iPSC-Derived Airway Epithelial Cells: Progress, Promise, and Challenges.

Induced pluripotent stem cells (iPSCs) generated from somatic cell sources are pluripotent and capable of indefinite expansion in vitro. They provide an unlimited source of cells that can be differentiated into lung progenitor cells for potential clinical use in pulmonary regenerative medicine. This review gives a comprehensive overview of recent progress toward the use of iPSCs to generate proximal and distal airway epithelial cells and mix lung organoids. Furthermore, their potential applications and future challenges for the field are discussed, with a focus on the technological hurdles that must be cleared before stem cell therapeutics can be used for clinical treatment.

Noncoding RNAs of Extracellular Vesicles in Tumor Angiogenesis: From Biological Functions to Clinical Significance.

Extracellular vesicles (EVs) act as multifunctional regulators of intercellular communication and are involved in diverse tumor phenotypes, including tumor angiogenesis, which is a highly regulated multi-step process for the formation of new blood vessels that contribute to tumor proliferation. EVs induce malignant transformation of distinct cells by transferring DNAs, proteins, lipids, and RNAs, including noncoding RNAs (ncRNAs). However, the functional relevance of EV-derived ncRNAs in tumor angiogenesis remains to be elucidated. In this review, we summarized current research progress on the biological functions and underlying mechanisms of EV-derived ncRNAs in tumor angiogenesis in various cancers. In addition, we comprehensively discussed the potential applications of EV-derived ncRNAs as cancer biomarkers and novel therapeutic targets to tailor anti-angiogenic therapy.

Circular RNAs in stem cells: from basic research to clinical implications.

Circular RNAs (circRNAs) are a special class of endogenous RNAs with a wide variety of pathophysiological functions via diverse mechanisms, including transcription, microRNA (miRNA) sponge, protein sponge/decoy, and translation. Stem cells are pluripotent cells with unique properties of self-renewal and differentiation. Dysregulated circRNAs identified in various stem cell types can affect stem cell self-renewal and differentiation potential by manipulating stemness. However, the emerging roles of circRNAs in stem cells remain largely unknown. This review summarizes the major functions and mechanisms of action of circRNAs in stem cell biology and disease progression. We also highlight circRNA-mediated common pathways in diverse stem cell types and discuss their diagnostic significance with respect to stem cell-based therapy.

Efficacy and safety of umbilical cord mesenchymal stem cells for the treatment of patients with COVID-19.

OBJECTIVES: The coronavirus disease (COVID-19) outbreak has catastrophically threatened public health worldwide and presented great challenges for clinicians. To date, no specific drugs are available against severe acute respiratory syndrome coronavirus 2. Mesenchymal stem cells (MSCs) appear to be a promising cell therapy owing to their potent modulatory effects on reducing and healing inflammation-induced lung and other tissue injuries. The present pilot study aimed to explore the therapeutic potential and safety of MSCs isolated from healthy cord tissues in the treatment of patients with COVID-19. METHODS: Twelve patients with COVID-19 treated with MSCs plus conventional therapy and 13 treated with conventional therapy alone (control) were included. The efficacy of MSC infusion was evaluated by changes in oxygenation index, clinical chemistry and hematology tests, immunoglobulin (Ig) levels, and pulmonary computerized tomography (CT) imaging. The safety of MSC infusion was evaluated based on the occurrence of allergic reactions and serious adverse events. RESULTS: The MSC-treated group demonstrated significantly improved oxygenation index. The area of pulmonary inflammation decreased significantly, and the CT number in the inflammatory area tended to be restored. Decreased IgM levels were also observed after MSC therapy. Laboratory biomarker levels at baseline and after therapy showed no significant changes in either the MSC-treated or control group. CONCLUSION: Intravenous infusion of MSCs in patients with COVID-19 was effective and well tolerated. Further studies involving a large cohort or randomized controlled trials are warranted.

Characterization and Establishment of a Novel EBV Strain Simultaneously Associated With Nasopharyngeal Carcinoma and B-Cell Lymphoma.

Epstein-Barr virus (EBV)-the prototypical human tumor virus-is responsible for 1-2% of the global cancer burden, but divergent strains seem to exist in different geographical regions with distinct predilections for causing lymphoid or epithelial malignancies. Here we report the establishment and characterization of Yu103, an Asia Pacific EBV strain with a highly remarkable provenance of being derived from nasopharyngeal carcinoma biopsy but subsequently propagated in human B-lymphoma cells and xenograft models. Unlike previously characterized EBV strains which are either predominantly B-lymphotropic or epitheliotropic, Yu103 evinces an uncanny capacity to infect and transform both B-lymphocytes and nasopharyngeal epithelial cells. Genomic and phylogenetic analyses indicated that Yu103 EBV lies midway along the spectrum of EBV strains known to drive lymphomagenesis or carcinogenesis, and harbors molecular features which likely account for its unusual properties. To our knowledge, Yu103 EBV is currently the only EBV isolate shown to drive human nasopharyngeal carcinoma and B-lymphoma, and should therefore provide a powerful novel platform for research on EBV-driven hematological and epithelial malignancies.

