Changes of HPC surface marker expression in human iPS cell-derived hepatic progenitor-like colonies.

<p>(<b>A</b>) After 12 days of culture with cytokines, CD13^highCD133⁺ cells were sorted onto GFP-MEFs. After 10–12 days, cells were trypsinized and stained with antibodies against CD13 and CD133. Expression of CD13 and CD133 was analyzed by flow cytometry. (<b>B–E</b>) Colony forming activity of CD13⁺CD133⁺, CD13⁺CD133⁻, CD13⁻CD133⁺, and CD13⁻CD133⁻ fractions of human iPS cell-derived HPCs. At every replating step, cells stained with antibodies against CD13 and CD133 were sorted onto new GFP-MEFs, and their colony-forming activity was analyzed. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067541#s3" target="_blank">Results</a> are represented as the mean colony count ± SD (duplicate samples). N.D. shows “not determined”.</p

Expressions of hepatic functional genes in differentiated HPCs.

<p>The levels of mRNAs encoding phase 1 and 2 enzymes in human iPS cell-derived HPCs from the 3rd culture, and spheroids derived from human iPS cell-derived HPCs from the 3rd culture are shown as the fold values relative to the levels in uncultured human hepatocytes. Spheroid formation was induced by hanging drop culture in the presence or absence of OSM. The results are represented as the mean colony counts ± SD (spheroid culture, n = 6; HPCs, n = 3; uncultured human hepatocytes, n = 2).</p

Expressions of undifferentiated and differentiated cell markers in human iPS cell-derived cells.

<p>The expressions of marker genes for mesodermal and endodermal cells (GSC, CXCR4, Sox17, MIXL1, T, HNF3β, and hHex) and hepatic cells (AFP, HNF4α, and ONECUT1) were examined in normal human iPS cells (iPS) and differentiated iPS cells. For quantitative PCR analyses, mRNAs were purified from MEF, HepG2 cells, normal iPS cells, and iPS cells stimulated with activin A on days 2 and 4, basic FGF and BMP-4 on days 2 and 4, and HGF on days 2 and 4. The results are represented as the mean colony counts ± SD (triplicate samples).</p

<i>In vitro</i> long-term expansion of CD13^highCD133⁺ cells.

<p>(<b>A</b>) Colonies derived from CD13^highCD133⁺ cells were trypsinized and replated onto MEFs. The number of cells was counted at each replating step. The cells continued to proliferate for more than 1 month. A representative growth curve is shown. Similar results were obtained in two independent experiments. (<b>B</b>) Expression of the proliferation marker Ki67 in human iPS cell-derived hepatocytic colonies. After the 4th passage, the colonies were stained with antibodies against Ki67, HNF4α, and Oct3/4. (<b>C</b>) Human iPS cells were stained with an antibody against Oct3/4.</p

Differentiation from human iPS cells toward hepatic lineage cells.

<p>(<b>A</b>) Schematic of the experimental procedure. Human iPS cells were sequentially stimulated with various cytokines: (1) activin A, (2) basic FGF and BMP-4, and (3) HGF. The cells were cultured in 10% O₂ for days 0–4 and 5% O₂ for days 5–12. (<b>B</b>) After 12 days of culture with or without cytokines, cells were stained with antibodies against AFP and HNF4α. Nuclei were counterstained with DAPI. (<b>C</b>) Expression of cell surface markers in human iPS cell-derived hepatic lineage cells.</p

An <i>In Vitro</i> Expansion System for Generation of Human iPS Cell-Derived Hepatic Progenitor-Like Cells Exhibiting a Bipotent Differentiation Potential

<div><p>Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult <i>ex vivo</i>. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments <i>in vitro</i>. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13^highCD133⁺ cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an <i>in vitro</i> expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.</p></div

Isolation of HPCs from human iPS cell-derived hepatic lineage cells.

