Stem cells, cell therapies, and bioengineering in lung biology and disease 2021.

The 9th biennial conference titled "Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases" was hosted virtually, due to the ongoing COVID-19 pandemic, in collaboration with the University of Vermont Larner College of Medicine, the National Heart, Lung, and Blood Institute, the Alpha-1 Foundation, the Cystic Fibrosis Foundation, and the International Society for Cell & Gene Therapy. The event was held from July 12th through 15th, 2021 with a pre-conference workshop held on July 9th. As in previous years, the objectives remained to review and discuss the status of active research areas involving stem cells (SCs), cellular therapeutics, and bioengineering as they relate to the human lung. Topics included 1) technological advancements in the in situ analysis of lung tissues, 2) new insights into stem cell signaling and plasticity in lung remodeling and regeneration, 3) the impact of extracellular matrix in stem cell regulation and airway engineering in lung regeneration, 4) differentiating and delivering stem cell therapeutics to the lung, 5) regeneration in response to viral infection, and 6) ethical development of cell-based treatments for lung diseases. This selection of topics represents some of the most dynamic and current research areas in lung biology. The virtual workshop included active discussion on state-of-the-art methods relating to the core features of the 2021 conference, including in situ proteomics, lung-on-chip, induced pluripotent stem cell (iPSC)-airway differentiation, and light sheet microscopy. The conference concluded with an open discussion to suggest funding priorities and recommendations for future research directions in basic and translational lung biology.

Notch3-Jagged signaling controls the pool of undifferentiated airway progenitors.

Basal cells are multipotent airway progenitors that generate distinct epithelial cell phenotypes crucial for homeostasis and repair of the conducting airways. Little is known about how these progenitor cells expand and transition to differentiation to form the pseudostratified airway epithelium in the developing and adult lung. Here, we show by genetic and pharmacological approaches that endogenous activation of Notch3 signaling selectively controls the pool of undifferentiated progenitors of upper airways available for differentiation. This mechanism depends on the availability of Jag1 and Jag2, and is key to generating a population of parabasal cells that later activates Notch1 and Notch2 for secretory-multiciliated cell fate selection. Disruption of this mechanism resulted in aberrant expansion of basal cells and altered pseudostratification. Analysis of human lungs showing similar abnormalities and decreased NOTCH3 expression in subjects with chronic obstructive pulmonary disease suggests an involvement of NOTCH3-dependent events in the pathogenesis of this condition.

Telomere dysfunction induces metabolic and mitochondrial compromise.

Telomere dysfunction activates p53-mediated cellular growth arrest, senescence and apoptosis to drive progressive atrophy and functional decline in high-turnover tissues. The broader adverse impact of telomere dysfunction across many tissues including more quiescent systems prompted transcriptomic network analyses to identify common mechanisms operative in haematopoietic stem cells, heart and liver. These unbiased studies revealed profound repression of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha and beta (PGC-1α and PGC-1ß, also known as Ppargc1a and Ppargc1b, respectively) and the downstream network in mice null for either telomerase reverse transcriptase (Tert) or telomerase RNA component (Terc) genes. Consistent with PGCs as master regulators of mitochondrial physiology and metabolism, telomere dysfunction is associated with impaired mitochondrial biogenesis and function, decreased gluconeogenesis, cardiomyopathy, and increased reactive oxygen species. In the setting of telomere dysfunction, enforced Tert or PGC-1α expression or germline deletion of p53 (also known as Trp53) substantially restores PGC network expression, mitochondrial respiration, cardiac function and gluconeogenesis. We demonstrate that telomere dysfunction activates p53 which in turn binds and represses PGC-1α and PGC-1ß promoters, thereby forging a direct link between telomere and mitochondrial biology. We propose that this telomere-p53-PGC axis contributes to organ and metabolic failure and to diminishing organismal fitness in the setting of telomere dysfunction.

Bcl2L12-mediated inhibition of effector caspase-3 and caspase-7 via distinct mechanisms in glioblastoma.

