Challenges and strategies: Scalable and efficient production of mesenchymal stem cells-derived exosomes for cell-free therapy.

Exosomes are small membrane vesicles secreted by most cell types, and widely exist in cell supernatants and various body fluids. They can transmit numerous bioactive elements, such as proteins, nucleic acids, and lipids, to affect the gene expression and function of recipient cells. Mesenchymal stem cells (MSCs) have been confirmed to be a potentially promising therapy for tissue repair and regeneration. Accumulating studies demonstrated that the predominant regenerative paradigm of MSCs transplantation was the paracrine effect but not the differentiation effect. Exosomes secreted by MSCs also showed similar therapeutic effects as their parent cells and were considered to be used for cell-free regenerative medicine. However, the inefficient and limited production has hampered their development for clinical translation. In this review, we summarize potential methods to efficiently promote the yield of exosomes. We mainly focus on engineering the process of exosome biogenesis and secretion, altering the cell culture conditions, cell expansion through 3D dynamic culture and the isolation of exosomes. In addition, we also discuss the application of MSCs-derived exosomes as therapeutics in disease treatment.

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells.

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

Enhanced metanephric specification to functional proximal tubule enables toxicity screening and infectious disease modelling in kidney organoids.

While pluripotent stem cell-derived kidney organoids are now being used to model renal disease, the proximal nephron remains immature with limited evidence for key functional solute channels. This may reflect early mispatterning of the nephrogenic mesenchyme and/or insufficient maturation. Here we show that enhanced specification to metanephric nephron progenitors results in elongated and radially aligned proximalised nephrons with distinct S1 - S3 proximal tubule cell types. Such PT-enhanced organoids possess improved albumin and organic cation uptake, appropriate KIM-1 upregulation in response to cisplatin, and improved expression of SARS-CoV-2 entry factors resulting in increased viral replication. The striking proximo-distal orientation of nephrons resulted from localized WNT antagonism originating from the organoid stromal core. PT-enhanced organoids represent an improved model to study inherited and acquired proximal tubular disease as well as drug and viral responses.

Intermediary Role of Lung Alveolar Type 1 Cells in Epithelial Repair upon Sendai Virus Infection.

The lung epithelium forms the first barrier against respiratory pathogens and noxious chemicals; however, little is known about how more than 90% of this barrier, made of AT1 (alveolar type 1) cells, responds to injury. Using the Sendai virus to model natural infection in mice, we find evidence that AT1 cells have an intermediary role by persisting in areas depleted of AT2 cells, upregulating IFN responsive genes, and receding from invading airway cells. Sendai virus infection mobilizes airway cells to form alveolar SOX2+ (Sry-box 2+) clusters without differentiating into AT1 or AT2 cells. Large AT2 cell-depleted areas remain covered by AT1 cells, which we name "AT2-less regions", and are replaced by SOX2+ clusters spreading both basally and luminally. AT2 cell proliferation and differentiation are largely confined to topologically distal regions and form de novo alveolar surface, with limited contribution to in situ repairs of AT2-less regions. Time-course single-cell RNA sequencing profiling and RNAscope validation suggest enhanced immune responses and altered growth signals in AT1 cells. Our comprehensive spatiotemporal and genomewide study highlights the hitherto unappreciated role of AT1 cells in lung injury-repair.

Single cell biology-a Keystone Symposia report.

Single cell biology has the potential to elucidate many critical biological processes and diseases, from development and regeneration to cancer. Single cell analyses are uncovering the molecular diversity of cells, revealing a clearer picture of the variation among and between different cell types. New techniques are beginning to unravel how differences in cell state-transcriptional, epigenetic, and other characteristics-can lead to different cell fates among genetically identical cells, which underlies complex processes such as embryonic development, drug resistance, response to injury, and cellular reprogramming. Single cell technologies also pose significant challenges relating to processing and analyzing vast amounts of data collected. To realize the potential of single cell technologies, new computational approaches are needed. On March 17-19, 2021, experts in single cell biology met virtually for the Keystone eSymposium "Single Cell Biology" to discuss advances both in single cell applications and technologies.

A human three-dimensional neural-perivascular 'assembloid' promotes astrocytic development and enables modeling of SARS-CoV-2 neuropathology.

