Elsevier

The Annals of Thoracic Surgery

Volume 98, Issue 5, November 2014, Pages 1721-1729
The Annals of Thoracic Surgery

Original article
General thoracic
Enhanced Lung Epithelial Specification of Human Induced Pluripotent Stem Cells on Decellularized Lung Matrix

Presented at the Fiftieth Annual Meeting of The Society of Thoracic Surgeons, Orlando, FL, Jan 25–29, 2014.
https://doi.org/10.1016/j.athoracsur.2014.05.080Get rights and content

Background

Whole-lung scaffolds can be created by perfusion decellularization of cadaveric donor lungs. The resulting matrices can then be recellularized to regenerate functional organs. This study evaluated the capacity of acellular lung scaffolds to support recellularization with lung progenitors derived from human induced pluripotent stem cells (iPSCs).

Methods

Whole rat and human lungs were decellularized by constant-pressure perfusion with 0.1% sodium dodecyl sulfate solution. Resulting lung scaffolds were cryosectioned into slices or left intact. Human iPSCs were differentiated to definitive endoderm, anteriorized to a foregut fate, and then ventralized to a population expressing NK2 homeobox 1 (Nkx2.1). Cells were seeded onto slices and whole lungs, which were maintained under constant perfusion biomimetic culture. Lineage specification was assessed by quantitative polymerase chain reaction and immunofluorescent staining. Regenerated left lungs were transplanted in an orthotopic position.

Results

Activin-A treatment, followed by transforming growth factor-β inhibition, induced differentiation of human iPSCs to anterior foregut endoderm as confirmed by forkhead box protein A2 (FOXA2), SRY (Sex Determining Region Y)-Box 17 (SOX17), and SOX2 expression. Cells cultured on decellularized lung slices demonstrated proliferation and lineage commitment after 5 days. Cells expressing Nkx2.1 were identified at 40% to 60% efficiency. Within whole-lung scaffolds and under perfusion culture, cells further upregulated Nkx2.1 expression. After orthotopic transplantation, grafts were perfused and ventilated by host vasculature and airways.

Conclusions

Decellularized lung matrix supports the culture and lineage commitment of human iPSC-derived lung progenitor cells. Whole-organ scaffolds and biomimetic culture enable coseeding of iPSC-derived endothelial and epithelial progenitors and enhance early lung fate. Orthotopic transplantation may enable further in vivo graft maturation.

Section snippets

Human iPSC Culture

All experiments used iPSCs generated by messenger RNA transfection (Kruppel-like factor 4 [Klf4], c-Myc, octamer-binding transcription factor 4 [Oct4], Sox2, LIN28) of postnatal fibroblasts [25]. Undifferentiated cells were maintained in feeder-free culture in mTeSR media (STEMCELL Technologies Inc, Vancouver, BC, Canada) on Geltrex-coated plates (Life Technologies Corp, Carlsbad, CA), and passaged with Accutase (STEMCELL Technologies Inc) at 70% confluency. Differentiation was initiated at 80%

Results

Human fibroblast–derived iPSCs were differentiated in vitro toward a lung epithelial progenitor phenotype through defined developmental stages (Fig 1A). First, activin A treatment generated definitive endoderm at day 4, as confirmed by loss of OCT4 and induction SOX17 and FOXA2 (Fig 1B, C). Next, inhibition of TGF-β signalling for 4 days facilitated anteriorization to a foregut fate, indicated by SOX2 upregulation (Fig 1D, E). Stimulation with FGF-2 and BMP-4, in combination with glycogen

Comment

There is continued need for alternate and novel therapies for patients with end-stage lung disease. Although advances in organ preservation, recipient management, and immunosuppression represent significant therapeutic advancements, lung transplantation remains the only effective treatment option 1, 27. Progress in donor management and organ reconditioning have increased the usable fraction of the donor pool to nearly 50% [28], yet half of all donor lungs are not transplanted, often for

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