Colin R. Butler

Stem-cell-based, tissue engineered tracheal replacement in a child: A 2-year follow-up study

BACKGROUND Stem-cell-based, tissue engineered transplants might offer new therapeutic options for patients, including children, with failing organs. The reported replacement of an adult airway using stem cells on a biological scaffold with good results at 6 months supports this view. We describe the case of a child who received a stem-cell-based tracheal replacement and report findings after 2 years of follow-up. METHODS A 12-year-old boy was born with long-segment congenital tracheal stenosis and pulmonary sling. His airway had been maintained by metal stents, but, after failure, a cadaveric donor tracheal scaffold was decellularised. After a short course of granulocyte colony stimulating factor, bone marrow mesenchymal stem cells were retrieved preoperatively and seeded onto the scaffold, with patches of autologous epithelium. Topical human recombinant erythropoietin was applied to encourage angiogenesis, and transforming growth factor β to support chondrogenesis. Intravenous human recombinant erythropoietin was continued postoperatively. Outcomes were survival, morbidity, endoscopic appearance, cytology and proteomics of brushings, and peripheral blood counts. FINDINGS The graft revascularised within 1 week after surgery. A strong neutrophil response was noted locally for the first 8 weeks after surgery, which generated luminal DNA neutrophil extracellular traps. Cytological evidence of restoration of the epithelium was not evident until 1 year. The graft did not have biomechanical strength focally until 18 months, but the patient has not needed any medical intervention since then. 18 months after surgery, he had a normal chest CT scan and ventilation-perfusion scan and had grown 11 cm in height since the operation. At 2 years follow-up, he had a functional airway and had returned to school. INTERPRETATION Follow-up of the first paediatric, stem-cell-based, tissue-engineered transplant shows potential for this technology but also highlights the need for further research. FUNDING Great Ormond Street Hospital NHS Trust, The Royal Free Hampstead NHS Trust, University College Hospital NHS Foundation Trust, and Region of Tuscany.

Rapid Expansion of Human Epithelial Stem Cells Suitable for Airway Tissue Engineering

RATIONALE Stem cell-based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell-seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts. OBJECTIVES To define a scalable cell culture system to deliver airway epithelium to clinical grafts. METHODS Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air-liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air-liquid interface cultures. MEASUREMENTS AND MAIN RESULTS 3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model. CONCLUSIONS Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical transplantation, suggesting its suitability for use in tracheal reconstruction.

Outcomes of slide tracheoplasty in 101 children: A 17-year single-center experience

OBJECTIVE Our study describes the results from surgical slide tracheoplasty (STP) in children with long segment tracheal stenosis. METHODS Demographic and preoperative conditions, operative details, and outcome measures, including the need for endoscopic airway intervention and mortality, were collected for children undergoing STP between February 1995 and December 2012. RESULTS One hundred one patients (median age, 5.8 months; range, 5 days-15 years) underwent STP. Seventy-two patients (71.3%) had associated cardiovascular anomalies. Preoperative ventilation was necessary in 56 patients (55.4%), whereas extracorporeal membrane oxygenation was required in 10 patients (9.9%). Abnormal bronchial arborization was present in 39 children (38.6%), which included 13 patients (12.8%) with an anomalous right upper lobe bronchus and 17 patients (16.8%) with tracheal trifurcation. Airway stenosis extended into 1 or both bronchi in 24 patients (23.7%) and preoperative malacia was present in 24 patients (23.7%). STP was extended into the bronchus in 47 patients (46.5%). Overall survival was 88.2% (mortality in 12 patients). Post-STP balloon dilation was necessary in 45 patients (44.6%) and stenting was required in 22 patients (21.8%). Multivariate analysis revealed preoperative extracorporeal membrane oxygenation (P < .05), preoperative malacia (P < .001), and bronchial stenosis (P < .05) to be adverse predictors of survival. Preoperative malacia was a significant risk factor for stenting (P < .05). CONCLUSIONS STP is a versatile and reliable technique associated with low morbidity and mortality when compared with previous strategies for children with long segment tracheal stenosis. The presence of preoperative bronchomalacia is a significant risk factor for death and postoperative stenting.

β‐Catenin determines upper airway progenitor cell fate and preinvasive squamous lung cancer progression by modulating epithelial–mesenchymal transition

Human lung cancers, including squamous cell carcinoma (SCC) are a leading cause of death and, whilst evidence suggests that basal stem cells drive SCC initiation and progression, the mechanisms regulating these processes remain unknown. In this study we show that β‐catenin signalling regulates basal progenitor cell fate and subsequent SCC progression. In a cohort of preinvasive SCCs we established that elevated basal cell β‐catenin signalling is positively associated with increased disease severity, epithelial proliferation and reduced intercellular adhesiveness. We demonstrate that transgene‐mediated β‐catenin inhibition within keratin 14‐expressing basal cells delayed normal airway repair while basal cell‐specific β‐catenin activation increased cell proliferation, directed differentiation and promoted elements of early epithelial‐mesenchymal transition (EMT), including increased Snail transcription and reduced E‐cadherin expression. These observations are recapitulated in normal human bronchial epithelial cells in vitro following both pharmacological β‐catenin activation and E‐cadherin inhibition, and mirrored our findings in preinvasive SCCs. Overall, the data show that airway basal cell β‐catenin determines cell fate and its mis‐expression is associated with the development of human lung cancer. Copyright

Rac1 deletion causes thymic atrophy.