Efficacy and safety of umbilical cord mesenchymal stem cells for the treatment of patients with COVID-19

OBJECTIVES: The coronavirus disease (COVID-19) outbreak has catastrophically threatened public health worldwide and presented great challenges for clinicians. To date, no specific drugs are available against severe acute respiratory syndrome coronavirus 2. Mesenchymal stem cells (MSCs) appear to be a promising cell therapy owing to their potent modulatory effects on reducing and healing inflammation-induced lung and other tissue injuries. The present pilot study aimed to explore the therapeutic potential and safety of MSCs isolated from healthy cord tissues in the treatment of patients with COVID-19. METHODS: Twelve patients with COVID-19 treated with MSCs plus conventional therapy and 13 treated with conventional therapy alone (control) were included. The efficacy of MSC infusion was evaluated by changes in oxygenation index, clinical chemistry and hematology tests, immunoglobulin (Ig) levels, and pulmonary computerized tomography (CT) imaging. The safety of MSC infusion was evaluated based on the occurrence of allergic reactions and serious adverse events. RESULTS: The MSC-treated group demonstrated significantly improved oxygenation index. The area of pulmonary inflammation decreased significantly, and the CT number in the inflammatory area tended to be restored. Decreased IgM levels were also observed after MSC therapy. Laboratory biomarker levels at baseline and after therapy showed no significant changes in either the MSC-treated or control group. CONCLUSION: Intravenous infusion of MSCs in patients with COVID-19 was effective and well tolerated. Further studies involving a large cohort or randomized controlled trials are warranted.

Epstein-Barr Virus Infection of Pseudostratified Nasopharyngeal Epithelium Disrupts Epithelial Integrity.

Epstein-Barr virus (EBV) is a human oncogenic virus that causes several types of tumor, such as Burkitt's lymphoma and nasopharyngeal carcinoma (NPC). NPC tumor cells are clonal expansions of latently EBV-infected epithelial cells. However, the mechanisms by which EBV transforms the nasopharyngeal epithelium is hampered, because of the lack of good in vitro model to pursue oncogenic process. Our primary nasopharyngeal epithelial cell cultures developed pseudostratified epithelium at the air-liquid interface, which was susceptible to EBV infection. Using the highly sensitive RNA in situ hybridization technique, we detected viral infection in diverse cell types, including ciliated cells, goblet cells, and basal cells. EBV-encoded small RNA-positive cells were more frequently detected in the suprabasal layer than in the basal layer. We established the most physiologically relevant EBV infection model of nasopharyngeal epithelial cells. This model will advance our understanding of EBV pathogenesis in the development of NPC.

SARS-CoV-2 infection and stem cells: Interaction and intervention.

The emergence of the novel severe acute respiratory coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread have created a global health emergency. The resemblance with SARS-CoV in spike protein suggests that SARS-CoV-2 employs spike-driven entry into angiotensin-converting enzyme 2 (ACE2)-expressing cells. From a stem cell perspective, this review focuses on the possible involvement of ACE2+ stem/progenitor cells from both the upper and lower respiratory tracts in coronavirus infection. Viral infection-associated acute respiratory distress syndrome and acute lung injury occur because of dysregulation of the immune response. Mesenchymal stem cells appear to be a promising cell therapy given that they favorably modulate the immune response to reduce lung injury. The use of exogenous stem cells may lead to lung repair. Therefore, intervention by transplantation of exogenous stem cells may be required to replace, repair, remodel, and regenerate lung tissue in survivors infected with coronavirus. Ultimately, vaccines, natural killer cells and induced-pluripotent stem cell-derived virus-specific cytotoxic T lymphocytes may offer off-the-shelf therapeutics for preventing coronavirus reemergence.

Presence of lytic Epstein-Barr virus infection in nasopharyngeal carcinoma.