<p>(<b>A</b>) Expression of hepatic progenitor markers in undifferentiated human iPS cells and differentiated cells. After 12 days of culture with or without cytokines, cells were stained with antibodies against CD13 and CD133, and then analyzed by flow cytometry. Ratios of CD13^highCD133⁺ cells are shown. (<b>B</b>) Representative images of a colony derived from a single CD13^highCD133⁺ cell. Colonies were stained with antibodies against AFP and HNF4α. Nuclei were counterstained with DAPI. (<b>C</b>) Culture condition of the human iPS cell-derived hepatic progenitor colony assay. CD13^highCD133⁺ cells were sorted and cultured on MEFs in the presence or absence of A-83-01 (ALK inhibitor) and Y-27632 (ROCK inhibitor). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067541#s3" target="_blank">Results</a> are represented as the mean colony count ± SD (triplicate samples). (<b>D</b>) CD13⁻CD133⁻, CD13 weakly single positive, CD13^mid single positive and CD13^highCD133⁺ cells were sorted onto MEFs. The cells were cultured in standard culture media in the presence of A-83-01 and Y-27632. Large colonies (containing more than 100 cells) derived from individual sorted cells were counted. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067541#s3" target="_blank">Results</a> are represented as the mean colony count ± SD (triplicate samples).</p

Phlorizin increased myocardial injury after ischemia-reperfusion.

<p>(A) CPK profiles in the effluent collected during the reperfusion period. (B) Area under the curve (AUC) was calculated from the CPK profile shown in (A). (phlorizin-perfused hearts, n = 11; control hearts, n = 10). (C) Micrograph showing representative TTC staining of cardiac sections obtained from the control (top row) and phlorizin-perfused hearts (bottom row). (D) Effects on quantitated cumulative infarct area size in the phlorizin-perfused hearts (n = 9) compared with that observed in the control group (n = 8). %MI, myocardial infarct area/ventricular area. *P<0.05 and **P<0.01 versus control.</p

SGLT1 is highly expressed in human hearts.

<p>(A) Results of the immunohistochemical analysis of the SGLT1 expression in the various parts of the myocardium (in each panel: left, HE staining; right, immunostaining with an SGLT1 antibody) obtained from the human autopsied hearts (x40). Representative data from five independent patients are shown. Bars: 20 μm. (B) Immunohistochemical analysis of the SGLT1 expression using the same antibody in the brush border membrane of the human small intestine (left panel, x10, bar: 100 μm) and proximal tubule straight segment in the deep cortex and medullary rays (Cortico-medullary junction) of human kidneys (mid panel, x10, bar: 100 μm; right panel, x2, bar: 1mm) obtained from intraoperative samples shown as positive controls. (C) Representative immunoblots of SGLT1 in the membrane fraction from the indicated regions in the human autopsied hearts from four independent patients are shown. Total lysates extracted from the human autopsied small intestine and kidneys were immunoblotted as positive controls. Immunoblots of Na⁺/K⁺ ATPase from the same membrane are shown as a loading control for the membrane fraction.</p

Phlorizin reduced tissue ATP content in the heart, associated with decreased glucose uptake and glycolytic flux during IRI.

<p>(A) Ischemic contracture was observed as a sigmoid increase in end-diastolic pressure, the onset and extent of which was recorded. The definitions of the individual parameters are shown. (B) The tissue ATP content in the hearts measured at the indicated time points (Ischemia 0 minutes: n = 4 each, Ischemia 5 minutes: n = 4 each, Ischemia 20 minutes: phlorizin-perfused; n = 4, control; n = 7, Reperfusion 10 minutes: phlorizin-perfused; n = 7, control; n = 8, Reperfusion 40 minutes: phlorizin-perfused; n = 7, control; n = 8). *P<0.05 versus control. (C) Glucose uptake in the hearts perfused with or without phlorizin under the pre-ischemic baseline condition (n = 6 each) and post-ischemic condition measured at 10-minute reperfusion following 20-minute global ischemia (phlorizin-perfused; n = 7, control; n = 6). **P<0.01 versus the control hearts at baseline; ^†P<0.01 versus the phlorizin-perfused hearts at baseline; ^$P<0.05 versus the control hearts at post-ischemia. (D) Glycogen content in the perfused hearts under the baseline condition prior to global ischemia (n = 4 each). (E) Lactate output profiles in the effluent collected during the reperfusion period (n = 10 each). *P<0.05 versus control. (F) AUC was calculated from the lactate output profiles shown in (E) (n = 10 each). *P<0.05 versus control.</p