Grantee Research Project Results
2017 Progress Report: Liver MAPs
EPA Grant Number: R835737C001Subproject: this is subproject number 001 , established and managed by the Center Director under grant R835737
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: The Morgridge Institute for Research
Center Director: Thomson, James
Title: Liver MAPs
Investigators: Thomson, James
Institution: The Morgridge Institute for Research
Current Institution: The Morgridge Institute for Research , University of Wisconsin - Madison
EPA Project Officer: Aja, Hayley
Project Period: December 1, 2014 through November 30, 2018 (Extended to November 30, 2019)
Project Period Covered by this Report: December 1, 2016 through November 30,2017
RFA: Organotypic Culture Models for Predictive Toxicology Center (2013) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Objective:
OBJECTIVE 1: Identify regulatory networks that control liver maturation.
OBJECTIVE 2: Screen for factors that promote human ES/iPS cell-derived hepatocyte maturation
OBJECTIVE 3: Optimize 3D organotypic cultures of human ES/iPS cell-derived hepatocytes with mature metabolic function.
Progress Summary:
OBJECTIVE 1: Identify regulatory networks that control liver maturation.
A. Due to the high cell number requirement of DNase-seq, we focused on ATAC-seq, which is based on DNA fragmentation using a transposase instead of DNase I. ATAC-seq is robust and requires only 5 X 104 input cells compared to the 2 X 107 input cells needed for DNAse-seq. We successfully optimized the protocol and identified areas of open chromatin in hepatocytes (Objective 1: Figure 1). This figure illustrates the area around the albumin gene in our hepatocytes compared to data generated from ATAC-seq experiments done on neonatal hepatocytes (Bing Ren, P0) and a DNAse I seq experiment done on adult hepatocytes (John Stamatoyannopoulos). In replication 2 (rep 2), we identified areas of open chromatin that align very well with areas of open chromatin identified in P0 hepatocytes as well as the DNAse seq data. Interestingly, we also identified a peak in P0 hepatocytes upstream of the alpha feto-protein gene (expected to be expressed only in fetal and neonatal livers) not found in the DNAse I seq data. We completed collection of cells from a time course experiment of dedifferentiating hepatocytes every 2 hours for the first 24 hours; these cells will undergo ATAC-seq in an effort to gain detailed insight into the changes seen in open chromatin during this dedifferentiation. We will look for transcription factor binding sites in areas of open chromatin both appearing or disappearing.
B. We performed small molecule screens to identify compounds that will extend the expression of albumin in primary hepatocytes in culture using a mouse with CBR luciferase inserted into the albumin locus and CBG luciferase inserted into the alpha feto-protein locus (Objective 1 Figure 2 top image). We isolated and cultured hepatocytes from this mouse for 48 hours in media containing the small molecule of interest on a drug screening robot. We identified 6 compounds, (3 Janus kinase inhibitors, 2 aurora kinase inhibitors and ascorbic acid) that significantly inhibited decrease in CBR luminescence after 48 hours in culture. We tested these compounds in a dilution series and confirmed the activity of the three janus kinase inhibitors in a dose response assay (Objective 1: Figure 2).
OBJECTIVE 2: Screen for factors that promote human ES/iPS cell-derived hepatocyte maturation.
A. We previously reported generation of a dual reporter human ES cell clone that expresses tdTomato driven by the alpha-fetoprotein locus along with GFP driven by the albumin locus and identification of a small molecule, stauprimide, in our robotic screen to increase albumin reporter expression. We performed RNA-Seq on hepatocytes derived from the dual clone after treatment with Stauprimide or DMSO control for 72 hours. We found Stauprimide to significantly increase expression of mature hepatic genes involved in Phase I and II metabolism (Objective 2: Figure 1) simultaneously reducing the expression of the fetal marker, AFP. Gene expression values on Y-axis are in TPM.
B. In an effort to mature ES/iPS-derived hepatocytes, we also performed RNA-Seq on dedifferentiating primary human hepatocytes and pathway analysis to identify pathways that may be important in maturing ES/iPS derived hepatocytes. We identified the MAP Kinase pathway to be upregulated during dedifferentiation and treated iCell hepatocytes (Cellular Dynamics International, Madison, WI) with a MAPK inhibitor, UO126, and control DMSO for 72 hours. Objective 2: Figure 2 shows upregulation of multiple mature liver genes by UO126 (Y-axis represents TPM obtained by RNA-Seq).
C. We are in the process of optimizing a defined hepatocyte differentiation protocol from mouse ES cells. We will use these cells to screen for small molecules that may increase cell maturation, using the previously reported double knock-in mouse ES cell line wherein the P. pyralis luciferase gene is regulated by the AFP locus and the R. reniformis luciferase gene regulated by the ALB locus.
OBJECTIVE 3: Optimize 3D organotypic cultures of human ES/iPS cell-derived hepatocytes with mature metabolic function.
A. We studied the effect of heterotypic cell-cell interaction on human iPS-derived hepatocyte (iCell hepatocyte) maturation in a 3D organotypic culture using human sinusoidal endothelial cells (sEC) and hepatic stellate cells (hSC). We measured both xenobiotic metabolism (measured by Promega CYP450 Glo assay) and albumin secretion in the media by ELISA. We found iCell Hepatocyte CYP3A4 metabolism to be significantly increased by co-culture with sEC, with or without hSC (Objective 3: Figure 1, left); co-culture with hSC alone did not increase metabolic activity, indicating the increase to be due to sEC. We similarly found albumin synthesis to be upregulated in the presence of sEC with or without hSC but not by hSC alone (Objective 3: Figure 1, right). We have thus identified sEC to increase the maturity of iCell Hepatocytes in 3D aggregate co-cultures.