BACKGROUND: Chromogenic Epstein-Barr virus-encoded RNA (EBER) in situ hybridization (EBER-ISH) is the gold standard to detect Epstein-Barr virus (EBV) but it is difficult to use in conjunction with immunohistochemistry (IHC). In this study, our purpose was to validate the sensitivity and specificity of RNAscope in detection of EBV infection in nasal epithelium and its stroma. METHODS: Fluorescence-based RNAscope EBER-ISH, BRLF1-ISH, and lineage marker-IHC were performed on archived formalin-fixed paraffin-embedded tissues from normal nasal cavity (n = 5), nasopharynx (n = 8), and nasopharyngeal carcinoma (NPC) specimens (n = 10). RESULTS: The EBERs were detected in 10 of 10 NPC samples but was absent in all normal tissues from the nasal cavity and nasopharynx. The EBERs were exclusively located in pan-cytokeratin (pan-CK)-positive tumor epithelial cells but not in CD45-positive leukocytes and vimentin-positive stromal fibroblasts. The level of EBER expression varied in tumor cells within patient and between patients as well. Additionally, 5 of 10 patients had positive BRLF-ISH. CONCLUSION: We developed a simple and reproducible method to simultaneously detect mRNA and protein in formalin-fixed paraffin-embedded tissues of NPC. As a single staining, traditional EBER continues to be useful; however, for interpretation of the phenotype of EBV-infected cells, RNAscope is superior. Significantly, we showed that lytic EBV infection took place in NPC tumors.

Pan-HDAC inhibition by panobinostat mediates chemosensitization to carboplatin in non-small cell lung cancer via attenuation of EGFR signaling.

Accumulating evidence has implicated the aberrant regulation of histone deacetylases (HDACs) as a nexus for multiple cancer hallmarks and in mediating tumor adaptation and resistance to genotoxic chemotherapy, suggesting a rational pairing of HDAC inhibitors with DNA damaging chemotherapeutic agents in the treatment of human malignancies. Here we report that panobinostat (LBH589), a potent pan-HDAC inhibitor, effectively curbed the proliferation of non-small cell lung cancer (NSCLC) cell lines A549, Calu-1, H226, H460, H838 and SKMES-1 at IC50 concentrations between 4 and 31 nmol/L via pleiotropic mechanisms, including crosstalk with EGFR signal transduction cascades. Combination therapy with carboplatin elicited rapid tumor cell kill and effectively restrained anchorage-independent clonogenic survival to a considerably greater extent over either monotherapy. The administration of carboplatin and panobinostat at clinically relevant doses to NOD-SCID xenograft mice drastically stalled disease progression by 92% as compared with negative control (P = .0026), which was greater than the 28% and 54% achieved with either carboplatin (P = .220) or panobinostat (P = .017) alone. These data demonstrate that panobinostat has strong anti-NSCLC activity and chemosensitizes tumors to carboplatin, thus justifying further evaluation of this combination approach in clinical trials.

Non-malignant epithelial cells preferentially proliferate from nasopharyngeal carcinoma biopsy cultured under conditionally reprogrammed conditions.

Nasopharyngeal carcinoma (NPC) is an invasive cancer with particularly high incidence in Southern China and Southeast Asia. The study of NPC is greatly hampered by the lack of reliable cell lines due to the loss of EBV genome and HeLa cell contamination. Conditional reprogramming (CR) cell culture technique has been reported for rapid and efficient establishment of patient-derived normal and tumor cell cultures. The purpose of this study was to assess this method to culture NPC patient-derived primary tumor cells. Using CR protocol, we demonstrated that epithelial cells could be efficiently cultured from normal (70%) and cancerous nasopharyngeal (46%) biopsies. However, by comparing with original tumors in terms of mutation and methylation profiles, epithelial cells derived from cancerous biopsy represented non-malignant cells. Further, they exhibited stem-like characteristics based on their cell surface proteins and could differentiate into pseudostratified epithelium in an air-liquid interface culture system. We conclude that CR method is a highly selective and useful method for growing non-malignant nasopharyngeal epithelial cells.

Super-Enhancers Promote Transcriptional Dysregulation in Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is an invasive cancer with particularly high incidence in Southeast Asia and Southern China. The pathogenic mechanisms of NPC, particularly those involving epigenetic dysregulation, remain largely elusive, hampering clinical management of this malignancy. To identify novel druggable targets, we carried out an unbiased high-throughput chemical screening and observed that NPC cells were highly sensitive to inhibitors of cyclin-dependent kinases (CDK), especially THZ1, a covalent inhibitor of CDK7. THZ1 demonstrated pronounced antineoplastic activities both in vitro and in vivo An integrative analysis using both whole-transcriptome sequencing and chromatin immunoprecipitation sequencing pinpointed oncogenic transcriptional amplification mediated by super-enhancers (SE) as a key mechanism underlying the vulnerability of NPC cells to THZ1 treatment. Further characterization of SE-mediated networks identified many novel SE-associated oncogenic transcripts, such as BCAR1, F3, LDLR, TBC1D2, and the long noncoding RNA TP53TG1. These transcripts were highly and specifically expressed in NPC and functionally promoted NPC malignant phenotypes. Moreover, DNA-binding motif analysis within the SE segments suggest that several transcription factors (including ETS2, MAFK, and TEAD1) may help establish and maintain SE activity across the genome. Taken together, our data establish the landscape of SE-associated oncogenic transcriptional network in NPC, which can be exploited for the development of more effective therapeutic regimens for this disease. Cancer Res; 77(23); 6614-26. ©2017 AACR.

