Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs.

CRISPR-Cas9 deletion (CRISPR-del) is the leading approach for eliminating DNA from mammalian cells and underpins a variety of genome-editing applications. Target DNA, defined by a pair of double-strand breaks (DSBs), is removed during nonhomologous end-joining (NHEJ). However, the low efficiency of CRISPR-del results in laborious experiments and false-negative results. By using an endogenous reporter system, we show that repression of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-an early step in NHEJ-yields substantial increases in DNA deletion. This is observed across diverse cell lines, gene delivery methods, commercial inhibitors, and guide RNAs, including those that otherwise display negligible activity. We further show that DNA-PKcs inhibition can be used to boost the sensitivity of pooled functional screens and detect true-positive hits that would otherwise be overlooked. Thus, delaying the kinetics of NHEJ relative to DSB formation is a simple and effective means of enhancing CRISPR-deletion.

Mesenchyme-derived vertebrate lonesome kinase controls lung organogenesis by altering the matrisome.

Vertebrate lonesome kinase (VLK) is the only known secreted tyrosine kinase and responsible for the phosphorylation of a broad range of secretory pathway-resident and extracellular matrix proteins. However, its cell-type specific functions in vivo are still largely unknown. Therefore, we generated mice lacking the VLK gene (protein kinase domain containing, cytoplasmic (Pkdcc)) in mesenchymal cells. Most of the homozygous mice died shortly after birth, most likely as a consequence of their lung abnormalities and consequent respiratory failure. E18.5 embryonic lungs showed a reduction of alveolar type II cells, smaller bronchi, and an increased lung tissue density. Global mass spectrometry-based quantitative proteomics identified 97 proteins with significantly and at least 1.5-fold differential abundance between genotypes. Twenty-five of these had been assigned to the extracellular region and 15 to the mouse matrisome. Specifically, fibromodulin and matrilin-4, which are involved in extracellular matrix organization, were significantly more abundant in lungs from Pkdcc knockout embryos. These results support a role for mesenchyme-derived VLK in lung development through regulation of matrix dynamics and the resulting modulation of alveolar epithelial cell differentiation.

Interactome Analysis of iPSC Secretome and Its Effect on Macrophages In Vitro.

Induced pluripotent stem cell secretome (iPSC-CM) mitigate organ injury and help in repair. Macrophages play a critical role in tissue repair and regeneration and can be directed to promote tissue repair by iPSC-CM, although the exact mechanisms are not known. In the current investigative study, we evaluated the possible mechanism by which iPSC-CM regulates the phenotype and secretory pattern of macrophages in vitro. Macrophages were obtained from human peripheral blood mononuclear cells and differentiated to various subpopulations and treated with either iPSC-CM or control media in vitro. Macrophage phenotype was assessed by flow cytometry, gene expression changes by qRT PCR and secretory pattern by multiplex protein analysis. The protein and gene interaction network revealed the involvement of Amyloid precursor protein (APP) and ELAV-like protein 1 (ELAVL-1) both present in the iPSC-CM to play an important role in regulating the macrophage phenotype and their secretory pattern. This exploratory study reveals, in part, the possible mechanism and identifies two potential targets by which iPSC-CM regulate macrophages and help in repair and regeneration.