Glioblastoma multiforme (GBM) is a highly aggressive brain cancer that is characterized by the paradoxical features of intense apoptosis resistance yet a marked propensity to undergo necrosis. Bcl2L12 (for Bcl2-Like12) is a nuclear and cytoplasmic oncoprotein that is universally overexpressed in primary GBM and functions to block postmitochondrial apoptosis signaling by neutralizing effector caspase-3 and caspase-7 maturation. This postmitochondrial block in apoptosis engenders the alternate cell fate of cellular necrosis, thus providing a molecular explanation for GBM's classical features. Whereas Bcl2L12-mediated neutralization of caspase-7 maturation involves physical interaction, the mechanism governing Bcl2L12-mediated inhibition of caspase-3 activity is not known. The nuclear localization of Bcl2L12 prompted expression profile studies of primary astrocytes engineered to overexpress Bcl2L12. The Bcl2L12 transcriptome revealed a striking induction of the small heat shock protein alpha-basic-crystallin (alphaB-crystallin/HspB5), a link reinforced by robust alphaB-crystallin expression in Bcl2L12-expressing orthotopic glioma and strong coexpression of alphaB-crystallin and Bcl2L12 proteins in human primary GBMs. On the functional level, enforced alphaB-crystallin or Bcl2L12 expression enhances orthotopic tumor growth. Conversely, RNAi-mediated knockdown of alphaB-crystallin in Bcl2L12-expressing astrocytes and glioma cell lines with high endogenous alphaB-crystallin showed enhanced apoptosis, yet decreased necrotic cell death with associated increased caspase-3 but not caspase-7 activation. Mirroring this specific effect on effector caspase-3 activation, alphaB-crystallin selectively binds pro-caspase-3 and its cleavage intermediates in vitro and in vivo. Thus, alphaB-crystallin is a Bcl2L12-induced oncoprotein that enables Bcl2L12 to block the activation of both effector caspases via distinct mechanisms, thereby contributing to GBM pathogenesis and its hallmark biological properties.

Repeated injury promotes tracheobronchial tissue stem cell attrition.

Chronic lung disease has been attributed to stem cell aging and/or exhaustion. We investigated these mechanisms using mouse and human tracheobronchial tissue-specific stem cells (TSC). In mouse, chromatin labeling and flow cytometry demonstrated that naphthalene (NA) injury activated a subset of TSC. These activated TSC continued to proliferate after the epithelium was repaired and a clone study demonstrated that ~96% of activated TSC underwent terminal differentiation. Despite TSC attrition, epithelial repair after a second NA injury was normal. The second injury accelerated proliferation of previously activated TSC and a nucleotide-label retention study indicated that the second injury recruited TSC that were quiescent during the first injury. These mouse studies indicate that (a) injury causes selective activation of the TSC pool; (b) activated TSC are predisposed to further proliferation; and (c) the activated state leads to terminal differentiation. In human TSC, repeated proliferation also led to terminal differentiation and depleted the TSC pool. A clone study identified long- and short-lived TSC and showed that short-lived TSC clones had significantly shorter telomeres than their long-lived counterparts. The TSC pool was significantly depleted in dyskeratosis congenita donors, who harbor mutations in telomere biology genes. The remaining TSC had short telomeres and short lifespans. Collectively, the mouse and human studies support a model in which epithelial injury increases the biological age of the responding TSC. When applied to chronic lung disease, this model suggests that repeated injury accelerates the biological aging process resulting in abnormal repair and disease initiation.

Transcriptional analysis of cystic fibrosis airways at single-cell resolution reveals altered epithelial cell states and composition.

Cystic fibrosis (CF) is a lethal autosomal recessive disorder that afflicts more than 70,000 people. People with CF experience multi-organ dysfunction resulting from aberrant electrolyte transport across polarized epithelia due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF-related lung disease is by far the most important determinant of morbidity and mortality. Here we report results from a multi-institute consortium in which single-cell transcriptomics were applied to define disease-related changes by comparing the proximal airway of CF donors (n = 19) undergoing transplantation for end-stage lung disease with that of previously healthy lung donors (n = 19). Disease-dependent differences observed include an overabundance of epithelial cells transitioning to specialized ciliated and secretory cell subsets coupled with an unexpected decrease in cycling basal cells. Our study yields a molecular atlas of the proximal airway epithelium that will provide insights for the development of new targeted therapies for CF airway disease.

Author Correction: Cytoplasmic E2f4 forms organizing centres for initiation of centriole amplification during multiciliogenesis.

This corrects the article DOI: 10.1038/ncomms15857.

Cytoplasmic E2f4 forms organizing centres for initiation of centriole amplification during multiciliogenesis.

