Enhanced Direct Conversion into Hepatocyte-like Cells by Accelerated Sequential Epigenetic Events using Additional Stem Cell Factors
ABSTRACT: Recent studies have shown that defined sets of transcription factors could directly convert fibroblasts into induced hepatocytes (iHeps). However, the underlying mechanism of direct conversion process toward a hepatic lineage is largely unknown. Here, we report that the direct conversion kinetics from fibroblasts into iHeps throughout screening multiple additional factors that potentially rescue the delayed kinetics of MET and hepatic program. Mouse embryonic fibroblasts (MEFs) were efficiently converted into iHeps in the presence of c-Myc and Klf4 (CK), the activators for MET process, with the previously defined sets of hepatic transcription factors, resulting in remarkably improved generation of iHeps. Furthermore, in the presence of CK, Hnf4α alone could convert fibroblasts into iHeps within 5 days with a relatively higher efficiency. Cells transduced with different combinations of factors were cultured in standard Hep medium. Epithelial colonies were observed within 5 days with much higher numbers in the presence of additional factor, c-Myc and Klf4, compared to control group, indicating the number of epithelial colony was dramatically increased in the presence of additional stem cell factors
Project description:Previous studies demonstrated that hepatocyte-specific transcription factors could directly convert fibroblasts into functional hepatocytes-like cells, namely induced hepatocytes (iHeps) using viral systems. However, viral integration into host genome causes insertional mutation and risk of tumorigenecity. we showed iHeps could be generated from MEFs using the integration-free system. They were expandable in vitro and showed hepatic features, similar to primary hepatocytes. iHeps_G4H1F3 transfected (Episomal vectors) and iHeps_G4H1F3 transduced (pMXs) were duplicate, respectively. MEFs and primary hepatocytes were used as negative and positive controls, respectively.
Project description:Induced pluripotent stem cell-derived human hepatocyte-like cells (iHeps) could provide a powerful tool for studying the mechanisms underlying human liver development and disease, testing the efficacy and safety of pharmaceuticals across different patients (i.e. personalized medicine), and enabling cell-based therapies in the clinic. However, current in vitro protocols that rely upon growth factors and extracellular matrices (ECM) alone yield iHeps with low levels of liver functions relative to adult primary human hepatocytes (PHHs). Moreover, these low hepatic functions in iHeps are difficult to maintain for prolonged times (weeks to months) in culture. Here, we engineered a micropatterned co-culture (iMPCC) platform in a multi-well format that, in contrast to conventional confluent cultures, significantly enhanced the functional maturation and longevity of iHeps in culture for 4 weeks in vitro when benchmarked against multiple donors of PHHs. In particular, iHeps were micropatterned onto collagen-coated domains of empirically optimized dimensions, surrounded by 3T3-J2 murine embryonic fibroblasts, and then sandwiched with a thin layer of ECM gel (Matrigel™). We assessed iHep maturity via global gene expression profiles, hepatic polarity, secretion of albumin and urea, basal CYP450 activities, phase-II conjugation, drug-mediated CYP450 induction, and drug-induced hepatotoxicity. Conclusion: Controlling both homotypic interactions between iHeps and heterotypic interactions with stromal fibroblasts significantly matures iHep functions and maintains them for several weeks in culture. In the future, iMPCCs could prove useful for drug screening, studying molecular mechanisms underlying iHep differentiation, modeling liver diseases, and integration into human-on-a-chip systems being designed to assess multi-organ responses to compounds. We used Affymetrix microarrays to profile the global gene expression of co-culture stabilized iHeps (iMPCCs) relative to freshly isolated and co-culture stabilized primary human hepatocytes (2 donors). To assess the transcriptomic stability of iHeps in iMPCCs, RNA was extracted following 9 and 21 days of culture for hybridization to Affymetrix microarrays. The hepatic maturation state of iHeps was assessed by comparing gene expression against microarrays containing data from two primary human hepatocyte donors, both following hepatocyte isolation (day 0) and after stabilization in the micropatterened co-culture platform (day 6 and day 42 MPCCs), as previously described.
Project description:We developed in vivo reprogramming of myofibroblasts (MFs) into induced hepatocytes (MF-iHeps) using adeno-associated virus serotype 6 (AAV6) vectors expressing hepatic transcription factors in MF fate tracing (Lrat-Cre;R26R-ZsGreen) mice with carbon tetrachloride (CCl4)-induced liver fibrosis. To determine whether MF-iHeps acquire full hepatocyte differentiation, we used microarrays to profile their global gene expression. We isolated MF-iHeps and primary hepatocytes (Heps) from the same mice by laser-capture microdissection (5 and 3 biological replicates, respectively) and hepatic MFs from CCl4-treated littermates isolated by fluorescence-activated cell sorting (3 biological replicates). Total RNA was extracted, transcribed, amplified and biotin labeled. Labeled cDNA targets were hybridized to GeneChip Mouse Gene 1.0 ST arrays (Affymetrix).