Transcriptomic profiling reveals disease-specific characteristics of epithelial cells in idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is an incurable disease characterized by progressive lung fibrosis ultimately resulting in respiratory failure and death. Recurrent micro-injuries to the alveolar epithelium and aberrant alveolar wound healing with impaired re-epithelialization define the initial steps of the pathogenic trajectory. Failure of timely alveolar epithelial repair triggers hyper-proliferation of mesenchymal cells accompanied by increased deposition of extracellular matrix into the lung interstitium. METHODS: We previously isolated fibrosis-specific mesenchymal stem cell (MSC)-like cells from lung tissue of patients with interstitial lung diseases. These cells produced factors bearing anti-fibrotic potential and changed their morphology from mesenchymal to epithelial upon culture in an epithelial cell (EC)-specific growth medium. Here, we set out to molecularly characterize these MSC-like cell-derived ECs using global gene expression profiling by RNA-sequencing. Moreover, we aimed at characterizing disease-specific differences by comparing the transcriptomes of ECs from IPF and non-IPF sources. RESULTS: Our results suggest that differentially expressed genes are enriched for factors related to fibrosis, hypoxia, bacterial colonization and metabolism, thus reflecting many of the hallmark characteristics of pulmonary fibrosis. IPF-ECs showed enrichment of both pro- and anti-fibrotic genes, consistent with the notion of adaptive, compensatory regulation. CONCLUSIONS: Our findings support the hypothesis of a functional impairment of IPF-ECs, which could possibly explain the poor clinical outcome of IPF that roughly compares to those of advanced-stage cancers. Our study provides a valuable resource for downstream mechanistic investigation and the quest for novel therapeutic IPF targets.

Alpha-Klotho Enrichment in Induced Pluripotent Stem Cell Secretome Contributes to Antioxidative Protection in Acute Lung Injury.

Induced pluripotent stem cells (iPSCs) have been reported to alleviate organ injury, although the mechanisms of action remain unclear and administration of intact cells faces many limitations. We hypothesized that cell-free conditioned media (CM) containing the secretome of iPSCs possess antioxidative constituents that can alleviate pulmonary oxidant stress damage. We derived iPSCs from human dermal fibroblasts and harvested the CM. Addition of iPSC CM to cultured human alveolar type-1 epithelial cells mitigated hyperoxia-induced depletion of endogenous total antioxidant capacity while tracheal instillation of iPSC CM into adult rat lungs enhanced hyperoxia-induced increase in TAC. In both the in vitro and in vivo models, iPSC CM ameliorated oxidative damage to DNA, lipid, and protein, and activated the nuclear factor (erythroid 2)-related factor 2 (Nrf2) network of endogenous antioxidant proteins. Compared with control fibroblast-conditioned or cell-free media, iPSC CM is highly enriched with αKlotho at a concentration up to more than 10-fold of that in normal serum. αKlotho is an essential antioxidative cell maintenance and protective factor and an activator of the Nrf2 network. Immunodepletion of αKlotho reduced iPSC CM-mediated cytoprotection by ∼50%. Thus, the abundant αKlotho content significantly contributes to iPSC-mediated antioxidation and cytoprotection. Results uncover a major mechanism of iPSC action, suggest a fundamental role of αKlotho in iPSC maintenance, and support the translational potential of airway delivery of cell-free iPSC secretome for protection against lung injury. The targeted cell-free secretome-based approach may also be applicable to the amelioration of injury in other organs. Stem Cells 2018;36:616-625.

Generation of an alveolar epithelial type II cell line from induced pluripotent stem cells.

Differentiation of primary alveolar type II epithelial cells (AEC II) to AEC type I in culture is a major barrier in the study of the alveolar epithelium in vitro. The establishment of an AEC II cell line derived from induced pluripotent stem cells (iPSC) represents a novel opportunity to study alveolar epithelial cell biology, for instance, in the context of lung injury, fibrosis, and repair. In the present study, we generated long-lasting AEC II from iPSC (LL-iPSC-AEC II). LL-iPSC-AEC II displayed morphological characteristics of AEC II, including growth in a cobblestone monolayer, the presence of lamellar bodies, and microvilli, as shown by electron microscopy. Also, LL-iPSC-AEC II expressed AEC type II proteins, such as cytokeratin, surfactant protein C, and LysoTracker DND 26 (a marker for lamellar bodies). Furthermore, the LL-iPSC-AEC II exhibited functional properties of AEC II by an increase of transepithelial electrical resistance over time, secretion of inflammatory mediators in biologically relevant quantities (IL-6 and IL-8), and efficient in vitro alveolar epithelial wound repair. Consistent with the AEC II phenotype, the cell line showed the ability to uptake and release surfactant protein B, to secrete phospholipids, and to differentiate into AEC type I. In summary, we established a long-lasting, but finite AEC type II cell line derived from iPSC as a novel cellular model to study alveolar epithelial cell biology in lung health and disease.