The other side of the coin: Leveraging Epstein-Barr virus in research and therapy.

Epstein-Barr virus is (EBV) a ubiquitous virus prevalent in 90% of the human population. Transmitted through infected saliva, EBV is the causative agent of infectious mononucleosis (IM) and is further implicated in malignancies of lymphoid and epithelial origins. In the past few decades, research efforts primarily focused on dissecting the mechanism of EBV-induced oncogenesis. Here, we present an alternate facet of the oncovirus EBV, on its applications in research and therapy. Finally, discussions on the prospective utilization of EBV in nasopharyngeal carcinoma (NPC) diagnosis and therapy will also be presented.

Primitive neural stem cells in the adult mammalian brain give rise to GFAP-expressing neural stem cells.

Adult forebrain definitive neural stem cells (NSCs) comprise a subpopulation of GFAP-expressing subependymal cells that arise from embryonic fibroblast growth factor (FGF)-dependent NSCs that are first isolated from the developing brain at E8.5. Embryonic FGF-dependent NSCs are derived from leukemia inhibitory factor (LIF)-responsive, Oct4-expressing primitive NSCs (pNSCs) that are first isolated at E5.5. We report the presence of a rare population of pNCSs in the periventricular region of the adult forebrain. Adult-derived pNSCs (AdpNSCs) are GFAP(-), LIF-responsive stem cells that display pNSC properties, including Oct4 expression and the ability to integrate into the inner cell mass of blastocysts. AdpNSCs generate self-renewing, multipotent colonies that give rise to definitive GFAP(+) NSCs in vitro and repopulate the subependyma after the ablation of GFAP(+) NSCs in vivo. These data support the hypothesis that a rare population of pNSCs is present in the adult brain and is upstream of the GFAP(+) NSCs.

NogoR1 and PirB signaling stimulates neural stem cell survival and proliferation.

Neural stem cells (NSCs) and neural progenitors (NPs) in the mammalian neocortex give rise to the main cell types of the nervous system. The biological behavior of these NSCs and NPs is regulated by extracellular niche derived autocrine-paracrine signaling factors on a developmental timeline. Our previous reports [Plos One 2010;5:e15341; J Neurochem 2011;117:565-578] have shown that chondroitin sulfate proteoglycan and ApolipoproteinE are autocrine-paracrine survival factors for NSCs. NogoA, a myelin related protein, is expressed in the cortical ventricular zones where NSCs reside. However, the functional role of Nogo signaling proteins in NSC behavior is not completely understood. In this study, we show that NogoA receptors, NogoR1 and PirB, are expressed in the ventricular zone where NSCs reside between E10.5 and 14.5 but not at E15.5. Nogo ligands stimulate NSC survival and proliferation in a dosage-dependent manner in vitro. NogoR1 and PirB are low and high affinity Nogo receptors, respectively and are responsible for the effects of Nogo ligands on NSC behavior. Inhibition of autocrine-paracrine Nogo signaling blocks NSC survival and proliferation. In NSCs, NogoR1 functions through Rho whereas PirB uses Shp1/2 signaling pathways to control NSC behavior. Taken together, this work suggests that Nogo signaling is an important pathway for survival of NSCs.

Malignant cells derived from 3T3 fibroblast feeder layer in cell culture for nasopharyngeal carcinoma.

In this study, we discovered a subpopulation of 3T3 feeder cells were malignantly transformed by nasopharyngeal carcinoma (NPC) tumor cells during co-culture. The transformed 3T3 cells acquired an accelerated growth rate, displayed loosely attached multilayer growth in vitro and highly tumorigenic in vivo. Most strikingly, instead of forming sarcomas, they developed into carcinoma-like tumors somewhat resembling the original NPC. We further demonstrated the transformation is not a single isolated event, rather a common reproducible, cell contact dispensable phenomena among NPC tumor cells. However, NPC tumor cells alone were not sufficient to confer the transformed characteristics onto normal human cells.

Induced pluripotent stem cells for the treatment of stroke: the potential and the pitfalls.