Project description:Direct induction of induced hepatocytes (iHeps) from fibroblasts holds potential as a strategy for regenerative medicine, but until now has only been shown in culture settings. Here, we describe in vivo iHep formation using transcription factor induction and genetic fate tracing in mouse models of chronic liver disease. We show that ectopic expression of the transcription factors FOXA3, GATA4, HNF1A and HNF4A from a polycistronic lentiviral vector converts mouse myofibroblasts into cells with a hepatocyte phenotype. In vivo expression of the same set of transcription factors from a p75 neurotrophin receptor peptide (p75NTRp)-tagged adenovirus enabled the generation of hepatocyte-like cells from myofibroblasts in fibrotic mouse livers and reduced liver fibrosis. We have therefore been able to convert profibrogenic myofibroblasts in the liver into hepatocyte-like cells with positive functional benefits. This direct in vivo reprogramming approach may open new avenues for the treatment of chronic liver disease. Whole Mouse Genome Oligo Microarray v2 (4x44K) (Agilent Technologies) was used to characterize global gene expression profiles of iHeps compared to myofibroblasts and primary mouse hepatocytes. All microarrays were performed at the Research Core Unit Transcriptomics of the Hanover Medical School. Briefly, total RNA was used to prepare the aminoallyl-UTPmodified (aaUTP) cRNAs (Amino Allyl MessageAmp™ II Kit; #AM1753; Life Technologies) as directed by the company. The aaUTP-cRNAs were labelled with Alexa Fluor 555 Reactive Dye (#A32756; LifeTechnologies). Prior to the reverse transcription reaction, 1μl of a 1:5000 dilution of Agilent’s One-Color spike-in Kit stock solution (#5188-5282, Agilent Technologies) was added to 100ng of total RNA of each analyzed sample. The cRNA fragmentation, hybridization, and washing steps were carried out according to Agilent’s One-Color Microarray-Based Gene Expression Analysis Protocol V5.7 except that 500ng of each labelled cRNA sample were used for hybridization. Slides were scanned on the Agilent Micro Array Scanner G2565 CA (pixel resolution 5 μm, bit depth 20). Data extraction was performed with the Feature Extraction Software V10.7.3.1. 12 samples were analyzed: Pr-mHSC: Primary myofibroblasts derived from hepatic stellate cells (HSCs), 2 replicates; In-vivo iHep: In-vivo myofibroblasts-derived from induced hepatocytes (iHep), 3 replicates; In-vivo eHep: In-vivo endogenous hepatocytes (eHep), 3 replicates; In-vitro iHep: In-vitro myofibroblasts-derived from induced hepatocytes (iHep), 3 replicates; PH24h: Primary hepatocytes (PH) cultured for 24 hours, 1 replicate.
Project description:Forced expression of Bmi1 accelerated the self-renewal of hepatic stem/progenitor cells and eventually induced their transformation in an in vivo transplant model. The Ink4a/Arf locus, which encodes a cyclin-dependent kinase inhibitor, p16Ink4a, and a tumor suppressor, p19Arf, is a pivotal target of Bmi1. Therefore, it would be of importance to understand the contribution of the Ink4a/Arf locus to Bmi1 oncogenic functions in cancer and search for as-yet-unknown Bmi1 target genes other than Ink4a/Arf. We used microarrays to explore novel candidate downstream targets for Bmi1 in hepatic stem/progenitor cells Experiment Overall Design: Purified Dlk-positive hepatoblasts at day 28 of culture were subjected to RNA extraction and hybridization on Affymetrix microarrays. Data were obtained for quadrant samples from four independent experiments.
Project description:We report the generation of induced oligodendrocyte precursor cells (iOPCs) by direct lineage conversion. Forced expression of the three transcription factors Sox10, Olig2 and Zfp536 was sufficient to convert mouse and rat fibroblasts into iOPCs with morphologies and gene expression signatures that resemble OPCs. We compared the global gene expression pattern of iOPCs, fibroblasts, primary OPCs from the neonatal rat brain, and their differentiated progeny. We purified iOPCs by O4 immunopanning three weeks after infection and extracted total RNA. Acutely isolated rat cortical OPCs were either used directly for RNA extraction or expanded in mitogen-containing media for 24h before switching into differentiation medium, lacking PDGF/NT-3 and containing T3. Cells were harvested for microarray analysis 3 and 6 days after induction of differentiation.
