Induced pluripotent stem cells derived from human amnion in chemically defined conditions

<p>Fetal stem cells are a unique type of adult stem cells that have been suggested to be broadly multipotent with some features of pluripotency. Their clinical potential has been documented but their upgrade to full pluripotency could open up a wide range of cell-based therapies particularly suited for pediatric tissue engineering, longitudinal studies or disease modeling. Here we describe episomal reprogramming of mesenchymal stem cells from the human amnion to pluripotency (AM-iPSC) in chemically defined conditions. The AM-iPSC expressed markers of embryonic stem cells, readily formed teratomas with tissues of all three germ layers present and had a normal karyotype after around 40 passages in culture. We employed novel computational methods to determine the degree of pluripotency from microarray and RNA sequencing data in these novel lines alongside an iPSC and ESC control and found that all lines were deemed pluripotent, however, with variable scores. Differential expression analysis then identified several groups of genes that potentially regulate this variability in lines within the boundaries of pluripotency, including metallothionein proteins. By further studying this variability, characteristics relevant to cell-based therapies, like differentiation propensity, could be uncovered and predicted in the pluripotent stage.</p

HDL insudation in the engineered artery equivalent.

<p>25 µg/ml of HDL was circulated for 24 hours. The insudation of HDL (white) was assessed after cryosection of the tissue on the luminal side (A) and in the tissue (B) (blue: nuclei). HDL localization in the tissue was further analysed by confocal microscopy at 2.5 um (C) and 16 um (D) deep in the tissue. The arrow shows the intracellular vesicular localisation. The up-take of HDL (white) into HUVECs (E) and UCMFB (F) was analysed in regular cell culture after 24 hours incubation with 25 µg/ml. Bars represent 50 µm and L: Lumen.</p

Insudation of LDL into the engineered artery.

<p>The insudation of LDL into the tissue (blue: nuclei) was analysed by confocal microscopy after incubation with 20 µg/ml of fluorescent LDL (white) for 24 hours and demonstrated the vesicular localization on the upper cell layer (A). The specificity of the signal was assessed by competition with 40-fold excess of non-labelled LDL (B). LDL localization in the tissue was further analysed by microscopy of tissue cryosections and demonstrated the time-dependent uptake and the sub-endothelial localization of the LDL (C: 2.5 hours and F: 24 hours). Zoom in of interesting regions shown with the arrows are presented in D and E for 2.5 hours and F and G for 24 hours. Bars represent 200 µm (A-C,F) and 20 µm (D-E, G-H) and L: Lumen.</p

Integrity and functionality of endothelial cells in the engineered artery.

<p>The endothelial integrity was analysed using Evan’s blue staining (0.5% for 10 minutes). In the absence of endothelial cell (A) the tissue appeared evenly stained in blue. Two weeks after endothelialization (B) the endothelial barrier retained the absorption of Evan’s blue and tissue appeared non-coloured. The integrity of the endothelium was further analysed by microscopy of cryosections for the expression of the tight junction protein (ZO)-1 (red) by endothelial cells (nuclei: blue). (C & D). Nitric oxide was measured using L-³H-arginine as the substrate and in presence of 10 nM acetylcholine (Ach) or 0.5 mg/ml HDL as the stimulator or 1 mM L-NAME as the inhibitor of endothelial nitric oxide synthase (eNOS). After 30 minutes L-³H-citruline was separated from L-³H-arginine by ion exchange column. Bar represents 100 (C) and 25 µm (D) and L: Lumen. *** p<0.001, ** p<0.01 and * p<0.05</p

Histological structure of an engineered artery equivalent.

