Regenerative potential of human airway stem cells in lung epithelial engineering
Graphical abstract
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
References (52)
- et al.
Perfusion decellularization of human and porcine lungs: bringing the matrix to clinical scale
J. heart Lung Transplant. Off. Publ. Int. Soc. Heart Transplant.
(2014) - et al.
Comparative decellularization and recellularization of normal versus emphysematous human lungs
Biomaterials.
(2014) - et al.
Efficient derivation of purified lung and thyroid progenitors from embryonic stem cells
Cell Stem Cell.
(2012) - et al.
Generation of multipotent lung and airway progenitors from mouse ESCs and patient-specific cystic fibrosis iPSCs
Cell Stem Cell.
(2012) - et al.
Murine epithelial cells: isolation and culture
J. Cyst. Fibros. Off. J. Eur. Cyst. Fibros. Soc.
(2004) - et al.
Distal airway stem cells yield alveoli in vitro and during lung regeneration following H1N1 influenza infection
Cell.
(2011) - et al.
Basal cells are a multipotent progenitor capable of renewing the bronchial epithelium
Am. J. Pathol.
(2004) - et al.
Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method
Methods.
(2001) - et al.
ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells
Am. J. Pathol.
(2012) - et al.
Design and validation of a clinical-scale bioreactor for long-term isolated lung culture
Biomaterials.
(2015)
Ex vivo non-invasive assessment of cell viability and proliferation in bio-engineered whole organ constructs
Biomaterials.
Clonal dynamics reveal two distinct populations of basal cells in slow-turnover airway epithelium
Cell Rep.
Comparative biology of decellularized lung matrix: implications of species mismatch in regenerative medicine
Biomaterials.
Whole-organ tissue engineering: decellularization and recellularization of three-dimensional matrix scaffolds
Annu. Rev. Biomed. Eng.
Perfusion decellularization of whole organs
Nat. Protoc.
Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart
Nat. Med.
Regeneration and orthotopic transplantation of a bioartificial lung
Nat. Med.
Tissue-engineered lungs for in vivo implantation
Science.
Using Nature's platform to engineer bio-artificial lungs
Ann. Am. Thorac. Soc.
The in vitro generation of lung and airway progenitor cells from human pluripotent stem cells
Nat. Protoc.
Lung regeneration: mechanisms, applications and emerging stem cell populations
Nat. Med.
Airway basal stem cells: a perspective on their roles in epithelial homeostasis and remodeling
Dis. Models Mech.
Epithelial stem cells of the lung: privileged few or opportunities for many?
Development.
Cell number and distribution in human and rat airways
Am. J. Respir. Cell Mol. Biol.
The differentiation potential of tracheal basal cells
Lab. Investig. J. Tech. Methods Pathol.
Basal cells as stem cells of the mouse trachea and human airway epithelium
Proc. Natl. Acad. Sci. U. S. of America.
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