Clinical evidence suggests the central nervous system is frequently impacted by SARS-CoV-2 infection, either directly or indirectly, although the mechanisms are unclear. Pericytes are perivascular cells within the brain that are proposed as SARS-CoV-2 infection points. Here we show that pericyte-like cells (PLCs), when integrated into a cortical organoid, are capable of infection with authentic SARS-CoV-2. Before infection, PLCs elicited astrocytic maturation and production of basement membrane components, features attributed to pericyte functions in vivo. While traditional cortical organoids showed little evidence of infection, PLCs within cortical organoids served as viral 'replication hubs', with virus spreading to astrocytes and mediating inflammatory type I interferon transcriptional responses. Therefore, PLC-containing cortical organoids (PCCOs) represent a new 'assembloid' model that supports astrocytic maturation as well as SARS-CoV-2 entry and replication in neural tissue; thus, PCCOs serve as an experimental model for neural infection.

The Transcription Factor TCF1 in T Cell Differentiation and Aging.

The transcription factor T cell factor 1 (TCF1), a pioneer transcription factor as well as a downstream effector of WNT/ß-catenin signaling, is indispensable for T cell development in the thymus. Recent studies have highlighted the additional critical role of TCF1 in peripheral T cell responses to acute and chronic infections as well as cancer. Here, we review the regulatory functions of TCF1 in the differentiation of T follicular helper cells, memory T cells and recently described stem-like exhausted T cells, where TCF1 promotes less differentiated stem-like cell states by controlling common gene-regulatory networks. These studies also provide insights into the mechanisms of defective T cell responses in older individuals. We discuss alterations in TCF1 expression and related regulatory networks with age and their consequences for T cell responses to infections and vaccination. The increasing understanding of the pathways regulating TCF1 expression and function in aged T cells holds the promise of enabling the design of therapeutic interventions aiming at improving T cell responses in older individuals.

Role of iron and iron-related proteins in mesenchymal stem cells: Cellular and clinical aspects.

Mesenchymal stem cells (MSCs) are located in various tissues where these cells show niche-dependent multilineage differentiation and secrete immunomodulatory molecules to support numerous physiological processes. Due to their regenerative and reparative properties, MSCs are extremely valuable for cell-based therapy in tackling several pathological conditions including COVID-19. Iron is essential for MSC processes but iron-loading, which is common in several chronic conditions, hinders normal MSC functionality. This not only aggravates disease pathology but can also affect allogeneic and autologous MSC therapy. Thus, understanding MSCs from an iron perspective is of clinical significance. Accordingly, this review highlights the roles of iron and iron-related proteins in MSC physiology. It describes the contribution of iron and endogenous iron-related effectors like hepcidin, ferroportin, transferrin receptor, lactoferrin, lipocalin-2, bone morphogenetic proteins and hypoxia inducible factors in MSC biology. It summarises the excess-iron-induced alterations in MSC components, processes and discusses signalling pathways involving ROS, PI3K/AKT, MAPK, p53, AMPK/MFF/DRP1 and Wnt. Additionally, it evaluates the endogenous and exogenous saviours of MSCs against iron-toxicity. Lastly, it elaborates on the involvement of MSCs in the pathology of clinical conditions of iron-excess, namely, hereditary hemochromatosis, diabetes, ß-thalassaemia and myelodysplastic syndromes. This unique review integrates the distinct fields of iron regulation and MSC physiology. Through an iron-perspective, it describes both mechanistic and clinical aspects of MSCs and proposes an iron-linked MSC-contribution to physiology, pathology and therapeutics. It advances the understanding of MSC biology and may aid in identifying signalling pathways, molecular targets and compounds for formulating adjunctive iron-based therapies for excess-iron conditions, and thereby inform regenerative medicine.

Downregulated Gene Expression Spectrum and Immune Responses Changed During the Disease Progression in Patients With COVID-19.

BACKGROUND: The World Health Organization characterizes novel coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as a pandemic. Here, we investigated the clinical, cytokine levels; T-cell proportion; and related gene expression occurring in patients with COVID-19 on admission and after initial treatment. METHODS: Eleven patients diagnosed with COVID-19 with similar initial treatment regimens were enrolled in the hospital. Plasma cytokine, peripheral T cell proportions, and microfluidic quantitative polymerase chain reaction analyses for gene expression were conducted. RESULTS: Five patients with mild and 6 with severe disease were included. Cough and fever were the primary symptoms in the 11 COVID-19 cases. Older age, higher neutrophil count, and higher C-reactive protein levels were found in severe cases. IL-10 level significantly varied with disease progression and treatment. Decreased T-cell proportions were observed in patients with COVID-19, especially in severe cases, and all were returned to normal in patients with mild disease after initial treatment, but only CD4+ T cells returned to normal in severe cases. The number of differentially expressed genes (DEGs) increased with the disease progression, and decreased after initial treatment. All downregulated DEGs in severe cases mainly involved Th17-cell differentiation, cytokine-mediated signaling pathways, and T-cell activation. After initial treatment in severe cases, MAP2K7 and SOS1 were upregulated relative to that on admission. CONCLUSIONS: Our findings show that a decreased T-cell proportion with downregulated gene expression related to T-cell activation and differentiation occurred in patients with severe COVID-19, which may help to provide effective treatment strategies for COVID-19.

