Fe65-engineered neuronal exosomes encapsulating corynoxine-B ameliorate cognition and pathology of Alzheimer's disease.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the predominant impairment of neurons in the hippocampus and the formation of amyloid plaques, hyperphosphorylated tau protein, and neurofibrillary tangles in the brain. The overexpression of amyloid-ß precursor protein (APP) in an AD brain results in the binding of APP intracellular domain (AICD) to Fe65 protein via the C-terminal Fe65-PTB2 interaction, which then triggers the secretion of amyloid-ß and the consequent pathogenesis of AD. Apparently, targeting the interaction between APP and Fe65 can offer a promising therapeutic approach for AD. Recently, exosome, a type of extracellular vesicle with diameter around 30-200 nm, has gained much attention as a potential delivery tool for brain diseases, including AD, due to their ability to cross the blood-brain barrier, their efficient uptake by autologous cells, and their ability to be surface-modified with target-specific receptor ligands. Here, the engineering of hippocampus neuron cell-derived exosomes to overexpress Fe65, enabled the development of a novel exosome-based targeted drug delivery system, which carried Corynoxine-B (Cory-B, an autophagy inducer) to the APP overexpressed-neuron cells in the brain of AD mice. The Fe65-engineered HT22 hippocampus neuron cell-derived exosomes (Fe65-EXO) loaded with Cory-B (Fe65-EXO-Cory-B) hijacked the signaling and blocked the natural interaction between Fe65 and APP, enabling APP-targeted delivery of Cory-B. Notably, Fe65-EXO-Cory-B induced autophagy in APP-expressing neuronal cells, leading to amelioration of the cognitive decline and pathogenesis in AD mice, demonstrating the potential of Fe65-EXO-Cory-B as an effective therapeutic intervention for AD.

FORMATION OF MALIGNANT, METASTATIC SMALL CELL LUNG CANCERS THROUGH OVERPRODUCTION OF cMYC PROTEIN IN TP53 AND RB1 DEPLETED PULMONARY NEUROENDOCRINE CELLS DERIVED FROM HUMAN EMBRYONIC STEM CELLS.

We recently described our initial efforts to develop a model for small cell lung cancer (SCLC) derived from human embryonic stem cells (hESCs) that were differentiated to form pulmonary neuroendocrine cells (PNECs), a putative cell of origin for neuroendocrine-positive SCLC. Although reduced expression of the tumor suppressor genes TP53 and RB1 allowed the induced PNECs to form subcutaneous growths in immune-deficient mice, the tumors did not display the aggressive characteristics of SCLC seen in human patients. Here we report that the additional, doxycycline-regulated expression of a transgene encoding wild-type or mutant cMYC protein promotes rapid growth, invasion, and metastasis of these hESC-derived cells after injection into the renal capsule. Similar to others, we find that the addition of cMYC encourages the formation of the SCLC-N subtype, marked by high levels of NEUROD1 RNA. Using paired primary and metastatic samples for RNA sequencing, we observe that the subtype of SCLC does not change upon metastatic spread and that production of NEUROD1 is maintained. We also describe histological features of these malignant, SCLC-like tumors derived from hESCs and discuss potential uses of this model in efforts to control and better understand this recalcitrant neoplasm.

Ring Finger Protein 125 Is an Anti-Proliferative Tumor Suppressor in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers worldwide and the only cancer with an increasing incidence in the United States. Recent advances in sequencing technology have enabled detailed profiling of liver cancer genomes and revealed extensive inter- and intra-tumor heterogeneity, making it difficult to identify driver genes for HCC. To identify HCC driver genes, we performed transposon mutagenesis screens in a mouse HBV model of HCC and discovered many candidate cancer genes (SB/HBV-CCGs). Here, we show that one of these genes, RNF125 is a potent anti-proliferative tumor suppressor gene in HCC. RNF125 is one of nine CCGs whose expression was >3-fold downregulated in human HCC. Depletion of RNF125 in immortalized mouse liver cells led to tumor formation in transplanted mice and accelerated growth of human liver cancer cell lines, while its overexpression inhibited their growth, demonstrating the tumor-suppressive function of RNF125 in mouse and human liver. Whole-transcriptome analysis revealed that RNF125 transcriptionally suppresses multiple genes involved in cell proliferation and/or liver regeneration, including Egfr, Met, and Il6r. Blocking Egfr or Met pathway expression inhibited the increased cell proliferation observed in RNF125 knockdown cells. In HCC patients, low expression levels of RNF125 were correlated with poor prognosis demonstrating an important role for RNF125 in HCC. Collectively, our results identify RNF125 as a novel anti-proliferative tumor suppressor in HCC.

