Fig 2: Loss of CD144 and CD31 expression over serial passages correlated with a loss of cellular proliferation. (A) Immunofluorescence confirmed the loss of CD144 and CD31, as well typical morphology, over serial passages of purified iPSC-ECs. (B) iPSC-ECs demonstrated a decrease in the percent of cells proliferating by EdU labeling over serial passages in culture.

Fig 3: Heterotopic implantation of co-culture BELs in a large animal model.a Schematic of heterotopic BEL implant surgical model. See text for details. b Post-operative 3D-reconstruction from CT imaging demonstrating BEL perfusion and devascularization of native liver. BEL is outlined in yellow and native liver is outlined in green. c Representative axial CT imaging of recipient animal at post-op, 24 h, and 48 h time points. BEL is outlined in yellow. d Kaplan–Meier curves showing animal survival times within portocaval shunt and BEL implant groups. Symbols are matched to ammonia values in (d). e Post-operative blood ammonia levels measured in BEL implant recipient animals (n = 3) and portocaval shunt animals (n = 2) over the duration of the experiment. Asterisks (*) denote data points that were above the upper limit of quantification of the assay (1 mM). f, g Representative histological section of BEL tissue explanted 48 h post-implant showing viable hepatocytes and endothelialized vasculature. h, i Representative immunostaining BEL tissue (h) pre-implant and (i) explanted 48 h post-implant showing maintenance of CD31 and albumin expression. j, k Representative immunostaining BEL tissue (j) pre-implant and (k) explanted 48 h post-implant showing maintenance of CD31 and FAH expression. l, m Representative immunostaining BEL tissue (l) pre-implant and (m) explanted 48 h post-implant showing maintenance of CYP3A4 expression. HA—hepatic artery; BEL—bioengineered liver; P/C—portocaval; CT—computed tomography; FAH—fumarylacetoacetate hydrolase; CYP3A4—cytochrome p450 3A4.

Fig 4: Histological and functional characterization of BEL constructs.a Representative photograph of a BEL seeded with HUVECs and porcine hepatocytes. b Hematoxylin and eosin staining of representative co-culture BEL tissue sections fixed 48 h after seeding hepatocytes. c–e Immunofluorescent staining of cell lineage markers in non-serial tissue sections 48 h after seeding hepatocytes: (c) CD31 & albumin; (d) CD31 & FAH; (e) CD31 and LYVE1. f Schematic depicting seeding and culture timeline for HUVEC only, hepatocyte only, and co-culture BEL constructs used in (g, h, j, k). g vWF production in grafts before and after hepatocyte seeding. Data from independent HUVEC only (n = 5), hepatocyte only (n = 7), and co-culture (n = 7) BEL constructs are shown. Error bars denote the mean and standard deviation at each time point. h 24-h average albumin production in co-culture grafts 48 h following hepatocyte seeding. Data from independent HUVEC only (n = 5), hepatocyte only (n = 7), and co-culture (n = 7) BELs are shown. Error bars denote the mean and standard deviation. i Schematic of in vitro ammonia clearance and urea production assay. Ammonium chloride is added to the bioreactor media at a concentration of 0.8 mM. Ammonia and urea levels are measured in media samples taken at t = 0, 1, 2 7, and 23 h following the addition of ammonium chloride. Error bars denote the mean and standard deviation each time point. j, k Ammonia clearance (j) and urea production kinetics (k) following the addition of ammonium chloride to the bioreactor perfusion media. Data from independent HUVEC only (n = 5), hepatocyte only (n = 7), and co-culture (n = 7) BELs, media only controls (n = 4) are shown. Error bars denote the mean and standard deviation each time point. BEL—bioengineered liver; FAH—fumarylacetoacetate hydrolase; CD31—cluster of differentiation 31; LYVE1—lymphatic vessel endothelial hyaluronan receptor 1; vWF—von Willebrand factor.

Fig 5: Analysis of rBEL culture kinetics and HUVEC phenotypic plasticity in decellularized liver matrix.(a) HUVECs are expanded in 2D tissue culture flasks, harvested and seeded through the graft SVC, followed by the PV 24 hours later. (b) Representative CD31+ flow cytometry demonstrating a phenotypically pure HUVEC population immediately prior to graft seeding. (c) Plots of glucose consumption rates over time from independently seeded and cultured rBEL constructs (n=14). Peak glucose consumption rates correlated with total endothelial cell coverage as characterized by H&E staining (d-f) and anti-CD31 immunostaining (g-i). (j) Quantitative RT-PCR analysis of CD31, LYVE1 and STAB2 mRNA levels in rBELs harvested at low (n=4), mid (n=4) and high (n=7) glucose consumption phases Data are plotted as fold change relative to HUVECs in 2D culture. Individual values represent biological replicates. Mean values ± standard deviation are shown. Statistical significance was determined using a one-way ANOVA test from dCT values prior to fold-change normalization. (k-m) CD31 and LYVE-1 immunostaining from rBELs harvested at low, mid and high glucose consumption phases.(n) Principal component analysis of RNA-seq gene expression profiles from rBELs harvested at low (n=2 biological replicates) and high (n=6 biological replicates) glucose consumption phases. (o) Similarity matrix of BEL samples from (n) comparing low and high glucose consumption phase rBEL samples with respect to panel of known liver endothelial cell markers (input genes: F8, CD31, STAB2, LYVE1, CD14, VWF, ENG, ICAM1).(p) RNA-seq expression profiles for liver sinusoidal endothelial markers LYVE1, VWF, and ICAM1 in low (n=2) and high (n=6) glucose consumption phase rBEL samples. HUVECs (n=1) and primary human LSECs (n=1) cultured in 2D are included for comparison. Biological replicates are plotted along with the mean ± standard deviation. (q-t) TEM images from native liver (q) and rBEL (r-t) samples. Red arrows indicate fenestrae-like structures within endothelial cells.