Cell differentiation and aging accompanied by depletion of the ACE2 protein.

ACE2 was observed as the cell surface receptor of the SARS-CoV-2 virus. Interestingly, we also found ACE2 positivity inside the cell nucleus. The ACE2 levels changed during cell differentiation and aging and varied in distinct cell types. We observed ACE2 depletion in the aortas of aging female mice, similarly, the aging caused ACE2 decrease in the kidneys. Compared with that in the heart, brain and kidneys, the ACE2 level was the lowest in the mouse lungs. In mice exposed to nicotine, ACE2 was not changed in olfactory bulbs but in the lungs, ACE2 was upregulated in females and downregulated in males. These observations indicate the distinct gender-dependent properties of ACE2. Differentiation into enterocytes, and cardiomyocytes, caused ACE2 depletion. The cardiomyogenesis was accompanied by renin upregulation, delayed in HDAC1-depleted cells. In contrast, vitamin D2 decreased the renin level while ACE2 was upregulated. Together, the ACE2 level is high in non-differentiated cells. This protein is more abundant in the tissues of mouse embryos and young mice in comparison with older animals. Mostly, downregulation of ACE2 is accompanied by renin upregulation. Thus, the pathophysiology of COVID-19 disease should be further studied not only by considering the ACE2 level but also the whole renin-angiotensin system.

Bargain with the tooth fairy - The savings accounts for dental stem cells.

Despite the hard times COVID-19 has imposed on us, the Biomedical Journal strives to provide fresh and compelling reading material - to be enjoyed safely from home. In this issue, we glance behind the scenes of dental stem cell preservation for potential therapeutic use, and discover that cancer cells hijack podoplanin expression to induce thrombosis. Moreover, we learn how the helicase DDX17 promotes tumour stemness, how genetic defects in meiosis and DNA repair cause premature ovarian insufficiency, and that the brain-derived neurotrophic factor is associated with several psychiatric diseases. Further accounts relate the role of miR-95-3p in colorectal cancer, the protective power of eggplants against mercury poisoning, and the predictive value of inhibin A for premature delivery. Finally, the very rare case of adenoid cystic carcinoma in the external auditory canal receives some attention, and we get to read up on how 3D imaging and modelling combines functional and aesthetic repair of cleft lip and palate cases.

Itolizumab, an anti-CD6 monoclonal antibody, as a potential treatment for COVID-19 complications.

INTRODUCTION: The globally rampant SARS CoV-2 pandemic requires novel medical strategies to control the severity of disease and death due to complications. Of the 15-20% patients that develop pulmonary symptoms, a sub-set develops an acute respiratory distress syndrome (ARDS) rapidly progressing into a critical condition. Marked elevation of cytokines/chemokines is observed with elevation of additional markers of inflammation, coagulation, and organ damage such as CRP, D-dimer, LDH, Ferritin and Troponin-I. This hyperinflammation leads to worsening of oxygen saturation due to pulmonary infiltration and exudation, organ damage, and dysfunction of coagulation pathway and may lead to multi-organ failure. AREAS COVERED: The role of anti-inflammatory monoclonal antibodies such as Itolizumab, in cytokine storm. EXPERT OPINION: Itolizumab, an anti-CD6 humanized IgG1 mAb, binds to domain-1 of CD-6 that is responsible for priming, activation, and differentiation of T-cells. Itolizumab significantly reduces T-cell proliferation along with substantial downregulation of the production of cytokines/chemokines. Approved for moderate to severe chronic plaque psoriasis in 2013 it is currently being studied for addressing COVID-19 related cytokine storm and its complications. This article reviews its use in COVID-19 infections; its dose, administration protocol, contra-indications, and safety in treating moderate-to-severe ARDS by preventing and treating the cytokine storm and its complications.

Stem Cell-Based Therapy for Coronavirus Disease 2019.

The novel coronavirus disease 2019 (COVID-19) has grown to be a global public-health emergency since patients were first detected in Wuhan, China, in December 2019. As of April 9, 2020, the novel coronavirus (named as SARS-CoV-2 by the International Committee on Taxonomy of Viruses on February 11) has infected 83,251 and 1,484,811 patients in China and the world, respectively. However, we have neither confirmed effective antiviral medications nor vaccines available to deal with this emergency. In this commentary, we offer an alternative promising therapy for COVID-19, that is, mesenchymal stem cell transplantation.