Abnormal development of multiciliated cells is a hallmark of a variety of human conditions associated with chronic airway diseases, hydrocephalus and infertility. Multiciliogenesis requires both activation of a specialized transcriptional program and assembly of cytoplasmic structures for large-scale centriole amplification that generates basal bodies. It remains unclear, however, what mechanism initiates formation of these multiprotein complexes in epithelial progenitors. Here we show that this is triggered by nucleocytoplasmic translocation of the transcription factor E2f4. After inducing a transcriptional program of centriole biogenesis, E2f4 forms apical cytoplasmic organizing centres for assembly and nucleation of deuterosomes. Using genetically altered mice and E2F4 mutant proteins we demonstrate that centriole amplification is crucially dependent on these organizing centres and that, without cytoplasmic E2f4, deuterosomes are not assembled, halting multiciliogenesis. Thus, E2f4 integrates nuclear and previously unsuspected cytoplasmic events of centriole amplification, providing new perspectives for the understanding of normal ciliogenesis, ciliopathies and cancer.

Predicting intervention in renal colic patients after emergency department evaluation.

OBJECTIVES: There is no set of prospectively validated criteria to identify the emergency department (ED) patients with renal colic who are most likely to eventually have to undergo an intervention. This study prospectively assessed predictors of intervention in this patient population. METHODS: This prospective cohort study included adult patients with renal colic who presented to 2 tertiary care hospital EDs. Patients had an 18-variable data form completed by an emergency physician and a radiological study to confirm urolithiasis. After discharge, patients were followed at 1 and 4 weeks to assess for intervention. The outcome criteria included the patient having had at least 1 of the following procedures performed: extracorporeal shockwave lithotripsy (ESWL), ureteroscopy, percutaneous nephrostomy or open surgery. Data were analyzed using appropriate univariate techniques, and those variables associated with intervention were combined using logistic regression analysis. RESULTS: Over an 8-month period, 245 patients with confirmed urolithiasis were followed; 20% (95% confidence interval [CI] 15%-25%) eventually had a procedure to remove their calculi. Three variables were significantly correlated with having a procedure: i) size of calculus >or= 6 mm (odds ratio [OR] 10.7, 95% CI 4.6-24.8), ii) location of calculus above mid-ureter (OR 6.9, 95% CI 3.0-15.9), and iii) Visual Analogue Scale score for pain at discharge from the ED >or= 2 cm (OR 2.6, 95% CI 1.0-6.8). The area under receiver operating characteristic curve was 0.77 (95% C I 0.70-0.84) (p < 0.001). If all variables were present there was a 90% probability of the patient having an intervention performed within 4 weeks of discharge from the ED. Conversely, if none of the variables were present there was only a 4% probability of an intervention. Overall, the model had a sensitivity of 92% (95% CI 89%-96%) and a specificity of 63% (95% CI 57%-69%). CONCLUSIONS: This study has identified variables that could potentially be used to identify those renal colic patients who require an intervention after ED evaluation. Future studies will prospectively validate this model.

Crumbs3-Mediated Polarity Directs Airway Epithelial Cell Fate through the Hippo Pathway Effector Yap.

Epithelial cells undergo dynamic polarity changes as organs pattern, but the relationship between epithelial polarity and cell fate is poorly understood. Using the developing lung as a model, we found that distinct alterations in apical-basal polarity dictate airway epithelial differentiation. We demonstrate that Crb3, a Crumbs isoform that determines epithelial apical domain identity, is required for airway differentiation by controlling the localization of the transcriptional regulator Yap. We show that Crb3 promotes the interaction between Yap and the Hippo pathway kinases Lats1/2 at apical cell junctions to induce Yap phosphorylation and cytoplasmic retention, which drive cell differentiation. Loss of Crb3 in developing mouse airways or isolated adult airway progenitors results in unrestricted nuclear Yap activity and consequent cell differentiation defects. Our findings demonstrate that polarity-dependent cues control airway cell differentiation, offering important molecular insights into organ patterning.

Measuring the minimum clinically important difference in BPH outcome measures.

