Bipotential Progenitor Cells Research Articles

Insulin resistance disrupts epithelial repair and niche-progenitor Fgf signaling during chronic liver injury.

Insulin provides important information to tissues about feeding behavior and energy status. Defective insulin signaling is associated with ageing, tissue dysfunction, and impaired wound healing. In the liver, insulin resistance leads to chronic damage and fibrosis, but it is unclear how tissue-repair mechanisms integrate insulin signals to coordinate an appropriate injury response or how they are affected by insulin resistance. In this study, we demonstrate that insulin resistance impairs local cellular crosstalk between the fibrotic stroma and bipotent adult liver progenitor cells (LPCs), whose paracrine interactions promote epithelial repair and tissue remodeling. Using insulin-resistant mice deficient for insulin receptor substrate 2 (Irs2), we highlight dramatic impairment of proregenerative fibroblast growth factor 7 (Fgf7) signaling between stromal niche cells and LPCs during chronic injury. We provide a detailed account of the role played by IRS2 in promoting Fgf7 ligand and receptor (Fgfr2-IIIb) expression by the two cell compartments, and we describe an insulin/IRS2-dependent feed-forward loop capable of sustaining hepatic re-epithelialization by driving FGFR2-IIIb expression. Finally, we shed light on the regulation of IRS2 and FGF7 within the fibrotic stroma and show—using a human coculture system—that IRS2 silencing shifts the equilibrium away from paracrine epithelial repair in favor of fibrogenesis. Hence, we offer a compelling insight into the contribution of insulin resistance to the pathogenesis of chronic liver disease and propose IRS2 as a positive regulator of communication between cell types and the transition between phases of stromal to epithelial repair.

Human ESC-Derived Chimeric Mouse Models of Huntington's Disease Reveal Cell-Intrinsic Defects in Glial Progenitor Cell Differentiation.

Huntington's disease (HD) is characterized by hypomyelination and neuronal loss. To assess the basis for myelin loss in HD, we generated bipotential glial progenitor cells (GPCs) from human embryonic stem cells (hESCs) derived from mutant Huntingtin (mHTT) embryos or normal controls and performed RNA sequencing (RNA-seq) to assess mHTT-dependent changes in gene expression. In human GPCs (hGPCs) derived from 3 mHTT hESC lines, transcription factors associated with glial differentiation and myelin synthesis were sharply downregulated relative to normal hESC GPCs; NKX2.2, OLIG2, SOX10, MYRF, and their downstream targets were all suppressed. Accordingly, when mHTT hGPCs were transplanted into hypomyelinated shiverer mice, the resultant glial chimeras were hypomyelinated; this defect could be rescued by forced expression of SOX10 and MYRF by mHTT hGPCs. The mHTT hGPCs also manifested impaired astrocytic differentiation and developed abnormal fiber architecture. White matter involution in HD is thus a product of the cell-autonomous, mHTT-dependent suppression of glial differentiation.

Hdac1 Regulates Differentiation of Bipotent Liver Progenitor Cells During Regeneration via Sox9b and Cdk8

Upon liver injury in which hepatocyte proliferation is compromised, liver progenitor cells (LPCs), derived from biliary epithelial cells (BECs), differentiate into hepatocytes. Little is known about the mechanisms of LPC differentiation. We used zebrafish and mouse models of liver injury to study the mechanisms. We used transgenic zebrafish, Tg(fabp10a:CFP-NTR), to study the effects of compounds that alter epigenetic factors on BEC-mediated liver regeneration. We analyzed zebrafish with disruptions of the histone deacetylase 1 gene (hdac1) or exposed to MS-275 (an inhibitor of Hdac1, Hdac2, and Hdac3). We also analyzed zebrafish with mutations in sox9b, fbxw7, kdm1a, and notch3. Zebrafish larvae were collected and analyzed by whole-mount immunostaining and in situ hybridization; their liver tissues were collected for quantitative reverse transcription polymerase chain reaction. We studied mice in which hepatocyte-specific deletion of β-catenin (Ctnnb1flox/flox mice injected withAdeno-associated virus serotype 8 [AAV8]-TBG-Cre) induces differentiation of LPCs into hepatocytes after a choline-deficient, ethionine-supplemented (CDE) diet. Liver tissues were collected and analyzed by immunohistochemistry and immunoblots. We performed immunohistochemical analyses of liver tissues from patients with compensated or decompensated cirrhosis or acute on chronic liver failure (n= 15). Loss of Hdac1 activity in zebrafish blocked differentiation of LPCs into hepatocytes by increasing levels of sox9b mRNA and reduced differentiation of LPCs into BECs by increasing levels of cdk8 mRNA, which encodes a negative regulator gene of Notch signaling. We identified Notch3 as the receptor that regulates differentiation of LPCs into BECs. Loss of activity of Kdm1a, a lysine demethylase that forms repressive complexes with Hdac1, produced the same defects in differentiation of LPCs into hepatocytes and BECs as observed in zebrafish with loss of Hdac1 activity. Administration of MS-275 to mice with hepatocyte-specific loss of β-catenin impaired differentiation of LPCs into hepatocytes after the CDE diet. HDAC1 was expressed in reactive ducts and hepatocyte buds of liver tissues from patients with cirrhosis. Hdac1 regulates differentiation of LPCs into hepatocytes via Sox9b and differentiation of LPCs into BECs via Cdk8, Fbxw7, and Notch3 in zebrafish with severe hepatocyte loss. HDAC1 activity was also required for differentiation of LPCs into hepatocytes in mice with liver injury after the CDE diet. These pathways might be manipulated to induce LPC differentiation for treatment of patients with advanced liver diseases.