Efficacy of In Vivo Electroporation-Mediated IL-10 Gene Delivery on Survival of Skin Flaps.

Despite advances in understanding the underlying mechanisms of flap necrosis and improvement in surgical techniques, skin flap necrosis after reconstructive surgery remains a crucial issue. We investigated the efficacy of electroporation-mediated IL-10 gene transfer to random skin flap with an aim to accelerate wound healing and improve skin flap survival. Nine male Wistar rats (300-330 g) were divided in two groups (a) control group (n = 5), only surgery no gene transfer, and (b) experimental group, received electroporation-mediated IL-10 gene transfer 24 h before the surgery as prophylaxis (n = 4). Random skin flap (McFarlane) was performed in both groups. Planimetry, Laser Doppler imaging, and immunohistochemistry were used to evaluate the effect of IL-10 gene transfer between study groups at day 7. Electroporation-mediated IL-10 gene transfer decreased percentage of flap necrosis (p value = 0.0159) and increased cutaneous perfusion compared to the control group (p value = 0.0159). In addition, Spearman's rank correlation showed a significant negative correlation between percentage of flap necrosis and Laser Index (p value = 0.0083, r -0.83, respectively). Furthermore, significantly higher mean CD31+ vessel density was detected in the experimental group compared to the control group (p value = 0.0159). Additionally, semi-quantitative image analysis showed lower inflammatory cell count in experimental group compared to control group (p value = 0.0317). In vivo electroporation-mediated IL-10 gene transfer reduced necrosis, enhanced survival and vascularity in the ischemic skin flap.

Culture of human alveolar epithelial type II cells by sprouting.

BACKGROUND: Type II alveolar epithelial cells (AT2) play a pivotal role in maintaining the integrity and function of the alveoli. Only recently, the role of impaired epithelial repair mechanisms after injury in the pathogenesis of idiopathic pulmonary fibrosis has been demonstrated, and has shifted the AT2 cell in the focus of interest. Therefore, using primary human AT2 cells instead of cell lines for in vitro experiments has become desirable. Several groups have developed methods to isolate human AT2 cells applying tissue digestion and consecutive filtration in their protocols. Here we present a technique to isolate primary human AT2 cells by sprouting directly from peripheral human lung tissue. METHODS: Epithelial cell cultures were established from lung tissue obtained from patients undergoing diagnostic or therapeutic video-assisted thoracoscopic surgery or undergoing flexible bronchoscopy with transbronchial biopsy. Lung tissue was cut into small pieces and those were placed into cell culture flasks containing supplemented epithelial growth medium for cell sprouting. Cells were characterized by immunofluorescence stainings for E-cadherin, pan-cytokeratin, surfactant protein C (SP-C), and for lysotracker; fluorescent surfactant associated protein B (SP-B) uptake and secretion was assessed by live cell imaging; RNA levels of SP-A, SP-B, SP-C, and SP-D were determined by real-time PCR; Electron microscopy was used to search for the presence of lamellar bodies. RESULTS: Sprouting of cells started two to four days after the start of culture. Epithelial differentiation was confirmed by positive staining for E-cadherin and pan-cytokeratin. Further characterization demonstrated positivity for the AT2 cell marker SP-C and for lysotracker which selectively labels lamellar bodies in cultured AT2 cells. The up-take and release of SP-B, a mechanism described for AT2 cells only, was demonstrated by live cell imaging. Real-time RT-PCR showed mRNA expression of all four surfactant proteins with highest levels for SP-B. The presence of lamellar bodies was demonstrated by electron microscopy. CONCLUSIONS: This study describes a novel method for isolating AT2 cells from human adult lung tissue by sprouting. The characterization of the cultured AT2 cells complies with current criteria for an alveolar type 2 cell phenotype. Compared to current protocols for the culture of AT2 cells, isolating the cells by sprouting is simple, avoids proteolytic tissue digestion, and has the advantage to be successful even from as few tissue as attained from a transbronchial forceps biopsy.