Differential effects of macrophage subtypes on SARS-CoV-2 infection in a human pluripotent stem cell-derived model.

Dysfunctional immune responses contribute critically to the progression of Coronavirus Disease-2019 (COVID-19), with macrophages as one of the main cell types involved. It is urgent to understand the interactions among permissive cells, macrophages, and the SARS-CoV-2 virus, thereby offering important insights into effective therapeutic strategies. Here, we establish a lung and macrophage co-culture system derived from human pluripotent stem cells (hPSCs), modeling the host-pathogen interaction in SARS-CoV-2 infection. We find that both classically polarized macrophages (M1) and alternatively polarized macrophages (M2) have inhibitory effects on SARS-CoV-2 infection. However, M1 and non-activated (M0) macrophages, but not M2 macrophages, significantly up-regulate inflammatory factors upon viral infection. Moreover, M1 macrophages suppress the growth and enhance apoptosis of lung cells. Inhibition of viral entry using an ACE2 blocking antibody substantially enhances the activity of M2 macrophages. Our studies indicate differential immune response patterns in distinct macrophage phenotypes, which could lead to a range of COVID-19 disease severity.

Energy Sources for Exosome Communication in a Cancer Microenvironment.

Exosomes are crucial extracellular vesicles (EVs) with a diameter of approximately 30-200 nm. They are released by most cell types in their extracellular milieu and carry various biomolecules, including proteins and nucleic acids. Exosomes are increasingly studied in various diseases, including cancer, due to their role in local and distant cell-cell communication in which they can promote tumor growth, cancer progression, and metastasis. Interestingly, a tremendous number of exosomes is released by malignant cancer cells, and these are then taken up by autologous and heterologous recipient stromal cells such as immune cells, cancer stem cells, and endothelial cells. All these events demand an enormous amount of energy and require that exosomes remain stable while having the capacity to reach distant sites and cross physical barriers. Nevertheless, there is a dearth of research pertaining to the energy sources of exosomes, and questions remain about how they maintain their motility in the tumor microenvironment (TME) and beyond. Moreover, exosomes can produce adenosine triphosphate (ATP), an important energy molecule required by all cells, and mitochondria have been identified as one of the exosomal cargoes. These findings strengthen the prospect of exosomal communication via transfer of mitochondria and the bioenergetics of target recipient cells. In the TME, the accumulation of ATP and lactate may facilitate the entry of exosomes into cancer cells to promote metastasis, as well as help to target cancer cells at the tumor site. This review highlights how exosomes obtain sufficient energy to thrive in the TME and communicate with distant physiological destinations.

The mini player with diverse functions: extracellular vesicles in cell biology, disease, and therapeutics.

Extracellular vesicles (EVs) are tiny biological nanovesicles ranging from approximately 30-1000 nm in diameter that are released into the extracellular matrix of most cell types and in biofluids. The classification of EVs includes exosomes, microvesicles, and apoptotic bodies, dependent on various factors such as size, markers, and biogenesis pathways. The transition of EV relevance from that of being assumed as a trash bag to be a key player in critical physiological and pathological conditions has been revolutionary in many ways. EVs have been recently revealed to play a crucial role in stem cell biology and cancer progression via intercellular communication, contributing to organ development and the progression of cancer. This review focuses on the significant research progress made so far in the role of the crosstalk between EVs and stem cells and their niche, and cellular communication among different germ layers in developmental biology. In addition, it discusses the role of EVs in cancer progression and their application as therapeutic agents or drug delivery vehicles. All such discoveries have been facilitated by tremendous technological advancements in EV-associated research, especially the microfluidics systems. Their pros and cons in the context of characterization of EVs are also extensively discussed in this review. This review also deliberates the role of EVs in normal cell processes and disease conditions, and their application as a diagnostic and therapeutic tool. Finally, we propose future perspectives for EV-related research in stem cell and cancer biology.