Contribution of Resident Stem Cells to Liver and Biliary Tree Regeneration in Human Diseases.

Two distinct stem/progenitor cell populations of biliary origin have been identified in the adult liver and biliary tree. Hepatic Stem/progenitor Cells (HpSCs) are bipotent progenitor cells located within the canals of Hering and can be differentiated into mature hepatocytes and cholangiocytes; Biliary Tree Stem/progenitor Cells (BTSCs) are multipotent stem cells located within the peribiliary glands of large intrahepatic and extrahepatic bile ducts and able to differentiate into hepatic and pancreatic lineages. HpSCs and BTSCs are endowed in a specialized niche constituted by supporting cells and extracellular matrix compounds. The actual contribution of these stem cell niches to liver and biliary tree homeostatic regeneration is marginal; this is due to the high replicative capabilities and plasticity of mature parenchymal cells (i.e., hepatocytes and cholangiocytes). However, the study of human liver and biliary diseases disclosed how these stem cell niches are involved in the regenerative response after extensive and/or chronic injuries, with the activation of specific signaling pathways. The present review summarizes the contribution of stem/progenitor cell niches in human liver diseases, underlining mechanisms of activation and clinical implications, including fibrogenesis and disease progression.

Small molecule-mediated reprogramming of human hepatocytes into bipotent progenitor cells

Currently, much effort is directed towards the development of new cell sources for clinical therapy using cell fate conversion by small molecules. Direct lineage reprogramming to a progenitor state has been reported in terminally differentiated rodent hepatocytes, yet remains a challenge in human hepatocytes. Human hepatocytes were isolated from healthy and diseased donor livers and reprogrammed into progenitor cells by 2 small molecules, A83-01 and CHIR99021 (AC), in the presence of EGF and HGF. The stemness properties of human chemically derived hepatic progenitors (hCdHs) were tested by standard in vitro and in vivo assays and transcriptome profiling. We developed a robust culture system for generating hCdHs with therapeutic potential. The use of HGF proved to be an essential determinant of the fate conversion process. Based on functional evidence, activation of the HGF/MET signal transduction system collaborated with A83-01 and CHIR99021 to allow a rapid expansion of progenitor cells through the activation of the ERK pathway. hCdHs expressed hepatic progenitor markers and could self-renew for at least 10 passages while retaining a normal karyotype and potential to differentiate into functional hepatocytes and biliary epithelial cells in vitro. Gene expression profiling using RNAseq confirmed the transcriptional reprogramming of hCdHs towards a progenitor state and the suppression of mature hepatocyte transcripts. Upon intrasplenic transplantation in several models of therapeutic liver repopulation, hCdHs effectively repopulated the damaged parenchyma. Our study is the first report of successful reprogramming of human hepatocytes to a population of proliferating bipotent cells with regenerative potential. hCdHs may provide a novel tool that permits expansion and genetic manipulation of patient-specific progenitors to study regeneration and the repair of diseased livers. Human primary hepatocytes were reprogrammed towards hepatic progenitor cells by a combined treatment with 2 small molecules, A83-01 and CHIR99021, and HGF. Chemically derived hepatic progenitors exhibited a high proliferation potential and the ability to differentiate into hepatocytes and biliary epithelial cells both in vitro and in vivo. This approach enables the generation of patient-specific hepatic progenitors and provides a platform for personal and stem cell-based regenerative medicine.

