RESUMO
A "universal strategy" replacing the full-length CFTR cDNA may treat >99% of people with cystic fibrosis (pwCF), regardless of their specific mutations. Cas9-based gene editing was used to insert the CFTR cDNA and a truncated CD19 (tCD19) enrichment tag at the CFTR locus in airway basal stem cells. This strategy restores CFTR function to non-CF levels. Here, we investigate the safety of this approach by assessing genomic and regulatory changes after CFTR cDNA insertion. Safety was first assessed by quantifying genetic rearrangements using CAST-seq. After validating restored CFTR function in edited and enriched airway cells, the CFTR locus open chromatin profile was characterized using ATAC-seq. The regenerative potential and differential gene expression in edited cells was assessed using scRNA-seq. CAST-seq revealed a translocation in â¼0.01% of alleles primarily occurring at a nononcogenic off-target site and large indels in 1% of alleles. The open chromatin profile of differentiated airway epithelial cells showed no appreciable changes, except in the region corresponding to the CFTR cDNA and tCD19 cassette, indicating no detectable changes in gene regulation. Edited stem cells produced the same types of airway cells as controls with minimal alternations in gene expression. Overall, the universal strategy showed minor undesirable genomic changes.
RESUMO
BACKGROUND: Airway epithelium defects are a hallmark of recurrent benign tracheal stenosis (RBTS). Reconstructing an intact airway epithelium is of great importance in airway homeostasis and epithelial wound healing and has great potential for treating tracheal stenosis. METHODS: An experimental study was conducted in canines to explore the therapeutic effect of autologous basal cell transplantation in restoring airway homeostasis. First, airway mucosae from human patients with recurrent tracheal stenosis were analyzed by single-cell RNA sequencing. Canines were then randomly divided into tracheal stenosis, Stent, Stentâ +â Cells, and Stentâ +â Cellsâ +â Biogel groups. Autologous airway basal cells of canines in the Stentâ +â Cells and Stentâ +â Cellsâ +â Biogel groups were transplanted onto the stenotic airway after modeling. A biogel was coated on the airway prior to basal cell transplantation in the Stentâ +â Cellsâ +â Biogel group. After bronchoscopic treatments, canines were followed up for 16 weeks. RESULTS: Single-cell RNA sequencing demonstrated packed airway basal cells and an absence of normal airway epithelial cells in patients with RBTS. Autologous airway basal cell transplantation, together with biogel coating, was successfully performed in the canine model. Follow-up observation indicated that survival time in the Stentâ +â Cellsâ +â Biogel group was significantly prolonged, with a higher (100%) survival rate compared with the other groups. In terms of pathological and bronchoscopic findings, canines that received autologous basal cell transplantation showed a reduction in granulation hyperplasia as well as airway re-epithelialization with functionally mature epithelial cells. CONCLUSIONS: Autologous airway basal cell transplantation might serve as a novel regenerative therapy for airway re-epithelialization and inhibit recurrent granulation hyperplasia in benign tracheal stenosis.
Assuntos
Estenose Traqueal , Transplante Autólogo , Animais , Cães , Epitélio/patologia , Hiperplasia/patologia , Traqueia , Estenose Traqueal/terapia , CicatrizaçãoRESUMO
The airway epithelium is the footstone to maintain the structure and functions of lung, in which resident basal cells (BCs) maintain homeostasis and functional regeneration of epithelial barrier in response to injury. In recent clinical researches, transplanting BCs has shown great inspiring achievements in therapy of various lung diseases. In this study, we report a noninvasive optical method to activate BCs for airway epithelium regeneration in vivo by fast scanning of focused femtosecond laser on BCs of airway epithelium to active Ca2+ signaling and subsequent ERK and Wnt pathways. The photoactivated BCs present high proliferative capacity and maintain high pluripotency, which enables them to plant in the injured airway epithelium and differentiate to club cells for regeneration of epithelium. This optical method can also work in situ to activate localized BCs in airway tissue. Therefore, our results provide a powerful technology for noninvasive BC activation in stem-cell therapy of lung diseases.