Exosomes: Small vesicles with big roles in cancer, vaccine development, and therapeutics.

Cancer is a deadly disease that is globally and consistently one of the leading causes of mortality every year. Despite the availability of chemotherapy, radiotherapy, immunotherapy, and surgery, a cure for cancer has not been attained. Recently, exosomes have gained significant attention due to the therapeutic potential of their various components including proteins, lipids, nucleic acids, miRNAs, and lncRNAs. Exosomes constitute a set of tiny extracellular vesicles with an approximate diameter of 30-100 nm. They are released from different cells and are present in biofluids including blood, cerebrospinal fluid (CSF), and urine. They perform crucial multifaceted functions in the malignant progression of cancer via autocrine, paracrine, and endocrine communications. The ability of exosomes to carry different cargoes including drug and molecular information to recipient cells make them a novel tool for cancer therapeutics. In this review, we discuss the major components of exosomes and their role in cancer progression. We also review important literature about the potential role of exosomes as vaccines and delivery carriers in the context of cancer therapeutics.

Management of adrenoleukodystrophy: From pre-clinical studies to the development of new therapies.

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurodegenerative disorder associated with mutations of the ABCD1 gene that encodes a peroxisomal transmembrane protein. It results in accumulation of very long chain fatty acids in tissues and body fluid. Along with other factors such as epigenetic and environmental involvement, ABCD1 mutation-provoked disorders can present different phenotypes including cerebral adrenoleukodystrophy (cALD), adrenomyeloneuropathy (AMN), and peripheral neuropathy. cALD is the most severe form that causes death in young childhood. Bone marrow transplantation and hematopoietic stem cell gene therapy are only effective when performed at an early stage of onsets in cALD. Nonetheless, current research and development of novel therapies are hampered by a lack of in-depth understanding disease pathophysiology and a lack of reliable cALD models. The Abcd1 and Abcd1/Abcd2 knock-out mouse models as well as the deficiency of Abcd1 rabbit models created in our lab, do not develop cALD phenotypes observed in human beings. In this review, we summarize the clinical and biochemical features of X-ALD, the progress of pre-clinical and clinical studies. Challenges and perspectives for future X-ALD studies are also discussed.

Clinical analysis and pluripotent stem cells-based model reveal possible impacts of ACE2 and lung progenitor cells on infants vulnerable to COVID-19.

Introduction: An increasing number of children with severe coronavirus disease 2019 (COVID-19) is being reported, yet the spectrum of disease severity and expression patterns of angiotensin-converting enzyme 2 (ACE2) in children at different developmental stages are largely unknow. Methods: We analysed clinical features in a cohort of 173 children with COVID-19 (0-15 yrs.-old) between January 22, 2020 and March 15, 2020. We systematically examined the expression and distribution of ACE2 in different developmental stages of children by using a combination of children's lung biopsies, pluripotent stem cell-derived lung cells, RNA-sequencing profiles, and ex vivo SARS-CoV-2 pseudoviral infections. Results: It revealed that infants (< 1yrs.-old), with a weaker potency of immune response, are more vulnerable to develop pneumonia whereas older children (> 1 yrs.-old) are more resistant to lung injury. The expression levels of ACE2 however do not vary by age in children's lung. ACE2 is notably expressed not only in Alveolar Type II (AT II) cells, but also in SOX9 positive lung progenitor cells detected in both pluripotent stem cell derivatives and infants' lungs. The ACE2+SOX9+ cells are readily infected by SARS-CoV-2 pseudovirus and the numbers of the double positive cells are significantly decreased in older children. Conclusions: Infants (< 1 yrs.-old) with SARS-CoV-2 infection are more vulnerable to lung injuries. ACE2 expression in multiple types of lung cells including SOX9 positive progenitor cells, in cooperation with an unestablished immune system, could be risk factors contributing to vulnerability of infants with COVID-19. There is a need to continue monitoring lung development in young children who have recovered from SARS-CoV-2 infection.