Long-Term Engraftment Promotes Differentiation of Alveolar Epithelial Cells from Human Embryonic Stem Cell Derived Lung Organoids.

Human embryonic stem cell (hESC) derived 3D human lung organoids (HLOs) provide a promising model to study human lung development and disease. HLOs containing proximal or/and immature distal airway epithelial cells have been successfully generated in vitro, such as early staged alveolar type 2 (AT2) cells (SPC+/SOX9+) and immature alveolar type 1 (AT1) cells (HOPX+/SOX9+). When HLOs were transplanted into immunocompromised mice for further differentiation in vivo, only few distal epithelial cells could be observed. In this study, we transplanted different stages of HLOs into immunocompromised mice to assess whether HLOs could expand and mature in vivo. We found that short-term transplanted HLOs contained lung progenitor cells (NKX2.1+, SOX9+, and P63+), but not SPC+ AT2 cells or AQP5+ AT1 cells. Meanwhile, long-term engrafted HLOs could differentiate into lung distal bipotent progenitor cells (PDPN+/SPC+/SOX9+), AT2 cells (SPC+, SPB+), and immature AT1 cells (PDPN+, AQP5-). However, HLOs at late in vitro stage turned into mature AT1-like cells (AQP5+/SPB-/SOX9-) in vivo. Immunofluorescence staining and transmission electron microscopy (TEM) results revealed that transplanted HLOs contained mesenchymal cells (collagen I+), vasculature (ACTA2+), neuroendocrine-like cells (PGP9.5+), and nerve fiber structures (myelin sheath structure). Together, these data reveal that hESC-derived HLOs would be useful for human lung development modeling, and transplanted HLOs could mimic lung organ-like structures in vivo by possessing vascular network and neuronal network.

Side branching and luminal lineage commitment by ID2 in developing mammary glands.

Mammary glands develop through primary ductal elongation and side branching to maximize the spatial area. Although primary ducts are generated by bifurcation of terminal end buds, the mechanism through which side branching occurs is still largely unclear. Here, we show that inhibitor of DNA-binding 2 (ID2) drives side branch formation through the differentiation of K6+ bipotent progenitor cells (BPs) into CD61+ luminal progenitor cells (LPs). Id2-null mice had side-branching defects, along with developmental blockage of the differentiation of K6+ BPs into CD61+ LPs. Notably, CD61+ LPs were found in budding and side branches, but not in terminal end buds. Hormone reconstitution studies using ovariectomized MMTV-hemagglutinin-nuclear localized sequence-tagged Id2 transgenic mice revealed that ID2 is a key mediator of progesterone, which drives luminal lineage differentiation and side branching. Our results suggest that CD61 is a marker of side branches and that ID2 regulates side branch formation by inducing luminal lineage commitment from K6+ BPs to CD61+ LPs.

A pivotal role of BEX1 in liver progenitor cell expansion in mice

BackgroundThe activation and expansion of bipotent liver progenitor cells (LPCs) are indispensable for liver regeneration after severe or chronic liver injury. However, the underlying molecular mechanisms regulating LPCs and LPC-mediated liver regeneration remain elusive.MethodsHepatic brain-expressed X-linked 1 (BEX1) expression was evaluated using microarray screening, real-time polymerase chain reaction, immunoblotting and immunofluorescence. LPC activation and liver injury were studied following a choline-deficient, ethionine-supplemented (CDE) diet in wild-type (WT) and Bex1−/− mice. Proliferation, apoptosis, colony formation and hepatic differentiation were examined in LPCs from WT and Bex1−/− mice. Peroxisome proliferator-activated receptor gamma was detected in Bex1-deficient LPCs and mouse livers, and was silenced to analyse the expansion of LPCs from WT and Bex1−/− mice.ResultsHepatic BEX1 expression was increased during CDE diet-induced liver injury and was highly elevated primarily in LPCs. Bex1−/− mice fed a CDE diet displayed impaired LPC expansion and liver regeneration. Bex1 deficiency inhibited LPC proliferation and enhanced LPC apoptosis in vitro. Additionally, Bex1 deficiency inhibited the colony formation of LPCs but had no effect on their hepatic differentiation. Mechanistically, BEX1 inhibited peroxisome proliferator-activated receptor gamma to promote LPC expansion.ConclusionOur findings indicate that BEX1 plays a pivotal role in LPC activation and expansion during liver regeneration, potentially providing novel targets for liver regeneration and chronic liver disease therapies.