Alveolar derecruitment and collapse induration as crucial mechanisms in lung injury and fibrosis.

Idiopathic pulmonary fibrosis (IPF) and bleomycin-induced pulmonary fibrosis are associated with surfactant system dysfunction, alveolar collapse (derecruitment), and collapse induration (irreversible collapse). These events play undefined roles in the loss of lung function. The purpose of this study was to quantify how surfactant inactivation, alveolar collapse, and collapse induration lead to degradation of lung function. Design-based stereology and invasive pulmonary function tests were performed 1, 3, 7, and 14 days after intratracheal bleomycin-instillation in rats. The number and size of open alveoli was correlated to mechanical properties. Active surfactant subtypes declined by Day 1, associated with a progressive alveolar derecruitment and a decrease in compliance. Alveolar epithelial damage was more pronounced in closed alveoli compared with ventilated alveoli. Collapse induration occurred on Day 7 and Day 14 as indicated by collapsed alveoli overgrown by a hyperplastic alveolar epithelium. This pathophysiology was also observed for the first time in human IPF lung explants. Before the onset of collapse induration, distal airspaces were easily recruited, and lung elastance could be kept low after recruitment by positive end-expiratory pressure (PEEP). At later time points, the recruitable fraction of the lung was reduced by collapse induration, causing elastance to be elevated at high levels of PEEP. Surfactant inactivation leading to alveolar collapse and subsequent collapse induration might be the primary pathway for the loss of alveoli in this animal model. Loss of alveoli is highly correlated with the degradation of lung function. Our ultrastructural observations suggest that collapse induration is important in human IPF.

Biogenic polymer-based patches for congenital cardiac surgery: a feasibility study.

Objective: Currently used patch materials in congenital cardiac surgery do not grow, renew, or remodel. Patch calcification occurs more rapidly in pediatric patients eventually leading to reoperations. Bacterial cellulose (BC) as a biogenic polymer offers high tensile strength, biocompatibility, and hemocompatibility. Thus, we further investigated the biomechanical properties of BC for use as patch material. Methods: The BC-producing bacteria Acetobacter xylinum were cultured in different environments to investigate optimal culturing conditions. For mechanical characterization, an established method of inflation for biaxial testing was used. The applied static pressure and deflection height of the BC patch were measured. Furthermore, a displacement and strain distribution analysis was performed and compared to a standard xenograft pericardial patch. Results: The examination of the culturing conditions revealed that the BC became homogenous and stable when cultivated at 29°C, 60% oxygen concentration, and culturing medium exchange every third day for a total culturing period of 12 days. The estimated elastic modulus of the BC patches ranged from 200 to 530 MPa compared to 230 MPa for the pericardial patch. The strain distributions, calculated from preloaded (2 mmHg) to 80 mmHg inflation, show BC patch strains ranging between 0.6% and 4%, which was comparable to the pericardial patch. However, the pressure at rupture and peak deflection height varied greatly, ranging from 67 to around 200 mmHg and 0.96 to 5.28 mm, respectively. The same patch thickness does not automatically result in the same material properties indicating that the manufacturing conditions have a significant impact on durability. Conclusions: BC patches can achieve comparable results to pericardial patches in terms of strain behavior as well as in the maximum applied pressure that can be withstood without rupture. Bacterial cellulose patches could be a promising material worth further research.

Anti-Fibrotic Effect of SDF-1ß Overexpression in Bleomycin-Injured Rat Lung.