<p>A) Haematoxylin-Sudan staining demonstrated the formation of tissue in and on the surface of a PGA/P4HB scaffold as well as the scaffold’s partial degradation. B) H&E staining revealed dense tissue formation composed of cells and extracellular matrix. C) The secretion of collagen was observed after Masson’s trichrome staining. The expression of α-smooth muscle actin (α-SMA) (D) confirmed the smooth muscle phenotype of the cells in the inner layer. Collagen IV positive staining (E) demonstrated the secretion of basement membrane and CD31 positive staining (F) confirmed the presence of an endothelial cell monolayer on the luminal side of the bioengineered artery equivalent. Bars represent 100 µm.</p

Quantification of extracellular matrix composition and cell number.

<p>Total cell numbers, represented as DNA and total collagen secretion of collagen, represented as hydroxyproline (HYP) represented ≈ 50% of the native human aorta. The glycosaminoglycan (GAG) content was similar to native aorta.</p

Bioreactor and macrostructure of the engineered artery equivalent.

<p>A) Schematic view of the bioreactor set up. B) Macroscopic picture of the bioengineered artery demonstrated the presence of an open lumen after 5 weeks in culture as well as the formation of tissue on the luminal side of the graft. Bar represents 1 cm.</p

Endothelial monocyte adhesion and transmigration migration in the engineered artery.

<p>After pre-treatment in the absence (A, D) or the presence of 3 hours TNFα (10 ng/ml) (B, D) or 24 hours LDL (20 µg/ml) (C-I) 1×10⁶ fluorescently labelled monocytes (white) per ml were injected into the circulation loop and circulated for 24 hours. Tissues were analyzed by confocal microscopy and after cryosectionning. In addition, monocytes remaining in the circulation were counted. More monocytes (white arrows) adhered after pre-treatment with TNFα (B) or LDL (C) compared to the not stimulated (A). Less monocyte remained into the circulation after TNFα or LDL pre-treatment compared to the absence of stimuli (D). Monocytes adhesion and migration in the tissue was further analyzed by microscopy of cryosections after LDL pre-treatment. Microscopic observations demonstrated adhesion and migration of monocytes through the endothelium (dash line) (E, G) and accumulation of monocytes into the tissue (F, H-I). Bars represent 200 µm (A-C, E-F) and 20 µm (G-I).</p

Labeling of human BMMSCs and ATMSCs with superparamagnetic microspheres (MPIOs; co-labeled with Dragon-green fluorochromes) and CM-Dil.

<p>Successful labeling of human BMMSCs and ATMSCs was demonstrated on Prussian Blue staining before (<b>A and B</b>) and after MPIO labeling (<b>C and D</b>) as well as on immunofluorescence clearly showing the presence of MPIOs (<b>E–G</b>). After labeling, CM-Dil⁺/MPIO⁺ cells displayed excellent cell labeling efficiency in excess of 95% on FACS analysis (<b>H</b>). <i>Scale Bar: 100 um (A and C), 50 um (B, D–G)</i>.</p

Detection of CM-DiI⁺/MPIO⁺ human ATMSCs in the pre-immune fetal sheep myocardium via immunohistochemistry.

<p>Exemplary image series post intramyocardial transplantation of human ATMSCs into the fetal myocardium. Morphologically CM-DiI⁺/MPIO⁺ human ATMSCs could be easily identified within the fetal heart tissue suggesting to be in good shape and viable. The cells appeared to be integrated within the fetal myocardium and could be found as clusters as well as in the interstitial and intravascular spaces (<b>A–I</b>; <i>black arrows</i>). The cells stained positive for human specific Major Histo Compatibility Complex 1 (MHC-1) clearly confirming the human origin (<b>A and B</b>; <i>black arrows</i>) and also stained positive for anti-FITC detecting the Dragon Green fluorochrome labelled MPIOs within the human cells (<b>C and D</b>; <i>black arrows</i>). In addition, double staining for ALU Sequence and anti-FITC further confirmed the presence of the injected CM-DiI⁺/MPIO⁺ human ATMSCs within healthy heart tissue (<b>E and F</b>; <i>black arrows</i>) as well as in infarcted myocardium (<b>G–I</b>; <i>black arrows) Scale Bar: 100 um (A–C, E, G, H), 50 um (D, F, I)</i>.</p