Mutational Analysis of AKT1 and PIK3CA in Intraductal Papillomas of the Breast with Special Reference to Cellular Components

The pathologic feature of intraductal papillomas is defined as a papillary structure composed of a fibrovascular stromal core lined by luminal epithelial cells and myoepithelial cells. We used droplet digital PCR for the mutational analysis of AKT1 (E17K) and PIK3CA (H1047R, E542K, and E545K) in 60 papillomas. AKT1 and PIK3CA mutations were detected in 12 (20%) and 17 (28%) of the papillomas, respectively. In five tumors harboring mutations, mutational analysis of AKT1 or PIK3CA was performed separately using luminal epithelial cells and myoepithelial cells sorted using anti-cytokeratin 19 antibody and anti-α smooth muscle actin antibody. The two types of cells from a given papilloma had the identical mutation. Three patients with the PIK3CA mutation-positive papilloma developed breast cancers at the resection site of the papilloma, but none of these subsequent breast cancers had the PIK3CA mutation. These results indicate that a papilloma stems from a bipotent progenitor cell that contains the AKT1 or PIK3CA mutation and proliferates and differentiates to form the papilloma. Papilloma can be a risk factor for developing breast cancer but is unlikely to be its obligate precursor.

Generation of Chemically Induced Liver Progenitors (CLiPs) from Rat Adult Hepatocytes.

Primary mature hepatocytes (MHs) or their progenitor cells are candidate cell sources for cell transplantation therapy in severe liver diseases. However, stable culture of these cells or generation of equivalent cells from pluripotent stem cells has been limited. Using a cocktail of small molecules that we previously found useful in stable culture of multiple types of stem/progenitor cells, we recently established a novel method to generate bipotent liver progenitor cells, named chemically induced liver progenitors (CLiPs), from adult rat MHs. Here, we describe a detailed protocol for the induction of rat CLiPs. We first describe the method to isolate primary rat MHs and then describe how to induce CLiPs from these MHs. In addition, we describe a method to evaluate the bipotentiality of generated CLiPs to differentiate into hepatocytes and biliary epithelial cells. We also describe how to establish stable CLiPs through long-term culture with detailed example data. Primary CLiPs can be generated within 2 weeks, and stable CLiPs, which undergo 10 passages, can be established within 2.5-4 months with batch-to-batch variability.

A pump-free microfluidic 3D perfusion platform for the efficient differentiation of human hepatocyte-like cells.

The practical application of microfluidic liver models for in vitro drug testing is partly hampered by their reliance on human primary hepatocytes, which are limited in number and have batch-to-batch variation. Human stem cell-derived hepatocytes offer an attractive alternative cell source, although their 3D differentiation and maturation in a microfluidic platform have not yet been demonstrated. We develop a pump-free microfluidic 3D perfusion platform to achieve long-term and efficient differentiation of human liver progenitor cells into hepatocyte-like cells (HLCs). The device contains a micropillar array to immobilize cells three-dimensionally in a central cell culture compartment flanked by two side perfusion channels. Constant pump-free medium perfusion is accomplished by controlling the differential heights of horizontally orientated inlet and outlet media reservoirs. Computational fluid dynamic simulation is used to estimate the hydrostatic pressure heads required to achieve different perfusion flow rates, which are experimentally validated by micro-particle image velocimetry, as well as viability and functional assessments in a primary rat hepatocyte model. We perform on-chip differentiation of HepaRG, a human bipotent progenitor cell, and discover that 3D microperfusion greatly enhances the hepatocyte differentiation efficiency over static 2D and 3D cultures. However, HepaRG progenitor cells are highly sensitive to the time-point at which microperfusion is applied. Isolated HepaRG cells that are primed as static 3D spheroids before being subjected to microperfusion yield a significantly higher proportion of HLCs (92%) than direct microperfusion of isolated HepaRG cells (62%). This platform potentially offers a simple and efficient means to develop highly functional microfluidic liver models incorporating human stem cell-derived HLCs. Biotechnol. Bioeng. 2017;114: 2360-2370. © 2017 Wiley Periodicals, Inc.