Rational: Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease and is associated with high mortality due to a lack of effective treatment. Excessive deposition of the extracellular matrix by activated myofibroblasts in the alveolar space leads to scar formation that hinders gas exchange. Therefore, selectively removing activated myofibroblasts with the aim to repair and remodel fibrotic lungs is a promising approach. Stromal-derived growth factor (SDF-1) is known to stimulate cellular signals which attract stem cells to the site of injury for tissue repair and remodeling. Here, we investigate the effect of overexpression of SDF-1ß on lung structure using the bleomycin-injured rat lung model. Methods: Intratracheal administration of bleomycin was performed in adult male rats (F344). Seven days later, in vivo electroporation-mediated gene transfer of either SDF-1ß or the empty vector was performed. Animals were sacrificed seven days after gene transfer and histology, design-based stereology, flow cytometry, and collagen measurement were performed on the tissue collected. For in vitro experiments, lung fibroblasts obtained from IPF patients were used. Results: Seven days after SDF-1ß gene transfer to bleomycin-injured rat lungs, reduced total collagen, reduced collagen fibrils, improved histology and induced apoptosis of myofibroblasts were observed. Furthermore, it was revealed that TNF-α mediates SDF-1ß-induced apoptosis of myofibroblasts; moreover, SDF-1ß overexpression increased alveolar epithelial cell numbers and proliferation in vivo and also induced their migration in vitro. Conclusions: Our study demonstrates a new antifibrotic mechanism of SDF-1ß overexpression and suggests SDF-1ß as a potential new approach for the treatment of lung fibrosis.

Basal-Like Cell-Conditioned Medium Exerts Anti-Fibrotic Effects In Vitro and In Vivo.

In idiopathic pulmonary fibrosis (IPF), basal-like cells are atypically present in the alveolar region, where they may affect adjacent stromal cells by paracrine mechanisms. We here aimed to confirm the presence of basal-like cells in peripheral IPF lung tissue in vivo, to culture and characterize the cells in vitro, and to investigate their paracrine effects on IPF fibroblasts in vitro and in bleomycin-injured rats in vivo. Basal-like cells are mainly localized in areas of pathological bronchiolization or honeycomb cysts in peripheral IPF lung tissue. Single-cell RNA sequencing (scRNA-seq) demonstrated an overall homogeneity, the expression of the basal cell markers cytokeratin KRT5 and KRT17, and close transcriptomic similarities to basal cells in the majority of cells cultured in vitro. Basal-like cells secreted significant levels of prostaglandin E2 (PGE2), and their conditioned medium (CM) inhibited alpha-smooth muscle actin (α-SMA) and collagen 1A1 (Col1A1) and upregulated matrix metalloproteinase-1 (MMP-1) and hepatocyte growth factor (HGF) by IPF fibroblasts in vitro. The instillation of CM in bleomycin-injured rat lungs resulted in reduced collagen content, improved lung architecture, and reduced α-SMA-positive cells. Our data suggested that basal-like cells may limit aberrant fibroblast activation and differentiation in IPF through paracrine mechanisms.

LRR-protein RNH1 dampens the inflammasome activation and is associated with COVID-19 severity.

Inflammasomes are cytosolic innate immune sensors of pathogen infection and cellular damage that induce caspase-1-mediated inflammation upon activation. Although inflammation is protective, uncontrolled excessive inflammation can cause inflammatory diseases and can be detrimental, such as in coronavirus disease (COVID-19). However, the underlying mechanisms that control inflammasome activation are incompletely understood. Here we report that the leucine-rich repeat (LRR) protein ribonuclease inhibitor (RNH1), which shares homology with LRRs of NLRP (nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing) proteins, attenuates inflammasome activation. Deletion of RNH1 in macrophages increases interleukin (IL)-1ß production and caspase-1 activation in response to inflammasome stimulation. Mechanistically, RNH1 decreases pro-IL-1ß expression and induces proteasome-mediated caspase-1 degradation. Corroborating this, mouse models of monosodium urate (MSU)-induced peritonitis and lipopolysaccharide (LPS)-induced endotoxemia, which are dependent on caspase-1, respectively, show increased neutrophil infiltration and lethality in Rnh1 -/- mice compared with wild-type mice. Furthermore, RNH1 protein levels were negatively related with disease severity and inflammation in hospitalized COVID-19 patients. We propose that RNH1 is a new inflammasome regulator with relevance to COVID-19 severity.

Effect of electroporation-mediated diphtheria toxin A expression on PSA positive human prostate xenograft tumors in SCID mice.

