondrocytes. Therefore, we conducted a large-scale quantitative proteomic and phosphoproteomic study of bovine primary articular chondrocytes after 30-minute treatment with inorganic polyphosphate and Ca2+. Mass spectrometry identified more than 6000 phosphorylation sites on [~]1600 chondrocyte phosphoproteins while proteomic analysis detected approximately 4100 proteins. Analysis of the data revealed a swift and dynamic response to polyphosphate after 30 minutes. What emerged from the list of proteins most affected by the treatment were proteins with key roles in chondrogenesis including TNC, IGFBP-5, and CTGF, indicating that polyphosphate plays an important role in chondrocyte metabolism. This phosphoproteome serves as a meaningful resource to help elucidate the molecular events that contribute to extracellular matrix production in cartilage.

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the Coronavirus disease 2019 (COVID-19), which was declared a pandemic by the World Health Organization (WHO) in March 2020. The disease has caused more than 5.1 million deaths worldwide. While cells in the respiratory system are frequently the initial target for SARS-CoV-2, clinical studies suggest that COVID-19 can become a multi-organ disease in the most severe cases. Still, the direct affinity of SARS-CoV-2 for cells in other organs such as the kidneys, which are often affected in severe COVID-19, remains poorly understood. MethodIn this study, we employed a human induced pluripotent stem (iPS) cell-derived model to investigate the affinity of SARS-CoV-2 for kidney glomerular podocytes. We studied uptake of the live SARS-CoV-2 virus as well as pseudotyped viral particles by human iPS cell derived podocytes using qPCR, western blot, and immunofluorescence. Global gene expression and qPCR analyses revealed that human iPS cell-derived podocytes express many host factor genes (including ACE2, BSG/CD147, PLS3, ACTR3, DOCK7, TMPRSS2, CTSL CD209, and CD33) associated with SARS-CoV-2 binding and viral processing. ResultInfection of podocytes with live SARS-CoV-2 or spike-pseudotyped lentiviral particles revealed viral uptake by the cells at low Multiplicity of Infection (MOI of 0.01) as confirmed by RNA quantification and immunofluorescence studies. Our results also indicate that direct infection of human iPS cell-derived podocytes by SARS-CoV-2 virus can cause cell death and podocyte foot process retraction, a hallmark of podocytopathies and progressive glomerular diseases including collapsing glomerulopathy observed in patients with severe COVID-19 disease. Additionally, antibody blocking experiments identified BSG/CD147 and ACE2 receptors as key mediators of spike binding activity in human iPS cell-derived podocytes. ConclusionThese results show that SARS-CoV-2 can infect kidney glomerular podocytes in vitro. These results also show that the uptake of SARS-CoV-2 by kidney podocytes occurs via multiple binding interactions and partners, which may underlie the high affinity of SARS-CoV-2 for kidney tissues. This stem cell-derived model is potentially useful for kidney-specific antiviral drug screening and mechanistic studies of COVID-19 organotropism. Significant statementMany patients with COVID19 disease exhibit multiorgan complications, suggesting that SARS-CoV-2 infection can extend beyond the respiratory system. Acute kidney injury is a common COVID-19 complication contributing to increased morbidity and mortality. Still, SARS-Cov-2 affinity for specialized kidney cells remain less clear. By leveraging our protocol for stem cell differentiation, we show that SARS-CoV-2 can directly infect kidney glomerular podocytes by using multiple Spike-binding proteins including ACE2 and BSG/CD147. Our results also indicate that infection by SARS-CoV-2 virus can cause podocyte cell death and foot process effacement, a hallmark of podocytopathies including collapsing glomerulopathy observed in patients with severe COVID-19 disease. This stem cell-derived model is potentially useful for kidney-specific antiviral drug screening and mechanistic studies of COVID-19 organotropism.

The lymphatic system has been proposed to play a crucial role in the development and progression of osteoarthritis (OA). The synovial fluid (SF) of arthritic joints contains mediators of the inflammatory response and products of the injury to articular tissues, while lymphatic system plays a critical role in resolving inflammation and overall joint homeostasis. Despite the importance of both the lymphatic system and SF in OA disease, their relationship is still poorly understood. Here, we utilized SF derived from osteoarthritis patients (OASF) and healthy individuals (HSF) to investigate potential effects of SF on migration of lymphatic endothelial cells (LECs) in vitro, and lymphatic contractility of femoral lymphatic vessels (LVs) ex vivo. Both OASF and HSF treatments led to an increased migratory response in vitro compared to LECs treatment with media without serum. Ex vivo, both OASF and HSF treatments to the lumen of isolated LVs led to significant differences in the tonic and phasic contractions and these observations were dependent on the SF treatment time. Specifically, OASF treatment transiently enhanced the RFLVs tonic contractions. Regarding the phasic contractions, OASF generated either an abrupt reduction after 1 hr of treatment or a complete cease of contractions after an overnight treatment, while HSF treatment displayed a gradual decrease in lymphatic contractility. The observed variations after SF treatments suggest that the pump function of lymphatic vessel draining the joint could be directly compromised in OA and thus might present a new therapeutic target.