P 195 - Nuclear factor (erythroid-derived-2)-like 2 (Nrf2) signalling is involved in transdifferentiation of hepatocyte-like cells

The identification of key regulators in hepatic progenitor cells differentiation is determinant for organ regeneration and may improve stem cell transplantation for end-stage liver disease. Mesenchymal, embryonic and pluripotent stem cells have been investigated as potential sources for hepatic differentiation, but their unstable function and atypical morphology limit the usefulness for cell treatment. The human bipotent liver progenitor cell line HepaRG is applied for toxicity screening, viral hepatitis research, hepatocyte differentiation and transplantation testing. Since redox-dependent signaling molecules are involved in the regulation of stem cell self-renewal and differentiation, we studied the role of Nrf2 – the main transcription factor involved in the oxidative stress response – in HepaRG transdifferentiation. Nrf2 was upregulated in undifferentiated HepaRG, which exhibited higher free radical production as compared to transdifferentiated cells. Pharmacological and genetic inhibition of Nrf2 led to morphological, phenotypical and functional patterns characteristic of transdifferentiated elements. To conclude, we show a determinant role of Nrf2 in the process of HepaRG transdifferentiation, suggesting that this redox-dependent transcription factor represents a potential target to regulate the commitment of undifferentiated hepatic progenitor cells into specific lineages.

Anti-ACSA-2 defines a novel monoclonal antibody for prospective isolation of living neonatal and adult astrocytes.

Astrocytes are the most abundant cell type of the central nervous system and cover a broad range of functionalities. We report here the generation of a novel monoclonal antibody, anti-astrocyte cell surface antigen-2 (Anti-ACSA-2). Flow cytometry, immunohistochemistry and immunocytochemistry revealed that Anti-ACSA-2 reacted specifically with a not yet identified glycosylated surface molecule of murine astrocytes at all developmental stages. It did not show any labeling of non-astroglial cells such as neurons, oligodendrocytes, NG2+ cells, microglia, endothelial cells, leukocytes, or erythrocytes. Co-labeling studies of GLAST and ACSA-2 showed largely overlapping expression. However, there were also notable differences in protein expression levels and frequencies of single-positive subpopulations of cells in some regions of the CNS such as cerebellum, most prominently at early postnatal stages. In the neurogenic niches, the dentate gyrus of the hippocampus and the subventricular zone (SVZ), again a general overlap with slight differences in expression levels were observed. ACSA-2 was unlike GLAST not sensitive to papain-based tissue dissociation and allowed for a highly effective, acute, specific, and prospective purification of viable astrocytes based on a new rapid sorting procedure using Anti-ACSA-2 directly coupled to superparamagnetic MicroBeads. In conclusion, ACSA-2 appears to be a new surface marker for astrocytes, radial glia, neural stem cells and bipotent glial progenitor cells which opens up the possibility of further dissecting the characteristics of astroglial subpopulations and lineages.

Longitudinal in vivo bioimaging of hepatocyte transcription factor activity following cholestatic liver injury in mice

Molecular mechanisms regulating liver repair following cholestatic injury remain largely unknown. We have combined a mouse model of acute cholestatic liver injury, partial bile duct ligation (pBDL), with a novel longitudinal bioimaging methodology to quantify transcription factor activity during hepatic injury and repair. We administered lentiviral transcription factor activated luciferase/eGFP reporter (TFAR) cassettes to neonatal mice enabling longitudinal TFAR profiling by continued bioimaging throughout the lives of the animals and following pBDL in adulthood. Neonatal intravascular injection of VSV-G pseudotyped lentivirus resulted in almost exclusive transduction of hepatocytes allowing analysis of hepatocyte-specific transcription factor activity. We recorded acute but transient responses with NF-κB and Smad2/3 TFAR whilst our Notch reporter was repressed over the 40 days of evaluation post-pBDL. The bipotent hepatic progenitor cell line, HepaRG, can be directed to differentiate into hepatocytes and biliary epithelia. We found that forced expression of the Notch inhibitor NUMB in HepaRG resulted in enhanced hepatocyte differentiation and proliferation whereas over-expressing the Notch agonist JAG1 resulted in biliary epithelial differentiation. In conclusion, our data demonstrates that hepatocytes rapidly upregulate NF-κB and Smad2/3 activity, whilst repressing Notch signalling. This transcriptional response to cholestatic liver injury likely promotes partial de-differentiation to allow pro-regenerative proliferation of hepatocytes.