BACKGROUND: Current therapies to treat prostate cancer are often limited. Since it has been shown that very low concentrations of diphtheria toxin A (DT-A) result in abrogation of protein synthesis and apoptosis of cells, DT-A might serve as an efficient killer in cancer gene therapy. For this purpose we investigated in a quantitative manner using a stereological approach the apoptotic effect of DT-A in androgen receptor (AR) and prostate specific antigen (PSA) expressing cells after tumor formation in both flanks of SCID mice. METHODS: First, DT-A plasmid transfection was evaluated, using the lipid formulation DMRIE-C in C4-2 prostate cancer xenografts. After detection of an overall high rate of apoptosis by DMRIE-C alone, plasmid delivery was performed in a second study by electroporation. Finally this method was used to specifically target the AR and PSA expressing cell line C4-2 using pDT-A driven by a prostate specific promoter and enhancer (PSE/PSA). PC-3 cells, being AR and PSA negative, served as controls. RESULTS: The experiments revealed evidence of a reduced growth rate of AR and PSA expressing C4-2 cells in vitro and in vivo compared to the AR and PSA negative prostate cancer cell line PC-3. The electroporation technology favored the response compared to DMRIE-C. CONCLUSION: These results suggest that the local delivery of DT-A plasmid by electroporation might present a favorable factor to treat prostate cancer.

Prolonged amelioration of acute lung allograft rejection by sequential overexpression of human interleukin-10 and hepatocyte growth factor in rats.

The effect of prolonged electroporation-mediated human interleukin-10 (hIL-10) overexpression 24 hours before transplantation, combined with sequential human hepatocyte growth factor (HGF) overexpression into skeletal muscle on day 5, on rat lung allograft rejection was evaluated. Left lung allotransplantation was performed from Brown-Norway to Fischer-F344 rats. Gene transfer into skeletal muscle was enhanced by electroporation. Three groups were studied: group I animals (n = 5) received 2.5 µg pCIK-hIL-10 (hIL-10/CMV [cytomegalovirus] early promoter enhancer) on day -1 and 80 µg pCIK-HGF (HGF/CMV early promoter enhancer) on day 5. Group II animals (n = 4) received 2.5 µg pCIK-hIL-10 and pUbC-hIL-10 (hIL-10/pUbC promoter) on day -1. Control group III animals (n = 4) were treated by sham electroporation on days -1 and 5. All animals received daily nontherapeutic intraperitoneal dose of cyclosporin A (2.5 mg/kg) and were sacrificed on day 15. Graft oxygenation and allograft rejection were evaluated. Significant differences were found between study groups in graft oxygenation (Pao(2)) (P = .0028; group I vs. groups II and III, P < .01 each). Pao(2) was low in group II (31 ± 1 mm Hg) and in group III controls (34 ± 10 mm Hg), without statistically significant difference between these 2 groups (P = .54). In contrast, in group I, Pao(2) of recipients sequentially transduced with IL-10 and HGF plasmids was much improved, with 112 ± 39 mm Hg (vs. groups II and III; P < .01 each), paralleled by reduced vascular and bronchial rejection (group I vs. groups II and III, P < .021 each). Sequential overexpression of anti-inflammatory cytokine IL-10, followed by sequential and overlapping HGF overexpression on day 5, preserves lung function and reduces acute lung allograft rejection up to day 15 post transplant as compared to prolonged IL-10 overexpression alone.

Electrospray Mediated Localized and Targeted Chemotherapy in a Mouse Model of Lung Cancer.