PURPOSE: To determine the minimum clinically important difference with respect to the different outcome measures used in benign prostatic hyperplasia (BPH) research. MATERIALS AND METHODS: Forty-two patients diagnosed with symptomatic BPH were prospectively recruited from community urology clinics. Patients were asked at follow-up visits to rate their improvement using a self-administrated questionnaire. The improvement score was then compared to the changes from baseline in each of the outcome measures. RESULTS: Maximum and mean flow-rates did not correlate significantly with the patient's improvement measure. The Boyarsky score correlated best with the patient's grading of overall improvement (R=.507, p<.001). The mean change in Boyarsky symptom score for patients with a small improvement was 3.23 (sd=3.52).

The hippo pathway effector Yap controls patterning and differentiation of airway epithelial progenitors.

How epithelial progenitor cells integrate local signals to balance expansion with differentiation during organogenesis is still little understood. Here, we provide evidence that the Hippo pathway effector Yap is a key regulator of this process in the developing lung. We show that when epithelial tubules are forming and branching, a nucleocytoplasmic shift in Yap localization marks the boundary between the airway and the distal lung compartments. At this transition zone, Yap specifies a transcriptional program that controls Sox2 expression and ultimately generates the airway epithelium. Without Yap, epithelial progenitors are unable to properly respond to local TGF-ß-induced cues and control levels and distribution of Sox2 to form airways. Yap levels and subcellular localization also markedly influence Sox2 expression and differentiation of adult airway progenitors. Our data reveal a role for the Hippo-Yap pathway in integrating growth-factor-induced cues in the developing and adult lung potentially key for homeostasis and regeneration repair.

Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment.

The existence of a haematopoietic stem cell niche as a spatially confined regulatory entity relies on the notion that haematopoietic stem and progenitor cells (HSPCs) are strategically positioned in unique bone marrow microenvironments with defined anatomical and functional features. Here, we employ a powerful imaging cytometry platform to perform a comprehensive quantitative analysis of HSPC distribution in bone marrow cavities of femoral bones. We find that HSPCs preferentially localize in endosteal zones, where most closely interact with sinusoidal and non-sinusoidal bone marrow microvessels, which form a distinctive circulatory system. In situ tissue analysis reveals that HSPCs exhibit a hypoxic profile, defined by strong retention of pimonidazole and expression of HIF-1α, regardless of localization throughout the bone marrow, adjacency to vascular structures or cell-cycle status. These studies argue that the characteristic hypoxic state of HSPCs is not solely the result of a minimally oxygenated niche but may be partially regulated by cell-specific mechanisms.

Identification of IGPR-1 as a novel adhesion molecule involved in angiogenesis.

Angiogenesis-the growth of new blood vessels from preexisting vessels-is an important physiological process and is considered to play a key role in tumor growth and metastasis. We identified the immunoglobulin-containing and proline-rich receptor-1 (IGPR-1, also called TMIGD2) gene as a novel cell adhesion receptor that is expressed in various human organs and tissues, mainly in cells with epithelium and endothelium origins. IGPR-1 regulates cellular morphology, homophilic cell aggregation, and cell-cell interaction. IGPR-1 activity also modulates actin stress fiber formation and focal adhesion and reduces cell migration. Silencing of expression of IGPR-1 by small interfering RNA (siRNA) and by ectopic overexpression in endothelial cells showed that IGPR-1 regulates capillary tube formation in vitro, and B16F melanoma cells engineered to express IGPR-1 displayed extensive angiogenesis in the mouse Matrigel angiogenesis model. Moreover, IGPR-1, through its proline-rich cytoplasmic domain, associates with multiple Src homology 3 (SH3)-containing signaling proteins, including SH3 protein interacting with Nck (SPIN90/WISH), bullous pemphigoid antigen-1, and calcium channel ß2. Silencing of expression of SPIN90/WISH by siRNA in endothelial cells showed that SPIN90/WISH is required for capillary tube formation. These features of IGPR-1 suggest that IGPR-1 is a novel receptor that plays an important role in cell-cell interaction, cell migration, and angiogenesis.

PEST motif serine and tyrosine phosphorylation controls vascular endothelial growth factor receptor 2 stability and downregulation.

