1. Volume 142, Issue 4, Pages 907-917 (April 2012)
Increased Reprogramming Capacity of Mouse Liver Progenitor Cells, Compared With Differentiated Liver Cells, Requires the BAF Complex 
Alexander Kleger, Pallavi U. Mahaddalkar, Sarah–Fee Katz, André Lechel, Jin Young Joo, Komal Loya, Qiong Lin, Daniel Hartmann, Stefan Liebau, Johann M. Kraus, Tobias Cantz, Hans A. Kestler, Holm Zaehres, Hans Schöler, Karl Lenhard Rudolph 
Gastroenterology 
Volume 142, Issue 4, Pages (April 2012)
DOI: /j.gastro
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2. Figure 1
Cellular reprogramming of progenitor and differentiated liver cells using 4 reprogramming factors. (A) Schematic outline of the experimental course of cellular reprogramming of LPCs into iPS cells. Two thousand liver cells were sorted into each well of 12-well plates. After adherence, cells were infected with OSK- or OSKM-expressing lentiviruses. Labeling below and above the horizontal axis indicates culture conditions. (B) Representative pictures indicating the time-course of reprogramming of LPCs infected by a lentivirus expressing OSKM. EGFP photographs indicate activation of endogenous Oct4 reporter; morphologic changes during reprogramming are shown in phase-contrast images. Pictures at day 10 show the EGFP signal and the alkaline phosphatase staining of a single (identical) iPS cell colony. Scale bars, 200 µm. (C) Representative AP stainings of iPS cell colonies of 4F reprogrammed LPCs, non-LPCs (marker-negative cells), and nonpurified cells from mouse fetal liver at day 10 after viral infection. (D) Number of colonies of OSKM-infected fetal liver progenitor cells showing Oct4-EGFP expression and/or AP staining on day 10.
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Cellular reprogramming of progenitor and differentiated liver cells using 3 reprogramming factors and calculated reprogramming efficiencies for OSK- and OSKM-mediated reprogramming. (A) Representative time-course of OSK (3F) reprogramming to the reprogrammed iPS state (days 3, 4, 6, and 10). Endogenous Oct4-EGFP signal (dashed square, green), mCherry expression encoded by the polycistronic lentivirus (red), morphologic changes during reprogramming (phase contrast). Day 10 shows the same colonies as on day 6 after staining with alkaline phosphatase. At this time point, some of the fully formed iPS colonies in LPC cultures already started to differentiate, which is a well-known response to an increasing size of colonies (white arrow). Pictures are representative of 4 independent experiments. Scale bars, 200 µm. (B) Representative AP stainings of a 12-well plate of 3F reprogrammed fetal LPCs and non-LPCs (marker-negative liver subpopulation and unsorted fetal liver) on day 10 (2000 cells/well). (C) Representative AP stainings of a 12-well plate of OSK reprogrammed adult LPCs and non-LPCs (marker-negative liver subpopulation and Percoll-purified hepatocytes (GE Healthcare Europe GmbH, [GE Healthcare Europe GmbH, Freiburg, Germany]) on day 10 (2000 cells/well). (D) Number of colonies of OSK-infected fetal liver progenitor cells showing Oct4-EGFP expression or alkaline phosphatase staining on day 10. (E) The histogram shows the infection efficiency-corrected reprogramming efficiencies of LPCs and non-LPCs from fetal liver after reprogramming with a 3-factor–expressing lentivirus (OSK, 10 days after infection, n = 4). Efficiencies were calculated as described in the Materials and Methods section. ***P <
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
In vivo pluripotency and global gene expression profile of LPC-derived iPS cells. (A) 3F-fetal-LPC-iPS cells form in vivo teratomas containing all 3 germ layers upon injection into immunocompromised mice (Il2-RG−/−). Four weeks later, animals were killed, and tumors were stained with H&E to identify derivates of all germ layers. (B) 3F-fetal-LPC-iPS cells were injected into blastocysts that were transferred into pseudopregnant mice. On day 13.5, foster mice were killed. Male chimeric embryos were analyzed for Oct4-EGFP–positive cells in the germline. Note that Oct4-EGFP expression confirmed robust germline contribution of LPC-derived iPS cells. (C) Expression pattern of genes that show a 5-fold difference in transcript levels between LPCs (4 preparations from this study) and fetal LPC-derived iPS cells (3 cell lines including 1 with Cre-mediated excision of virally transduced reprogramming factors; LPC-iPSC) were chosen to plot a heat map. The same set of genes was analyzed in published profiles of ES cells (2 murine ES cell lines; ESC). Note that LPC-derived iPS cells are very similar to ES cells but distinct from LPCs. (D) Scatter plots comparing global gene expression profiles between LPCs and LPC-derived iPS cells (left panel) and between ES cells (ESC) and LPC-derived iPS cells (right panel). The black lines indicate 2-fold differences in gene expression levels between the paired cell populations. The transcript expression levels are on the log2 scale. Samples were as in panel C. (E) Hierarchical clustering analysis of LPCs, LPC-derived iPS cells, MEFs, MEF-derived iPS cells, and ES cells based on genome-wide gene expression. Samples were as in panel C plus data sets on MEFs and MEF-derived iPS cells. Samples which have been generated within this study are depicted in red.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