Background: An advanced stage, centrally localized invasive tumor is a major cause of sudden death in lung cancer patients. Currently, chemotherapy, radiotherapy, laser ablation, or surgical resection if possible are the available state-of-the-art treatments but none of these guarantee remedy or long-term relief and are often associated with fatal complications. Allowing localized chemotherapy, by direct and confined drug delivery only at the tumor site, could be a promising option for preoperative down staging or palliative therapy. Here we report the localized and targeted application of intra tumor delivery of chemotherapeutics using a novel device based on the principle of electrospray. Methods: C57BL/6J mice were injected with Lewis lung carcinoma cells subcutaneously. After 15 days, the animals were anesthetized and the tumors were exposed by skin incision. Tumors were electrosprayed with 100 µg cisplatin on days 0 and 2, and tumor volumes were measured daily. Animals were sacrificed on day 7 after the first electrospray and tumors were analyzed by immunohistochemistry. Results: In this proof-of-concept study, we report that the tumor volume was reduced by 81.2% (22.46 ± 12.14 mm3) after two electrospray mediated Cisplatin deliveries, while the control tumor growth, at the same time point, increased by 200% (514.30 ± 104.50 mm3). Moreover, tunnel and Caspase-3 positive cells were increased after Cisplatin electrospray compared to other experimental groups of animals. Conclusion: Targeted drug delivery by electrospray is efficient in the subcutaneous mouse model of lung cancer and offers a promising opportunity for further development toward its clinical application.

Efficacy of Combined in-vivo Electroporation-Mediated Gene Transfer of VEGF, HGF, and IL-10 on Skin Flap Survival, Monitored by Label-Free Optical Imaging: A Feasibility Study.

Preventing surgical flaps necrosis remains challenging. Laser Doppler imaging and ultrasound can monitor blood flow in flap regions, but they do not directly measure the cellular response to ischemia. The study aimed to investigate the efficacy of synergistic in-vivo electroporation-mediated gene transfer of interleukin 10 (IL-10) with either hepatocyte growth factor (HGF) or vascular endothelial growth factor (VEGF) on the survival of a modified McFarlane flap, and to evaluate the effect of the treatment on cell metabolism, using label-free fluorescence lifetime imaging. Fifteen male Wistar rats (290-320 g) were randomly divided in three groups: group-A (control group) underwent surgery and received no gene transfer. Group-B received electroporation mediated hIL-10 gene delivery 24 h before and VEGF gene delivery 24 h after surgery. Group-C received electroporation mediated hIL-10 gene delivery 24 h before and hHGF gene delivery 24 h after surgery. The animals were assessed clinically and histologically. In addition, label-free fluorescence lifetime imaging was performed on the flap. Synergistic electroporation mediated gene delivery significantly decreased flap necrosis (P = 0.0079) and increased mean vessel density (P = 0.0079) in treatment groups B and C compared to control group-A. NADH fluorescence lifetime analysis indicated an increase in oxidative phosphorylation in the epidermis of the group-B (P = 0.039) relative to controls. These findings suggested synergistic in-vivo electroporation-mediated gene transfer as a promising therapeutic approach to enhance viability and vascularity of skin flap. Furthermore, the study showed that combinational gene therapy promoted an increase in tissue perfusion and a relative increase in oxidative metabolism within the epithelium.

Inhalational delivery of induced pluripotent stem cell secretome improves postpneumonectomy lung structure and function.

Cell-free secretory products (secretome) of human induced pluripotent stem cells (iPSCs) have been shown to attenuate tissue injury and facilitate repair and recovery. To examine whether iPSC secretome facilitates mechanically induced compensatory responses following unilateral pneumonectomy (PNX), litter-matched young adult female hounds underwent right PNX (removing 55%-58% of lung units), followed by inhalational delivery of either the nebulized-conditioned media containing induced pluripotent stem cell secretome (iPSC CM) or control cell-free media (CFM); inhalation was repeated every 5 days for 10 treatments. Lung function was measured under anesthesia pre-PNX and 10 days after the last treatment (8 wk post-PNX); detailed quantitative analysis of lung ultrastructure was performed postmortem. Pre-PNX lung function was similar between groups. Compared with CFM control, treatment with iPSC CM attenuated the post-PNX decline in lung diffusing capacity for carbon monoxide and membrane diffusing capacity, accompanied by a 24% larger postmortem lobar volume and distal air spaces. Alveolar double-capillary profiles were 39% more prevalent consistent with enhanced intussusceptive angiogenesis. Frequency distribution of the harmonic mean thickness of alveolar blood-gas barrier shifted toward the lowest values, whereas alveolar septal tissue volume and arithmetic septal thickness were similar, indicating septal remodeling and reduced diffusive resistance of the blood-gas barrier. Thus, repetitive inhalational delivery of iPSC secretome enhanced post-PNX alveolar angiogenesis and septal remodeling that are associated with improved gas exchange compensation. Results highlight the plasticity of the remaining lung units following major loss of lung mass that are responsive to broad-based modulation provided by the iPSC secretome.NEW & NOTEWORTHY To examine whether the secreted products of human induced pluripotent stem cells (iPSCs) facilitate innate adaptive responses following loss of lung tissue, adult dogs underwent surgical removal of one lung, then received repeated administration of iPSC secretory products via inhalational delivery compared with control treatment. Inhalation of iPSC secretory products enhanced capillary formation and beneficial structural remodeling in the remaining lung, leading to improved lung function.