The internalization and degradation of vascular endothelial growth factor receptor 2 (VEGFR-2), a potent angiogenic receptor tyrosine kinase, is a central mechanism for the regulation of the coordinated action of VEGF in angiogenesis. Here, we show that VEGFR-2 is ubiquitinated in response to VEGF, and Lys 48-linked polyubiquitination controls its degradation via the 26S proteosome. The degradation and ubiquitination of VEGFR-2 is controlled by its PEST domain, and the phosphorylation of Ser1188/Ser1191 is required for the ubiquitination of VEGFR-2. F-box-containing ß-Trcp1 ubiquitin E3 ligase is recruited to S1188/S1191 VEGFR-2 and mediates the ubiquitination and degradation of VEGFR-2. The PEST domain also controls the activation of p38 mitogen-activated protein kinase (MAPK) through phospho-Y1173. The activation of p38 stabilizes VEGFR-2, and its inactivation accelerates VEGFR-2 downregulation. The VEGFR-2-mediated activation of p38 is established through the protein kinase A (PKA)/MKK6 pathway. PKA is recruited to VEGFR-2 through AKAP1/AKAP149, and its phosphorylation requires Y1173 of VEGFR-2. The study has identified a unique mechanism in which VEGFR-2 stability and degradation is modulated. The PEST domain acts as a dual modulator of VEGFR-2; the phosphorylation of S1188/S1191 controls ubiquitination and degradation via ß-Trcp1, where the phosphorylation of Y1173 through PKA/p38 MAPK controls the stability of VEGFR-2.

Hand-assisted laparoscopic versus open nephrectomies in living donors.

UNLABELLED: Shortages of cadaveric kidneys for transplant into rising numbers of patients with end-stage renal failure have increased the demand for kidneys from live donors. The morbidity associated with traditional open donor nephrectomies (ODN) may discourage many candidates. The newer laparoscopic technique has been promoted as having less morbidity. OBJECTIVES: To evaluate outcomes of hand-assisted laparoscopic nephrectomies (HALN) and prospectively compare HALN and ODN. METHODS: After retrospectively reviewing donor and recipient outcomes in 33 HALN (December through August, 2000), we prospectively compared another 47 with 30 ODN performed from September 2000 through April 2001. RESULTS: All 80 HALN were successful, with no requirement to convert to an open procedure. Four donors experienced surgery-related complications: wound infection, retroperitoneal hematoma, prolonged ileus and early small-bowel obstruction, respectively. Two recipients had ureteral complications (1 stricture, 1 leak); 5 experienced delayed graft function, 2 requiring dialysis; and 2 kidneys were lost from infarction. The prospective comparison showed the operative time for HALN (mean 184 min, standard deviation [SD] 39 min) was significantly longer (143 [SD 27] min, p < 0.01), but resulted in less blood loss (p < 0.05). Lengths of time to warm ischemia/early graft function, resumption of oral intake/first bowel movement, and hospital discharge were similar. The abdominal-wall laxity and loss of cutaneous sensation from the flank incision experienced by many ODN patients after was uncommon in the HALN group. Three months after nephrectomy, donor complaints of incisional pain were less common after HALN (p < 0.01). CONCLUSIONS: HALN had good outcomes for donors and recipients, with quicker, more complete recoveries 3 months afterward.

Renal transplantation using non-heart-beating donors: a potential solution to the organ donor shortage in Canada.

INTRODUCTION: There is a chronic shortage of cadaveric organ donors for renal transplantation, which might be solved by the use of non-heart-beating donors (patients who suffer cardiac arrest and whose kidneys are harvested subsequently when irreversible heart and respiratory function occur). We carried out a chart review to determine whether the renal transplantation rate would improve if a non-heart-beating donor program was introduced at a Canadian centre. METHODS: We reviewed the charts of all 1547 patients who died in the emergency department or intensive care unit of the Ottawa Hospital, a tertiary care centre serving 1.2 million people in eastern Ontario, between January 1999 and May 2001. The number of potential non-heart-beating donors was determined by the use of predefined criteria. The number of additional kidneys that could be obtained with a non-heart-beating donor program was estimated and compared to the actual number of kidneys procured from conventional brain-dead donors during the same period. The potential increase in the renal transplantation rate was calculated. RESULTS: There were 83 potential non-heart-beating donors during the 29-month study period. The mean (and standard deviation) age of the donors was 40.6 (13.1) years, and 20% were female. The mean serum creatinine value was 75 (29) micromol/L; 44.6% of donors died secondary to trauma. We estimated that the use of non-heart-beating donors would have provided 14 to 41 additional donors during the study period (12-34 kidneys/yr). The cadaveric renal transplantation rate would have increased between 30% and 87%. CONCLUSION: The cadaveric renal transplantation rate could improve significantly if non-heart-beating donors were used in Canadian hospitals.