1. Enhanced Lung Epithelial Specification of Human Induced Pluripotent Stem Cells on Decellularized Lung Matrix 
Sarah E. Gilpin, PhD, Xi Ren, PhD, Tatsuya Okamoto, MD, PhD, Jacques P. Guyette, PhD, Hongmei Mou, PhD, Jayaraj Rajagopal, MD, Douglas J. Mathisen, MD, Joseph P. Vacanti, MD, Harald C. Ott, MD 
The Annals of Thoracic Surgery 
Volume 98, Issue 5, Pages (November 2014)
DOI: /j.athoracsur
Copyright © 2014 The Society of Thoracic Surgeons Terms and Conditions

2. Fig 1
Differentiation of human induced pluripotent stem cells (iPSCs) to lung epithelial progenitors. (A) In vitro differentiation protocol (BMP = bone morphogenic protein; Nkx2.1 = NK2 homeobox 1; RiPSC = RNA induced iPSCs; RPMI = Roswell Park Memorial Institute; TGF-β = transforming growth factor β.) (B) Generation of definitive endoderm indicated by loss of octamer-binding transcription factor 4 (OCT4; upper right), SRY (Sex Determining Region Y)-Box 17 (SOX17; lower right), and forkhead box protein A2 (FOXA2; upper left), and dual-immunofluorescence indicating single-cell coexpression (yellow, upper left). (DAPI = 4′,6-diamidino-2-phenylindole.) (C) Quantitative polymerase chain reaction (qPCR) analysis on day 4 of differentiation relative to undifferentiated day 0 cells. (D) Anteriorized endoderm population indicated by SOX2 expression on day 8 of differentiation by immunofluorescent staining (red) and (E) qPCR analysis relative to day 0 (light gray) and day 4 (dark gray) (DE = definitive endoderm.) (F) Differentiation to a lung epithelial progenitor population on day 12 indicated by nuclear NK2 homeobox 1 (Nkx2.1) expression (red), and (G) qPCR expressed as fold-increase from day 0 undifferentiated population (n = 3 separate experiments). Gene expression all normalized to 18S expression by ΔΔCt (n = 3 samples/differentiation in duplicate). The error bars represent the standard deviation. Scale bars = 100 μm.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2014 The Society of Thoracic Surgeons Terms and Conditions

3. Fig 2
In vitro differentiation of induced pluripotent stem cell (iPSCs)-derived lung progenitor cells on human decellularized lung slices. (A) Cell localization relative to matrix slice and normalized to total matrix area per image (n = 6). ***P = by Student t test. Error bars represent the standard deviation. (B) Viability of day 10 differentiated iPSCs seeded onto lung slices and cultured for 5 days. CalceinAM dye (green), matrix autofluorescence (magenta), 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar = 100 μm. (C) Proliferation of day 10+5 cell–matrix cultures by Ki67 (red). (D) Lung epithelial marker E-cadherin (E-cad; red). (E) Lung progenitor marker NK2 homeobox 1 (Nkx2.1; red). Matrix slice identified by autofluorescence (green) and nucleus by DAPI (blue). Scale bar = 50 μm. (F and G) Gene expression of day 10+5 cells on matrix slices. (F) Fold increase normalized to 18S expression and relative to undifferentiated cells (ΔΔCt). (G) Fold increase in Nkx2.1 normalized to 18S expression and relative to parallel no-slice cultures (ΔΔCt). Four independent differentiated cultures (labeled a, b, c, d) were analyzed in duplicate. Error bars represent the standard deviation.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2014 The Society of Thoracic Surgeons Terms and Conditions

4. Fig 3
Whole-lung scaffold recellularization and biomimetic culture. (A) Experimental design. (iPS = induced pluripotent stem; RiPSCs = RNA induced pluripotent stem cells; TGF-β = transforming growth factor β.) (B) Reseeded rat lung scaffold in the bioreactor. (C) Cell distribution and retention in recellularized, cultured lung constructs, as indicated by hematoxylin and eosin staining (scale bar = 25 μm). (D) Cell proliferation by Ki67 staining (red, left panel; scale bar = 48 μm), NK2 homeobox 1 (Nkx2.1) expression (red, middle panel; scale bar = 24 μm), and endothelial phenotype by cluster of differentiation (CD) 31 (red, right panel; scale bar = 24 μm) within cultured lung constructs. Matrix outlined by autofluorescence (green) and nucleus by 4′,6-diamidino-2-phenylindole (DAPI; blue). (E) Expression of T1α (red, left panel) and Clara cell secretory protein (CCSP; red, right panel) in lung constructs on day 10+5; scale bar = 32.5 μm. (F) Polymerase chain reaction quantification of Nkx2.1 expression in lung constructs normalized to 18S and relative to parallel in vitro cultured cells (ΔΔCt). Four tissue pieces/lung, analyzed in duplicate; n = 3 regenerated lungs. Error bars are the standard error.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2014 The Society of Thoracic Surgeons Terms and Conditions

5. Fig 4
Orthotopic left lung transplantation of recellularized constructs. (A) Experimental flow from cadaveric lung explant to transplantation of recellularized construct. (B) Implantation and reperfusion of recellularized lung construct. The right panel identifies the custom cuffs and sites of anastomosis (PA = pulmonary artery; PV = pulmonary vein.) (C) Blood gases of recipient animal at 20 and 60 minutes after reperfusion with positive-pressure ventilation with 100% oxygen. Results from two separate transplants (Tx) are presented. (Pco2 = partial pressure of carbon dioxode; Po2 = partial pressure of oxygen.) (D) Histologic analysis of the transplanted, recellularized constructs demonstrates blood perfusion of the alveolar capillaries; scale bars = 25 μm.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2014 The Society of Thoracic Surgeons Terms and Conditions