LPC-derived iPS cells can be differentiated into the endodermal lineage and into functional hepatocytes. (A) Alpha-fetoprotein (AFP; upper row) and albumin (lower row) staining of monolayer adult LPC-iPS cultures (adult LPC-iPS) at day 20 after induction of differentiation. Nuclei (4',6-diamidino-2-phenylindole [DAPI]) are shown in blue. Scale bars, 10 µm. (B) The metabolic activity of CYP3A11 was measured in hepatocyte-like cells that were differentiated from either fetal or adult LPC-iPS cells (day 20 after induction of differentiation; n = 3). (C–E) Comparison of hepatic differentiation capacity of MEF-iPS (described by Pfaff et al56; passage 15), 3F-adult-LPC-iPS (LPC-iPS cells; passage 20), and 3F-hepatocyte-iPS (non-LPC-iPS cells; passage 16) using the same protocol as in panels A and B. (C) Induction of definitive endoderm (day 6 of hepatic differentiation) was evaluated by Brachury, GATA binding protein 4 (Gata4), and AFP expression. mRNA levels are shown relative to undifferentiated ES cells as internal reference. (D–E) Maturation of hepatocyte-like cells was evaluated by albumin expression (day 20 of hepatic differentiation). (D) mRNA levels are shown relative to the hepatoma cell line 1–6 as internal reference. (E) Secretion of albumin into the culture supernatant after day 20 of hepatic differentiation is shown as the amount of albumin in 24 hours per 1000 cells (C–E; n = 3). *P = .01 to .05; **P = .001 to .01; ***P < .001.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
LPCs and non-LPCs show similar proliferation capacities. (A and B) LPCs and non-LPCs (nonpurified liver cells) were infected with the same amount of reprogramming OSKM virus and subsequently analyzed on day 4 after infection by either (A) bromodeoxyuridine staining or (B) cell-cycle analysis as determined by propidium iodide staining. No feeder cells were added (n = 3). (C) Reprogramming kinetics of LPCs and non-LPCs after OSKM infection (n = 4). (D) Representative AP staining at day 10 of one of the reprogramming kinetic experiments. (E) Mathematic curve-fitting analysis of cellular reprogramming in LPCs and non-LPCs (n = 5). All experimental data were fitted (R version 2.11, function “nls”) with sigmoidal functions of the form: f(d) = 1/(1 + exp [−a x + b]). Parameter values were as follows: LPCs, green: a = 0.372, b = 2.311; and non-LPCs, red: a = , b = Inflection points were calculated to be at day 6.21 and day Green line, reprogramming kinetics of LPCs. Blue line, predicted effect of a 50% decrease in cell proliferation on the delay in reprogramming of LPCs. Red line, calculated reprogramming kinetics of non-LPCs based on the measured data.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Expression of endogenous reprogramming factors and BAF-complex factors in LPCs. (A) Pairwise distance calculation of LPCs and non-LPCs from established ES cell whole-transcriptome profiles. Note the decreased transcriptional distance between non-LPCs and ES cells. (B) The bar graphs show the mRNA expression of the indicated reprogramming factors in ES cells, fetal marker-positive LPCs and non-LPCs (marker-negative cells). Data represent expression from 8 embryos (embryonic day 14.5) of the Oct4-EGFP reporter mouse strain. Similar data were obtained in CD1 mice (not shown). (C) Bar graphs show the mRNA expression of the indicated reprogramming factors in ES cells, adult LPCs (marker positive), and non-LPCs (hepatocytes [8 adult mice at 2 months of age]). (D) The bar graphs show the mRNA expression of the indicated BAF factors (Baf155, Brg1) in fetal LPCs compared with non-LPCs (unsorted fetal liver). (E) The bar graphs show the mRNA expression of the indicated BAF factors (Baf155, Brg1) in adult LPCs compared with non-LPCs (Percoll-purified hepatocytes). (D and E) Expression of the indicated factors was set to 1 in non-LPCs. (F) The bar graphs depict the iPS cell–forming capacity of fetal liver-derived LPCs and non-LPCs that were infected with a scramble short hairpin RNA (shRNA) or a mix of shRNAs targeting the BAF-complex members Brg1 and Baf155. Colony numbers of scramble-infected cultures were set to 1. Note that the shRNA-mediated knock-down of Baf155 and Brg1 reduces the iPS cell–formation capacity of LPCs but does not affect the iPS cell–forming capacity of non-LPCs (left panels). Representative AP-stained plates of shBaf155/shBrg1- and scramble-infected LPCs and non-LPCs after 10 days of reprogramming (right panel).
Gastroenterology  , DOI: ( /j.gastro )
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