Project description:Hepatocytes generated from human induced pluripotent stem cells (hiPSCs) are unprecedented resources for pharmaceuticals and cell therapy. However, little attention has so far been paid to variations among hiPSC lines in terms of their hepatic differentiation. We developed an improved hepatic differentiation protocol and compared multiple hiPSC lines. This comparison indicated that the hepatic differentiation propensity varies among sibling hiPSC clones derived from the same adult human dermal fibroblasts (aHDFs). In addition, hiPSC clones derived from peripheral blood cells (PB-iPSCs) consistently showed good hepatic differentiation efficiency, whereas many hiPSC clones from adult dermal fibroblasts (aHDF-iPSCs) showed poor hepatic differentiation. However, when we compared hiPSCs from blood and dermal fibroblasts from the same individuals, we found that variations in hepatic differentiation were largely attributable to donor differences, rather than to the types of the original cells. In order to understand the molecular mechanisms underlying the observed variations in hepatic differentiation, we performed microarray analyses of sibling aHDF-iPSC clones, and aHDF- and PB-iPSC clones from the same individuals. Undifferentiated aHDF- and PB-iPSCs from the same individuals (two Parkinson’s disease patients (PD #1 and PD #2) and one adult healthy donor (donor91))
Project description:Gene expression of mouse hepatoblasts (HBs) expressing IDH1 WT, IDH1 R132C, IDH2 WT, R172K and empty vector controls (N=2 cultures for each condition) grown on collagen-coated plates and IDH1 R132C and empty vector controls on uncoated plates were evaluated using Affymetrix Mouse 430Av2 DNA microarrays that were processed at the Dana-Farber Cancer Institute core facility (http://macf-web.dfci.harvard.edu/) using their standard protocol. Mutations in Isocitrate dehydrogenase 1 (IDH1) and IDH2 are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly primary liver cancer. Mutant IDH proteins in IHCC and other malignancies acquire an abnormal enzymatic activity allowing them to convert alphaketoglutarate (aKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple aKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extracellular matrix maturation. However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks primary liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF4a, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models (GEMMs) expressing mutant IDH in the adult liver show aberrant response to hepatic injury, characterized by HNF4a silencing, impaired hepatocyte differentiation and markedly elevated levels of cell proliferation. Moreover, mutant IDH and activated Kras, genetic alterations that co-exist in a subset of human IHCCs, cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional link between IDH mutations, hepatic cell fate, and IHCC pathogenesis and present a novel GEMM of IDH-driven malignancy. Gene expression of HBs expressing IDH1 WT, IDH1 R132C, IDH2 WT, R172K and empty vector controls under a doxycycline-inducible system (N=2 cultures for each condition) grown on collagen-coated plates and IDH1 R132C and empty vector controls on uncoated plates were evaluated using Affymetrix Mouse 430Av2 DNA microarrays.
Project description:Engineering clinically relevant cells in vitro holds promise for regenerative medicine, but most protocols fail to faithfully recapitulate target cell properties. To address this, we developed CellNet, a network biology platform that determines whether engineered cells are equivalent to their target tissues, diagnoses aberrant gene regulatory networks, and prioritizes candidate transcriptional regulators to enhance engineered conversions. Using CellNet, we improved B cell to macrophage conversion, transcriptionally and functionally, by knocking down predicted B cell regulators. Analyzing conversion of fibroblasts to induced hepatocytes (iHeps), CellNet revealed an unexpected intestinal program regulated by the master regulator Cdx2. We observed functional engraftment of mouse colon by iHeps, thereby establishing their broader potential as endoderm progenitors and demonstrating direct conversion of fibroblasts into intestinal epithelium. Our studies illustrate how CellNet can be employed to improve direct conversion and to uncover unappreciated properties of engineered cells. 15 samples
Project description:The overexpression of transcription factors Oct4, Sox2, Klf4, and c-Myc reprograms a somatic nucleus to one that is transcriptionally and epigenetically indistinguishable from an embryonic stem (ES) cell. However, it is still unclear if transcription factors can completely convert the nucleus of a differentiated cell into that of a distantly related cell type such that it maintains complete transcriptional and epigenetic reprogramming in the absence of exogenous factor expression. To test this idea, we screened a library of doxycycline-inducible vectors encoding neural stem cell (NSC)-expressed genes and found that stable, self-maintaining NSC-like cells could be induced under defined growth conditions after transduction of transcription factors. These induced NSCs (iNSCs) were characterized in the absence of exogenous factor induction and were shown to be transcriptionally, epigenetically, and functionally similar to endogenous embryonic cortical NSCs. Importantly, iNSCs could be generated from multiple adult cell types including liver cells and B-cells with genetic rearrangements. Our results show that self-maintaining proliferative neural cells can be induced from non-ectodermal cells by expressing specific combinations of transcription factors. Expression analysis was performed on mouse embryonic fibroblasts (MEFs), primary-derived neural stem cells (NSCs) and induced neural stem cells (iNSCs).