Non-viral Gene Delivery Methods for Bone and Joints.

Viral carrier transport efficiency of gene delivery is high, depending on the type of vector. However, viral delivery poses significant safety concerns such as inefficient/unpredictable reprogramming outcomes, genomic integration, as well as unwarranted immune responses and toxicity. Thus, non-viral gene delivery methods are more feasible for translation as these allow safer delivery of genes and can modulate gene expression transiently both in vivo, ex vivo, and in vitro. Based on current studies, the efficiency of these technologies appears to be more limited, but they are appealing for clinical translation. This review presents a summary of recent advancements in orthopedics, where primarily bone and joints from the musculoskeletal apparatus were targeted. In connective tissues, which are known to have a poor healing capacity, and have a relatively low cell-density, i.e., articular cartilage, bone, and the intervertebral disk (IVD) several approaches have recently been undertaken. We provide a brief overview of the existing technologies, using nano-spheres/engineered vesicles, lipofection, and in vivo electroporation. Here, delivery for microRNA (miRNA), and silencing RNA (siRNA) and DNA plasmids will be discussed. Recent studies will be summarized that aimed to improve regeneration of these tissues, involving the delivery of bone morphogenic proteins (BMPs), such as BMP2 for improvement of bone healing. For articular cartilage/osteochondral junction, non-viral methods concentrate on targeted delivery to chondrocytes or MSCs for tissue engineering-based approaches. For the IVD, growth factors such as GDF5 or GDF6 or developmental transcription factors such as Brachyury or FOXF1 seem to be of high clinical interest. However, the most efficient method of gene transfer is still elusive, as several preclinical studies have reported many different non-viral methods and clinical translation of these techniques still needs to be validated. Here we discuss the non-viral methods applied for bone and joint and propose methods that can be promising in clinical use.

In Vivo Electroporation-Mediated, Intrahepatic Alpha1 Antitrypsin Gene Transfer Reduces Pulmonary Emphysema in Pallid Mice.

RATIONALE: Mutation in the alpha1 antitrypsin (AAT) gene leads to low circulating levels of AAT, which is associated with several disease processes including pulmonary emphysema. The standard of care relies on substitution with plasma-purified AAT. We studied a novel approach to obtain sustained therapeutic levels of circulating AAT using nonviral in vivo electroporation-mediated gene transfer to the liver. METHODS: In vivo intrahepatic electroporation-mediated human AAT gene transfer was performed in C57 Bl/6J mice carrying a genetic deficiency of murine AAT (pallid mice) and suffering from pulmonary emphysema. The animals were evaluated for lung function using flexiVent and detailed stereological assessments. Lung neutrophilic burden was assessed. RESULTS: Pallid mice showed morphologically detectable pulmonary emphysema. Thirty days after in vivo electroporation-mediated gene transfer directly aimed at the liver, circulating human AAT was elevated and lung function was significantly improved compared to non-treated pallid mice. Stereological analysis revealed a reduction in pulmonary emphysema. CONCLUSION: Our data indicate that in vivo intrahepatic electroporation-mediated gene transfer of AAT is a safe and efficient procedure resulting in reduction of pulmonary emphysema in pallid mice.