1. Engineering Patient-Specific Valves Using Stem Cells Generated From Skin Biopsy Specimens 
David L. Simpson, PhD, Brody Wehman, MD, Yekaterina Galat, BS, Sudhish Sharma, PhD, Rachana Mishra, PhD, Vasiliy Galat, PhD, Sunjay Kaushal, MD, PhD 
The Annals of Thoracic Surgery 
Volume 98, Issue 3, Pages (September 2014)
DOI: /j.athoracsur
Copyright © 2014 The Society of Thoracic Surgeons Terms and Conditions

2. Fig 1
Derivation and characterization of induced pluripotent stem cells-mesenchymal stem cells (IPS-MSC). (A) Induced pluripotent stem cells (IPSCs) were allowed to undergo an epithelial-to-mesenchymal transition (EMT) via the addition of EGM 2-MV (Lonza, Basel, Switzerland) cell culture media for ∼25 days. Upon passage, the emergence of mesenchymal-like cells became apparent via flow cytometry. (CD = cluster of differentiation; i.) (B) To assess the phenotype of differentiated cells, flow cytometry was performed to quantitate the abundance of MSCs, endothelial cells, and hematopoietic cells. With increasing passage we saw a trend toward increased purity of iPSCs-MSC with concurrent reductions in other cell types. *p < 0.05 p1 compared to p4. (CD = cluster of differentiation; p1 = passage 1, green; p3 = passage 3, red; p4 = passage 4, blue.) (C) Differentiation assays were performed to further demonstrate a MSC phenotype. iPSCs-MSCs demonstrated the ability for osteogenesis (top; Von Kossa stain), adipogenesis (middle; Oil-Red-O stain), and chondrogenesis (bottom; Alcian blue stain). (D) When compared with bone marrow (BM)-MSCs, iPSC-MSC gene expression was comparable, with the majority of genes unchanged between the two groups (red = upregulated; green = downregulated; black = unchanged). (E) When the proliferative capacity of these two cell groups was compared, iPSCs-MSCs demonstrated a higher ability to incorporate 5-ethynyl-2'-deoxyuridine, suggesting an increased doubling time. *p < (hMSC = human mesenchymal stem cells.) Range bars represent standard error of the mean.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2014 The Society of Thoracic Surgeons Terms and Conditions

3. Fig 2
Characterization of induced pluripotent stem cells (iPSCs)-endothelial cells (ECs). Upon coculture of iPSCs with OP-9 stromal cells, iPSCs differentiated into ECs. iPSCs-ECs could take up (A) low-density lipoprotein (LDL) and stained positive for (B) von Willebrand factor (vWF) by fluorescence microscopy (original magnification ×10). (C) In addition, as a functional assay, iPS-ECs spontaneously formed tubes upon culture in Matrigel (BD Bioscience, San Jose, Calif). (DAPI = 4′,6-diamidino-2-phenylindole.)
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2014 The Society of Thoracic Surgeons Terms and Conditions

4. Fig 3
Optimization of cell seeding strategy. We seeded cells onto decellularized matrix at a concentration of 1 × 106 cell/cm3 of tissue using three strategies: static culture, static/rotary/static (SRS) culture, and valves coated in Matrigel (BD Bioscience, San Jose, Calif), followed by static culture. After 14 days, histologic evaluation demonstrated that the SRS strategy increased not only overall engraftment but also integration of induced pluripotent stem cells-mesenchymal stem cells into the pulmonary valve leaflet compared with the static and Matrigel conditions. Interestingly, Matrigel coating of the pulmonary valve had no effect on cell binding and integration. (Black = surface; gray = integrated.) Range bars represent standard error of the mean.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2014 The Society of Thoracic Surgeons Terms and Conditions

5. Fig 4
Characterization of decellularized pulmonary valve (PV) repopulated with induced pluripotent stem cells-mesenchymal stem cells (iPSCs-MSCs). Repopulated PV demonstrated (A) increased collagen (Masson trichrome [MT], red) and (B) glycosaminoglycans (Movat pentachrome [MP], blue) presence 14 days after the initial static/rotary/static seeding. These extracellular matrix components were typically found surrounding the leaflet with moderate integration within the leaflet. (C) The iPSCs-MSCs demonstrated phenotypic differences in their expression of α-smooth muscle actin (SMA) based on engraftment location. In particular, those cells on the PV leaflet surface were α-SMA–positive, whereas those cells integrated into the PV leaflet were α-SMA–negative. (DAPI = 4′,6-diamidino-2-phenylindole.) Original magnification ×10, inset ×20.
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )
Copyright © 2014 The Society of Thoracic Surgeons Terms and Conditions