The extraordinary discovery of induced pluripotent stem cells (iPSCs) has led to the very real possibility that patient-specific cell therapy can be realized. The potential to develop cell replacement therapies outside the ethical and legal limitations, has initiated a new era of hope for regenerative strategies to treat human neurological disease including stroke. In this article, we will review and compare the current approaches to derive iPSCs from different somatic cells, and the induction into neuronal phenotypes, considering the advantages and disadvantages to the methodologies of derivation. We will highlight the work relating to the use of iPSC-based therapies in models of stroke and their potential use in clinical trials. Finally, we will consider future directions and areas of exploration which may promote the realization of iPSC-based cell replacement strategies for the treatment of stroke.

Phylogenetic analysis reveals the coexistence of interfamily and interspecies horizontal gene transfer in Streptococcus thermophilus strains isolated from the same yoghurt.

Horizontal gene transfer (HGT) is an important evolutionary mechanism that has shaped prokaryotic genomes. For Streptococcus thermophilus, there is no direct evidence that the bacteria might acquire a second paralog from a different origin in the same niche. In this study, we found that four isolates of S. thermophilus (B, C, E and F) from the same yoghurt contained two putative homologs of the eno genes (eno-1 and eno-2) and two putative homologs of the guaB genes (guaB-1 and guaB-2). Both eno-1 and guaB-1 shared 100% nucleotide identity among the four isolates, and with isolate A and S. thermophilus ND03. Phylogenetic and nucleotide divergence analyses indicated that guaB-2 of these isolates may have been acquired from species in the genus Streptococcus, while eno-2 of isolates B and C may have been acquired from a donor in the genus Streptococcus. The eno-2 genes of isolates E and F may have been acquired from a donor in the Enterococcus genera. Relative synonymous codon usage analysis confirmed the eno-2 genes of isolates E and F as being acquired from a donor in genus Enterococcus. This study provides evidence that interfamily and interspecies HGT occur in S. thermophilus strains isolated from the same niche.

Identification of a subpopulation of nasopharyngeal carcinoma cells with cancer stem-like cell properties by high aldehyde dehydrogenase activity.

OBJECTIVES/HYPOTHESIS: Cancer stem cells have been reported as a new therapeutic target in many cancers, but their existence in nasopharyngeal carcinoma (NPC) is largely unknown. This study was conducted to determine cancer stem-like cells in NPC cell line. STUDY DESIGN: Basic science experimental study. METHODS: Aldehyde dehydrogenase (ALDH) activity, a putative functional marker for cancer stem cells, was assessed in Epstein-Barr virus-associated NPC cell line C666-1 cells. The ability of cells with high and low ALDH activity to proliferate, resist therapy, and initiate tumor formation was compared. RESULTS: Enrichment of cancer stem-like cells (with high ALDH activity) in C666-1 was associated with a significantly greater ability to proliferate, be clonogenic, resist chemotherapy drugs and radiation, reconstitute a heterogeneous population, and express pluripotent markers. Furthermore, subcutaneous injection of these cells into immunodeficient nude mice resulted in a tendency of tumor formation at a higher rate as compared to cells with low ALDH activity. CONCLUSIONS: These results provide evidence for the existence of cancer stem-like cells in the NPC cell line C666-1 cells.

Notch signaling imparts and preserves neural stem characteristics in the adult brain.

Although the role of Notch has been studied extensively in the developing nervous system, the embryonic lethality of Notch pathway mutants has hindered studies in the adult brain. The creation of cre/lox-mediated conditional gain- and loss-of-function mice has allowed us to investigate the role of Notch signaling in adult neural stem and progenitor cells. We have determined that Notch signaling is important for conferring stem cell characteristics upon neural precursor cells. Knocking-out Notch signaling in vivo results in neural progenitors, leaving the subependymal niche and migrating along the rostral migratory stream to the olfactory bulb, while overexpressing Notch results in retention of cells in the subependyma. Further, increased Notch signaling in progenitor cells resulted in the expression of stem cell markers in vivo as well as conferring the characteristics of self-renewal and multipotentiality upon subsequent isolation in vitro. Similar to what has been reported from the embryonic brain, the overexpression of Notch in neural precursor cells in vitro increased the numbers of neurospheres from the adult brain. Finally, overexpression of Notch1 in pure populations of progenitor cells (excluding neural stem cells) isolated by fluorescence activated cell sorting led to the formation of multipotent, self-renewing neurospheres from the non-neurosphere forming fraction. Hence, Notch overexpression confers stem cell properties upon progenitor cells and demonstrates that Notch signaling not only preserves stem cell characteristics, but that it can confer stem cell characteristics upon a subset of progenitor cells.