Distinct Disease Severity Between Children and Older Adults With Coronavirus Disease 2019 (COVID-19): Impacts of ACE2 Expression, Distribution, and Lung Progenitor Cells.

BACKGROUND: Children and older adults with coronavirus disease 2019 (COVID-19) display a distinct spectrum of disease severity yet the risk factors aren't well understood. We sought to examine the expression pattern of angiotensin-converting enzyme 2 (ACE2), the cell-entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the role of lung progenitor cells in children and older patients. METHODS: We retrospectively analyzed clinical features in a cohort of 299 patients with COVID-19. The expression and distribution of ACE2 and lung progenitor cells were systematically examined using a combination of public single-cell RNA-seq data sets, lung biopsies, and ex vivo infection of lung tissues with SARS-CoV-2 pseudovirus in children and older adults. We also followed up patients who had recovered from COVID-19. RESULTS: Compared with children, older patients (>50 years.) were more likely to develop into serious pneumonia with reduced lymphocytes and aberrant inflammatory response (P = .001). The expression level of ACE2 and lung progenitor cell markers were generally decreased in older patients. Notably, ACE2 positive cells were mainly distributed in the alveolar region, including SFTPC positive cells, but rarely in airway regions in the older adults (P < .01). The follow-up of discharged patients revealed a prolonged recovery from pneumonia in the older (P < .025). CONCLUSIONS: Compared to children, ACE2 positive cells are generally decreased in older adults and mainly presented in the lower pulmonary tract. The lung progenitor cells are also decreased. These risk factors may impact disease severity and recovery from pneumonia caused by SARS-Cov-2 infection in older patients.

Identification of SARS-CoV-2 inhibitors using lung and colonic organoids.

There is an urgent need to create novel models using human disease-relevant cells to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) biology and to facilitate drug screening. Here, as SARS-CoV-2 primarily infects the respiratory tract, we developed a lung organoid model using human pluripotent stem cells (hPSC-LOs). The hPSC-LOs (particularly alveolar type-II-like cells) are permissive to SARS-CoV-2 infection, and showed robust induction of chemokines following SARS-CoV-2 infection, similar to what is seen in patients with COVID-19. Nearly 25% of these patients also have gastrointestinal manifestations, which are associated with worse COVID-19 outcomes1. We therefore also generated complementary hPSC-derived colonic organoids (hPSC-COs) to explore the response of colonic cells to SARS-CoV-2 infection. We found that multiple colonic cell types, especially enterocytes, express ACE2 and are permissive to SARS-CoV-2 infection. Using hPSC-LOs, we performed a high-throughput screen of drugs approved by the FDA (US Food and Drug Administration) and identified entry inhibitors of SARS-CoV-2, including imatinib, mycophenolic acid and quinacrine dihydrochloride. Treatment at physiologically relevant levels of these drugs significantly inhibited SARS-CoV-2 infection of both hPSC-LOs and hPSC-COs. Together, these data demonstrate that hPSC-LOs and hPSC-COs infected by SARS-CoV-2 can serve as disease models to study SARS-CoV-2 infection and provide a valuable resource for drug screening to identify candidate COVID-19 therapeutics.

Human pluripotent stem cell-derived lung organoids: Potential applications in development and disease modeling.