B. Since endothelial cells (ECs) increased the maturity of iPS derived hepatocytes, we next undertook to colonize hepatic aggregates with ECs. Vascular network is observed in confocal images (Objective 3: Figure 2) of iCell Hepatocyte aggregates. These aggregates have the potential to be incorporated into preformed capillary beds that will allow administration of drugs through the vasculature mimicking in vivo liver conditions and thus generating a more accurate drug metabolism and toxic response.
To achieve assimilation of hepatic aggregates into capillary beds, we used, in the earliest attempt, a three-layer microfluidic platform where vascular networks were formed in the bottom layer and aggregates were placed in the top layer; vascular networks were encouraged to migrate to the top layer by VEGF gradient induction (Objective 3: Figure 3a). Preliminary results showed successful engagement of vascularized aggregates with the capillary bed and increased life span of aggregates. However, due to stacking nature of the device, it is difficult to image the top layer of the device where aggregates are located. We modified the design to have vascular networks and aggregates on the same layer (Objective 3: Figure 3b). Furthermore, instead of letting endothelial cells self-assemble and form vascular networks, we put guiding channels lined with ECs (mimicking a micro-vessel of about 400 mm in diameter). We will monitor the anastomosis between the ECs from the guiding channels and hepatocyte aggregates by time lapse imaging. We will also collect media from the aggregate chamber to perform ELISA assay.
Future Activities:
OBJECTIVE 1: Identify regulatory networks that control liver maturation.
We will look for transcription factor binding sites in areas of open chromatin both appearing or disappearing.
OBJECTIVE 2: Screen for factors that promote human ES/iPS cell-derived hepatocyte maturation.
We are in the process of optimizing a defined hepatocyte differentiation protocol from mouse ES cells. We will use these cells to screen for small molecules that may increase cell maturation, using the previously reported double knock-in mouse ES cell line wherein the P. pyralis luciferase gene is regulated by the AFP locus and the R. reniformis luciferase gene regulated by the ALB locus.
OBJECTIVE 3: Optimize 3D organotypic cultures of human ES/iPS cell-derived hepatocytes with mature metabolic function.
We will monitor the anastomosis between the ECs from the guiding channels and hepatocyte aggregates by time lapse imaging. We will also collect media from the aggregate chamber to perform ELISA assay.
Journal Articles on this Report : 4 Displayed | Download in RIS Format
Other subproject views: | All 8 publications | 4 publications in selected types | All 4 journal articles |
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Other center views: | All 215 publications | 82 publications in selected types | All 81 journal articles |
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Barry C, Schmitz MT, Propson NE, Hou Z, Zhang J, Nguyen BK, Bolin JM, Jiang P, McIntosh BE, Probasco MD, Swanson S, Stewart R, Thomson JA, Schwartz MP, Murphy WL. Uniform neural tissue models produced on synthetic hydrogels using standard culture techniques. Experimental Biology and Medicine 2017;242(17):1679-1689. |
R835737 (2017) R835737C001 (2017) |
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Zanotelli MR, Ardalani H, Zhang J, Hou Z, Nguyen EH, Swanson S, Nguyen BK, Bolin J, Elwell A, Bischel LL, Xie AW, Stewart R, Beebe DJ, Thomson JA, Schwartz MP, Murphy WL. Stable engineered vascular networks from human induced pluripotent stem cell-derived endothelial cells cultured in synthetic hydrogels. Acta Biomaterialia 2016;35:32-41. |
R835737 (2015) R835737 (2016) R835737 (2017) R835737C001 (2017) R835737C004 (2015) |
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Zhang J, Schwartz MP, Hou Z, Bai Y, Ardalani H, Swanson S, Steill J, Ruotti V, Elwell A, Nguyen BK, Bolin J, Stewart R, Thomson JA, Murphy WL. A genome-wide analysis of human pluripotent stem cell-derived endothelial cells in 2D or 3D culture. Stem Cell Reports 2017;8(4):907-918. |
R835737 (2016) R835737 (2017) R835737C001 (2017) |
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Zhang J, Chu LF, Hou Z, Schwartz MP, Hacker T, Vickerman V, Swanson S, Leng N, Nguyen BK, Elwell A, Bolin J, Brown ME, Stewart R, Burlingham WJ, Murphy WL, Thomson JA. Functional characterization of human pluripotent stem cell-derived arterial endothelial cells. Proceedings of the National Academy of Sciences of the United States of America 2017;114(30):E6072-E6078. |
R835737 (2017) R835737C001 (2017) |
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Progress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R835737 The Morgridge Institute for Research Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R835737C001 Liver MAPs
R835737C002 Brain MAPs
R835737C003 Cancer MAPs: A 3D Organotypic Microfluidic Culture System to
Identify Chemicals that Impact Progression and Development of Breast Cancer
R835737C004 Vascular MAPs: Vascular and Neurovascular Tissue Models
R835737C005 Pathway Analysis Core
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.
Project Research Results
4 journal articles for this subproject
Main Center: R835737
215 publications for this center
81 journal articles for this center