Elsevier

Biomaterials

Volume 108, November 2016, Pages 111-119
Biomaterials

Regenerative potential of human airway stem cells in lung epithelial engineering

https://doi.org/10.1016/j.biomaterials.2016.08.055Get rights and content

Abstract

Bio-engineered organs for transplantation may ultimately provide a personalized solution for end-stage organ failure, without the risk of rejection. Building upon the process of whole organ perfusion decellularization, we aimed to develop novel, translational methods for the recellularization and regeneration of transplantable lung constructs.

We first isolated a proliferative KRT5+TP63+ basal epithelial stem cell population from human lung tissue and demonstrated expansion capacity in conventional 2D culture. We then repopulated acellular rat scaffolds in ex vivo whole organ culture and observed continued cell proliferation, in combination with primary pulmonary endothelial cells. To show clinical scalability, and to test the regenerative capacity of the basal cell population in a human context, we then recellularized and cultured isolated human lung scaffolds under biomimetic conditions. Analysis of the regenerated tissue constructs confirmed cell viability and sustained metabolic activity over 7 days of culture. Tissue analysis revealed extensive recellularization with organized tissue architecture and morphology, and preserved basal epithelial cell phenotype. The recellularized lung constructs displayed dynamic compliance and rudimentary gas exchange capacity. Our results underline the regenerative potential of patient-derived human airway stem cells in lung tissue engineering. We anticipate these advances to have clinically relevant implications for whole lung bioengineering and ex vivo organ repair.

Introduction

Solid organ bioengineering based on native extracellular matrix scaffolds has fuelled recent enthusiasm for regenerative medicine approaches to treat end organ failure [1]. The main approach involves combining regenerative cell populations with corresponding biological matrices to form living, functional grafts. To this end, native solid organ extracellular matrix (ECM) scaffolds can be readily generated by perfusion decellularization with specific detergents, rendering a biocompatible framework as a foundation for regeneration [2], [3], [4], [5], [6], [7].

Clinically relevant organ recellularization presents significant challenges, both in terms of identifying an ideal cell source and in the establishment of functional biomimetic organ culture systems to support organ maturation prior to transplantation [8]. An optimal cell source would be easily obtained and expanded in vitro, while maintaining contact inhibition and cell cycle control. Directed differentiation of induced pluripotent stem cells through key developmental stages presents a promising option for obtaining lung-specified cell populations [9], [10], [11], but the length of in vitro culture and limited cell number and purity restricts their current utility for large-scale organ engineering. While largely quiescent, adult lung tissue has a remarkable capacity for regeneration, owing to a number of facultative stem/progenitor cell populations that become activated in response to tissue damage [12]. Airway basal cells, identified by the transcription factor TP63 and expression of cytokeratin 5 (KRT5), function as multipotent stem cells of the proximal airway epithelium, and are critical for maintaining airway homeostasis during physiological cell turnover and regeneration [13], [14]. This essential cell population comprises 30% of the cells in human airway epithelium [15], and early studies of airway regeneration have demonstrated the ability for isolated basal cells to recapitulate a fully differentiated airway epithelium when seeded onto denuded mouse tracheas [16]. In response to injury, basal epithelial stem cells can rapidly proliferate and give rise to both ciliated and club cell progeny, confirming their important function in tissue homeostasis and injury repair [17]. Basal cells can be isolated [18], [19] and propagated in culture [20], which makes them a useful candidate population for tissue and organ engineering applications. We demonstrate that this population can be readily derived from human cadaveric lung tissue following clinical organ donation and cold ischemia, and expanded in vitro. This isolated primary stem cell population also provides an important tool for studying basic biology and tissue regeneration [13], particularly given their role in lung repair and capacity for multi-lineage differentiation [21], [22]. Following injury, basal airways stem cells have been reported to undergo rapid proliferation, migration, and a surprising differentiation toward distal pneumocyte lineages, in order to reconstitute the damaged alveolar-capillary network [23]. When delivered to rodent lungs following injury, TP63+KRT5+ cells have been shown to differentiate to type I and type II pneumocytes, in addition to bronchiolar secretory cells [24]. Lung repair and remodelling after influenza or bleomycin injury may also involve a specialized subset of KRT5+ cells [25], [26]. Although this phenomenon has yet to be investigated in the context of epithelial tissue engineering, these studies highlight the evolving understanding of traditional cell identity, hierarchy, and regenerative ability.

In the present study, we aimed to exploit the capacity for lung basal stem cells to respond to injury and to re-establish epithelial integrity and functional organization [13], [27], by investigating the utility of human donor tissue-derived cells in the context of whole organ engineering. We propose that the architectural and biological niches retained within the native extracellular matrix may provide a valid template to guide cell engraftment and investigate mechanisms of lung tissue repair [28], [29], and in combination with extended biomimetic culture, provide an important platform for the regeneration of human lung constructs.

Section snippets

Study approval

Human donor lungs otherwise unsuitable for transplantation were obtained from the New England Organ Bank (see Supplementary Table 1), following informed consent. All experiments were approved by the Massachusetts General Hospital Internal Review Board (#2011P002433) and Animal Utilization Protocol (#2014N000261).

Cell isolation and expansion

Donor lung peripheral tissue was gently homogenized and digested in 0.1 mg/ml DNAse (Sigma) and 1.4 mg/ml Pronase (Roche, 11459643001) for 24 h/4 °C [30]. Digested tissue was plated

Results

We first isolated and characterized a highly proliferative cell population from human cadaveric lung tissue. Robust expansion of a KRT5+TP63+ basal epithelial stem cell population was reproducible over serial passages in culture, with increased expression of E-Cadherin and loss of pro-SP-B and αTubulin positive cells. (Fig. 1A–B). The proliferative capacity of the isolated cell population was maintained through 3 passages (KI67+ cells by staining, 63.4 ± 8.08%, n = 3 images quantified per

Discussion

We have isolated a highly proliferative basal stem cell population from an easily accessible tissue source and demonstrated rapid expansion in vitro. This cell population, identified by KRT5+TP63+ expression, has been studied in many animal models of lung repair [40], [41], [42] and in human disease [43].

Within the KRT5+TP63+ population, additional distinct subpopulations of basal stem cells may exist, each with a unique role in tissue homeostasis and repair. This includes the recently reported

Author contributions

SEG designed, conducted, and analyzed all experiments, and prepared the manuscript; JMC constructed the lung bioreactor; XR prepared the endothelial cell population for recellularization; LFT prepared lungs for decellularization and recellularization, and assisted with manuscript preparation; TW assisted with primary cell isolation; DJM assisted with experimental design and data interpretation; HCO oversaw all experimental design, data analysis, and manuscript preparation.

This study was

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