The pulmonary system is comprised of two main compartments, airways and alveolar space. Their tissue and cellular complexity ensure lung function and protection from external agents, for example, virus. Two-dimensional (2D) in vitro systems and animal models have been largely employed to elucidate the molecular mechanisms underlying human lung development, physiology, and pathogenesis. However, neither of these models accurately recapitulate the human lung environment and cellular crosstalk. More recently, human-derived three-dimensional (3D) models have been generated allowing for a deeper understanding of cell-to-cell communication. However, the availability and accessibility of primary human cell sources from which generate the 2D and 3D models may be limited. In the past few years, protocols have been developed to successfully employ human pluripotent stem cells (hPSCs) and differentiate them toward pulmonary fate in vitro. In the present review, we discuss the advantages and pitfalls of hPSC-derived lung 2D and 3D models, including the main characteristics and potentials for these models and their current and future applications for modeling development and diseases. Lung organoids currently represent the closest model to the human pulmonary system. We further focus on the applications of lung organoids for the study of human diseases such as pulmonary fibrosis, infectious diseases, and lung cancer. Finally, we discuss the present limitations and potential future applications of 3D lung organoids. This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Stem Cells and Disease Adult Stem Cells, Tissue Renewal, and Regeneration > Stem Cell Differentiation and Reversion.

Modeling COVID-19 with Human Pluripotent Stem Cell-Derived Cells Reveals Synergistic Effects of Anti-inflammatory Macrophages with ACE2 Inhibition Against SARS-CoV-2.

Dysfunctional immune responses contribute critically to the progression of Coronavirus Disease-2019 (COVID-19) from mild to severe stages including fatality, with pro-inflammatory macrophages as one of the main mediators of lung hyper-inflammation. Therefore, there is an urgent need to better understand the interactions among SARS-CoV-2 permissive cells, macrophage, and the SARS-CoV-2 virus, thereby offering important insights into new therapeutic strategies. Here, we used directed differentiation of human pluripotent stem cells (hPSCs) to establish a lung and macrophage co-culture system and model the host-pathogen interaction and immune response caused by SARS-CoV-2 infection. Among the hPSC-derived lung cells, alveolar type II and ciliated cells are the major cell populations expressing the viral receptor ACE2 and co-effector TMPRSS2, and both were highly permissive to viral infection. We found that alternatively polarized macrophages (M2) and classically polarized macrophages (M1) had similar inhibitory effects on SARS-CoV-2 infection. However, only M1 macrophages significantly up-regulated inflammatory factors including IL-6 and IL-18, inhibiting growth and enhancing apoptosis of lung cells. Inhibiting viral entry into target cells using an ACE2 blocking antibody enhanced the activity of M2 macrophages, resulting in nearly complete clearance of virus and protection of lung cells. These results suggest a potential therapeutic strategy, in that by blocking viral entrance to target cells while boosting anti-inflammatory action of macrophages at an early stage of infection, M2 macrophages can eliminate SARS-CoV-2, while sparing lung cells and suppressing the dysfunctional hyper-inflammatory response mediated by M1 macrophages.

A Human Pluripotent Stem Cell-based Platform to Study SARS-CoV-2 Tropism and Model Virus Infection in Human Cells and Organoids.

SARS-CoV-2 has caused the COVID-19 pandemic. There is an urgent need for physiological models to study SARS-CoV-2 infection using human disease-relevant cells. COVID-19 pathophysiology includes respiratory failure but involves other organ systems including gut, liver, heart, and pancreas. We present an experimental platform comprised of cell and organoid derivatives from human pluripotent stem cells (hPSCs). A Spike-enabled pseudo-entry virus infects pancreatic endocrine cells, liver organoids, cardiomyocytes, and dopaminergic neurons. Recent clinical studies show a strong association with COVID-19 and diabetes. We find that human pancreatic beta cells and liver organoids are highly permissive to SARS-CoV-2 infection, further validated using adult primary human islets and adult hepatocyte and cholangiocyte organoids. SARS-CoV-2 infection caused striking expression of chemokines, as also seen in primary human COVID-19 pulmonary autopsy samples. hPSC-derived cells/organoids provide valuable models for understanding the cellular responses of human tissues to SARS-CoV-2 infection and for disease modeling of COVID-19.

Identification of Candidate COVID-19 Therapeutics using hPSC-derived Lung Organoids.