Assuntos
Células Epiteliais , Pneumopatias , Humanos , Epitélio , Pulmão/metabolismo , Cicatrização , Diferenciação CelularRESUMO
BACKGROUND: Lysophosphatidic acid (LPA), generated extracellularly by the action of autotaxin and phospholipase A2, functions through LPA receptors (LPARs) or sphingosine-1-phosphate receptors (S1PRs) to induce pro-fibrotic signaling in the lower respiratory tract of patients with idiopathic pulmonary fibrosis (IPF). We hypothesized that LPA induces changes in small airway epithelial (SAE) basal cells (BC) that create cross-talk between the BC and normal human lung fibroblasts (NHLF), enhancing myofibroblast formation. METHODS: To assess LPA-induced signaling, BC were treated with LPA for 2.5 min and cell lysates were analyzed by phosphokinase array and Western blot. To assess transcriptional changes, BC were treated with LPA for 3 h and harvested for collection and analysis of RNA by quantitative polymerase chain reaction (qPCR). To assess signaling protein production and function, BC were washed thoroughly after LPA treatment and incubated for 24 h before collection for protein analysis by ELISA or functional analysis by transfer of conditioned medium to NHLF cultures. Transcription, protein production, and proliferation of NHLF were assessed. RESULTS: LPA treatment induced signaling by cAMP response element-binding protein (CREB), extracellular signal-related kinases 1 and 2 (Erk1/2), and epithelial growth factor receptor (EGFR) resulting in elevated expression of connective tissue growth factor (CTGF), endothelin-1 (EDN1/ET-1 protein), and platelet derived growth factor B (PDGFB) at the mRNA and protein levels. The conditioned medium from LPA-treated BC induced NHLF proliferation and increased NHLF expression of collagen I (COL1A1), smooth muscle actin (ACTA2), and autotaxin (ENPP2) at the mRNA and protein levels. Increased autotaxin secretion from NHLF correlated with increased LPA in the NHLF culture medium. Inhibition of CREB signaling blocked LPA-induced changes in BC transcription and translation as well as the pro-fibrotic effects of the conditioned medium on NHLF. CONCLUSION: Inhibition of CREB signaling may represent a novel target for alleviating the LPA-induced pro-fibrotic feedback loop between SAE BC and NHLF.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Células Epiteliais/patologia , Fibroblastos/fisiologia , Regulação da Expressão Gênica , Fibrose Pulmonar Idiopática/genética , Pulmão/patologia , Lisofosfolipídeos/farmacologia , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Pulmão/metabolismo , Proteínas do Tecido Nervoso , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Lentiviral-mediated integration of a CFTR transgene cassette into airway basal cells is a strategy being considered for cystic fibrosis (CF) cell-based therapies. However, CFTR expression is highly regulated in differentiated airway cell types and a subset of intermediate basal cells destined to differentiate. Since basal stem cells typically do not express CFTR, suppressing the CFTR expression from the lentiviral vector in airway basal cells may be beneficial for maintaining their proliferative capacity and multipotency. We identified miR-106b as highly expressed in proliferating airway basal cells and extinguished in differentiated columnar cells. Herein, we developed lentiviral vectors with the miR-106b-target sequence (miRT) to both study miR-106b regulation during basal cell differentiation and detarget CFTR expression in basal cells. Given that miR-106b is expressed in the 293T cells used for viral production, obstacles of viral genome integrity and titers were overcome by creating a 293T-B2 cell line that inducibly expresses the RNAi suppressor B2 protein from flock house virus. While miR-106b vectors effectively detargeted reporter gene expression in proliferating basal cells and following differentiation in the air-liquid interface and organoid cultures, the CFTR-miRT vector produced significantly less CFTR-mediated current than the non-miR-targeted CFTR vector following transduction and differentiation of CF basal cells. These findings suggest that miR-106b is expressed in certain airway cell types that contribute to the majority of CFTR anion transport in airway epithelium.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , MicroRNAs/genética , Mucosa Respiratória/metabolismo , Células-Tronco/metabolismo , Diferenciação Celular , Fibrose Cística/genética , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Vetores Genéticos , Células HEK293 , Humanos , Lentivirus/genéticaRESUMO
The mouse trachea is thought to contain two distinct stem cell compartments that contribute to airway repair-basal cells in the surface airway epithelium (SAE) and an unknown submucosal gland (SMG) cell type. Whether a lineage relationship exists between these two stem cell compartments remains unclear. Using lineage tracing of glandular myoepithelial cells (MECs), we demonstrate that MECs can give rise to seven cell types of the SAE and SMGs following severe airway injury. MECs progressively adopted a basal cell phenotype on the SAE and established lasting progenitors capable of further regeneration following reinjury. MECs activate Wnt-regulated transcription factors (Lef-1/TCF7) following injury and Lef-1 induction in cultured MECs promoted transition to a basal cell phenotype. Surprisingly, dose-dependent MEC conditional activation of Lef-1 in vivo promoted self-limited airway regeneration in the absence of injury. Thus, modulating the Lef-1 transcriptional program in MEC-derived progenitors may have regenerative medicine applications for lung diseases.
Assuntos
Células Epiteliais/citologia , Glândulas Exócrinas/citologia , Mucosa Respiratória/citologia , Células-Tronco/citologia , Traqueia/citologia , Animais , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos TransgênicosRESUMO
Human airway basal cells are the stem (or progenitor) population of the airway epithelium, and play a central role in anchoring the epithelium to the basement membrane. The anatomic position of basal cells allows for potential paracrine signaling between them and the underlying non-epithelial stromal cells. In support of this, we have previously demonstrated that endothelial cells support growth of basal cells during co-culture through vascular endothelial growth factor A (VEGFA)-mediated signaling. Building on these findings, we found, by RNA sequencing analysis, that basal cells expressed multiple fibroblast growth factor (FGF) ligands (FGF2, FGF5, FGF11 and FGF13) and that only FGF2 and FGF5 were capable of functioning in a paracrine manner to activate classical FGF receptor (FGFR) signaling. Antibody-mediated blocking of FGFR1 during basal-cell-endothelial-cell co-culture significantly reduced the endothelial-cell-dependent basal cell growth. Stimulation of endothelial cells with basal-cell-derived growth factors induced endothelial cell expression of matrix metallopeptidase 14 (MMP14), and short hairpin RNA (shRNA)-mediated knockdown of endothelial cell MMP14 significantly reduced the endothelial-cell-dependent growth of basal cells. Overall, these data characterize a new growth-factor-mediated reciprocal 'crosstalk' between human airway basal cells and endothelial cells that regulates proliferation of basal cells.