The thymic stroma supports T lymphocyte development and consists of an epithelium maintained by thymic epithelial progenitors. The molecular pathways that govern epithelial homeostasis are poorly understood. Here we demonstrate that deletion of Rac1 in Keratin 5/Keratin 14 expressing embryonic and adult thymic epithelial cells leads to loss of the thymic epithelial compartment. Rac1 deletion led to an increase in c-Myc expression and a generalized increase in apoptosis associated with a decrease in thymic epithelial proliferation. Our results suggest Rac1 maintains the epithelial population, and equilibrium between Rac1 and c-Myc may control proliferation, apoptosis and maturation of the thymic epithelial compartment. Understanding thymic epithelial maintenance is a step toward the dual goals of in vitro thymic epithelial cell culture and T cell differentiation, and the clinical repair of thymic damage from graft-versus-host-disease, chemotherapy or irradiation.

Surface modification of a POSS-nanocomposite material to enhance cellular integration of a synthetic bioscaffold

Polyhedral oligomeric silsesquioxane poly(carbonate-urea) urethane (POSS-PCU) is a versatile nanocomposite biomaterial with growing applications as a bioscaffold for tissue engineering. Integration of synthetic implants with host tissue can be problematic but could be improved by topographical modifications. We describe optimization of POSS-PCU by dispersion of porogens (sodium bicarbonate (NaHCO3), sodium chloride (NaCl) and sucrose) onto the material surface, with the principle aim of increasing surface porosity, thus providing additional opportunities for improved cellular and vascular ingrowth. We assess the effect of the porogens on the materials mechanical strength, surface chemistry, wettability and cytocompatibilty. Surface porosity was characterized by scanning electron microscopy (SEM). There was no alteration in surface chemistry and wettability and only modest changes in mechanical properties were detected. The size of porogens correlated well with the porosity of the construct produced and larger porogens improved interconnectivity of spaces within constructs. Using primary human bronchial epithelial cells (HBECs) we demonstrate moderate in vitro cytocompatibility for all surface modifications; however, larger pores resulted in cellular aggregation. These cells were able to differentiate on POSS-PCU scaffolds. Implantation of the scaffold in vivo demonstrated that larger pore sizes favor cellular integration and vascular ingrowth. These experiments demonstrate that surface modification with large porogens can improve POSS-PCU nanocomposite scaffold integration and suggest the need to strike a balance between the non-porous surfaces required for epithelial coverage and the porous structure required for integration and vascularization of synthetic scaffolds in future construct design.

Congenital tracheobronchial stenosis

Congenital tracheobronchial stenosis is a rare disease characterized by complete tracheal rings that can affect variable lengths of the tracheobronchial tree. It causes high levels of morbidity and mortality both due to the stenosis itself and to the high incidence of other associated congenital malformations. Successful management of this complex condition requires a highly individualized approach delivered by an experienced multidisciplinary team, which is best delivered within centralized units with the necessary diverse expertise. In such settings, surgical correction by slide tracheoplasty has become increasingly successful over the past 2 decades such that long-term survival now exceeds 88%, with normalization of quality of life scores for patients with non-syndrome-associated congenital tracheal stenosis. Careful assessment and planning of treatment strategies is of paramount importance for both successful management and the provision of patients and carers with accurate and realistic treatment counseling.

Vacuum-assisted decellularization: an accelerated protocol to generate tissue-engineered human tracheal scaffolds

Patients with large tracheal lesions unsuitable for conventional endoscopic or open operations may require a tracheal replacement but there is no present consensus of how this may be achieved. Tissue engineering using decellularized or synthetic tracheal scaffolds offers a new avenue for airway reconstruction. Decellularized human donor tracheal scaffolds have been applied in compassionate-use clinical cases but naturally derived extracellular matrix (ECM) scaffolds demand lengthy preparation times. Here, we compare a clinically applied detergent-enzymatic method (DEM) with an accelerated vacuum-assisted decellularization (VAD) protocol. We examined the histological appearance, DNA content and extracellular matrix composition of human donor tracheae decellularized using these techniques. Further, we performed scanning electron microscopy (SEM) and biomechanical testing to analyze decellularization performance. To assess the biocompatibility of scaffolds generated using VAD, we seeded scaffolds with primary human airway epithelial cells in vitro and performed in vivo chick chorioallantoic membrane (CAM) and subcutaneous implantation assays. Both DEM and VAD protocols produced well-decellularized tracheal scaffolds with no adverse mechanical effects and scaffolds retained the capacity for in vitro and in vivo cellular integration. We conclude that the substantial reduction in time required to produce scaffolds using VAD compared to DEM (approximately 9 days vs. 3–8 weeks) does not compromise the quality of human tracheal scaffold generated. These findings might inform clinical decellularization techniques as VAD offers accelerated scaffold production and reduces the associated costs.

A comparison of tracheal scaffold strategies for pediatric transplantation in a rabbit model

Despite surgical advances, childhood tracheal stenosis is associated with high morbidity and mortality. Various tracheal scaffold strategies have been developed as the basis for bioengineered substitutes, but there is no consensus on which may be superior in vivo. We hypothesized that there would be no difference in morbidity and mortality between three competing scaffold strategies in rabbits.

Airway tissue engineering for congenital laryngotracheal disease

Regenerative medicine offers hope of a sustainable solution for severe airway disease by the creation of functional, immunocompatible organ replacements. When considering fetuses and newborns, there is a specific spectrum of airway pathologies that could benefit from cell therapy and tissue engineering applications. While hypoplastic lungs associated with congenital diaphragmatic hernia (CDH) could benefit from cellular based treatments aimed at ameliorating lung function, patients with upper airway obstruction could take advantage from a de novo tissue engineering approach. Moreover, the international acceptance of the EXIT procedure as a means of securing the precarious neonatal airway, together with the advent of fetal surgery as a method of heading off postnatal co-morbidities, offers the revolutionary possibility of extending the clinical indication for tissue-engineered airway transplantation to infants affected by diverse severe congenital laryngotracheal malformations. This article outlines the necessary basic components for regenerative medicine solutions in this potential clinical niche.