The SARS-CoV-2 virus has caused already over 3.5 million COVID-19 cases and 250,000 deaths globally. There is an urgent need to create novel models to study SARS-CoV-2 using human disease-relevant cells to understand key features of virus biology and facilitate drug screening. As primary SARS-CoV-2 infection is respiratory-based, we developed a lung organoid model using human pluripotent stem cells (hPSCs) that could be adapted for drug screens. The lung organoids, particularly aveolar type II cells, express ACE2 and are permissive to SARS-CoV-2 infection. Transcriptomic analysis following SARS-CoV-2 infection revealed a robust induction of chemokines and cytokines with little type I/III interferon signaling, similar to that observed amongst human COVID-19 pulmonary infections. We performed a high throughput screen using hPSC-derived lung organoids and identified FDA-approved drug candidates, including imatinib and mycophenolic acid, as inhibitors of SARS-CoV-2 entry. Pre- or post-treatment with these drugs at physiologically relevant levels decreased SARS-CoV-2 infection of hPSC-derived lung organoids. Together, these data demonstrate that hPSC-derived lung cells infected by SARS-CoV-2 can model human COVID-19 disease and provide a valuable resource to screen for FDA-approved drugs that might be repurposed and should be considered for COVID-19 clinical trials.

Author Correction: A recellularized human colon model identifies cancer driver genes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Generation of pulmonary neuroendocrine cells and SCLC-like tumors from human embryonic stem cells.

Cancer models based on cells derived from human embryonic stem cells (hESCs) may reveal why certain constellations of genetic changes drive carcinogenesis in specialized lineages. Here we demonstrate that inhibition of NOTCH signaling induces up to 10% of lung progenitor cells to form pulmonary neuroendocrine cells (PNECs), putative precursors to small cell lung cancers (SCLCs), and we can increase PNECs by reducing levels of retinoblastoma (RB) proteins with inhibitory RNA. Reducing levels of TP53 protein or expressing mutant KRAS or EGFR genes did not induce or expand PNECs, but tumors resembling early-stage SCLC grew in immunodeficient mice after subcutaneous injection of PNEC-containing cultures in which expression of both RB and TP53 was blocked. Single-cell RNA profiles of PNECs are heterogeneous; when RB levels are reduced, the profiles resemble those from early-stage SCLC; and when both RB and TP53 levels are reduced, the transcriptome is enriched with cell cycle-specific RNAs. Our findings suggest that genetic manipulation of hESC-derived pulmonary cells will enable studies of this recalcitrant cancer.

Engineering a Bioartificial Human Colon Model Through Decellularization and Recellularization.

The tissue engineering method of decellularization and recellularization has been successfully used in a variety of regenerative medicine applications. The protocols used to de/recellularize various organs and tissues are largely different. Here we describe a method to effectively engineer a bioartificial colon by completely removing original cells from human intestinal tissues followed by repopulating the acellular tissue matrix with cell cultures. This method provides a novel approach for human intestinal regeneration and can be used to identify potential cancer driver genes.

A pumpless body-on-a-chip model using a primary culture of human intestinal cells and a 3D culture of liver cells.

We describe an expanded modular gastrointestinal (GI) tract-liver system by co-culture of primary human intestinal epithelial cells (hIECs) and 3D liver mimic. The two organ body-on-chip design consisted of GI and liver tissue compartments that were connected by fluidic medium flow driven via gravity. The hIECs and HepG2 C3A liver cells in the co-culture system maintained high viability for at least 14 days in which hIECs differentiated into major cell types found in native human intestinal epithelium and the HepG2 C3A cells cultured on 3D polymer scaffold formed a liver micro-lobe like structure. Moreover, the hIECs formed a monolayer on polycarbonate membranes with a tight junction and authentic TEER values of approximately 250 Ω cm2 for the native gut. The hIEC permeability was compared to a conventional permeability model using Caco-2 cell response for drug absorption by measuring the uptake of propranolol, mannitol and caffeine. Metabolic rates (urea or albumin production) of the cells in the co-culture GI-liver system were comparable to those of HepG2 C3A cells in a single-organ fluidic culture system, while induced CYP activities were significantly increased in the co-culture GI tract-liver system compared to the single-organ fluidic culture system. These results demonstrated potential of the low-cost microphysiological GI-liver model for preclinical studies to predict human response.