Optogenetic control of cell differentiation in channelrhodopsin-2-expressing OS3, a bipotential glial progenitor cell line

Alterations in the intracellular ion environment have been identified as one of the signals playing a critical role in the control of cellular proliferation and differentiation; however, the mechanisms responsible for signal transduction remain unclear. Recent studies have reported that channelrhodopsin-2 (ChR2) is a rapidly gated blue light (BL)-sensitive cation channel suitable for the non-invasive control of ion influx. We herein examined the expression of differentiation-associated markers by photo-activation and its signal transduction in ChR2-expressing OS3 (OS3ChR2) cells, which are clonal bipotential glial progenitor cells. Increases were observed in intracellular Na+ and Ca2+ concentrations in OS3ChR2 cells with BL exposure. Alterations in the intracellular ion environment, particularly in Ca2+, led to increases in the expression of oligodendrocyte markers including galactocerebrosides (GalC) and decreases in that of astrocyte markers such as glial fibrillary acidic protein (GFAP). These alterations also triggered activation of the ERK1/2 signaling pathway, which is involved in cell survival, and PI3K/Akt/mTOR signaling pathway, which is involved in oligodendrocyte differentiation, characterized by GalC expression. Moreover, when photo-activated OS3ChR2 cells were injected into mice with lysophosphatidyl choline (LPC)-induced demyelination, deficits in motor function were reduced. Our results demonstrated that signal transduction by ChR2-expressing glial progenitor cells may be controlled through alterations induced in the intracellular ion environment by photo-activation and results in oligodendrocyte differentiation from glial progenitor cells. Our results also suggest that ChR2-expressing glial progenitor cells have potential as a useful tool for therapeutic approaches to brain and spinal cord disorders associated with oligodendrocyte dysfunctions.

Nutritional modulation of mouse and human liver bud growth through a branched-amino acid metabolism

Liver bud progenitors experience a transient amplification during the early organ growth phase, yet the mechanism responsible is not fully understood. Collective evidence highlights the specific requirements in stem cell metabolism for expanding organ progenitors during organogenesis and regeneration. Here, transcriptome analyses show that progenitors of the mouse and human liver bud growth stage specifically express the gene branched chain aminotransferase 1, encoding a known breakdown enzyme of branched-chain amino acids (BCAAs) for energy generation. Global metabolome analysis confirmed the active consumption of BCAAs in the growing liver bud, but not in the later fetal or adult liver. Consistently, maternal dietary restriction of BCAAs during pregnancy significantly abrogated the conceptus liver bud growth capability through a striking defect in hepatic progenitor expansion. Under defined conditions, the supplementation of L-valine specifically among the BCAAs promoted rigorous growth of the human liver bud organoid in culture by selectively amplifying self-renewing bi-potent hepatic progenitor cells. These results highlight a previously underappreciated role of branched-chain amino acid metabolism in regulating mouse and human liver bud growth that can be modulated by maternal nutrition in vivo or cultural supplement in vitro.

Clinical features, histology, and histogenesis of combined hepatocellular-cholangiocarcinoma

Combined hepatocellular-cholangiocarcinoma (CHC) is a rare tumor with poor prognosis, with incidence ranging from 1.0%-4.7% of all primary hepatic tumors. This entity will be soon renamed as hepato-cholangiocarcinoma. The known risk factors for hepatocellular carcinoma (HCC) have been implicated for CHC including viral hepatitis and cirrhosis. It is difficult to diagnose this tumor pre-operatively. The predominant histologic component within the tumor largely determines the predominant radiographic features making it a difficult distinction. Heterogeneous and overlapping imaging features of HCC and cholangiocarcinoma should raise the suspicion for CHC and multiple core biopsies (from different areas of tumor) are recommended before administering treatment. Serum tumor markers CA19-9 and alpha-fetoprotein can aid in the diagnosis, but it remains a challenging diagnosis prior to resection. There is sufficient data to support bipotent hepatic progenitor cells as the cell of origin for CHC. The current World Health Organization classification categorizes two main types of CHC based on histo-morphological features: Classical type and CHC with stem cell features. Liver transplant is one of the available treatment modalities with other management options including transarterial chemoembolization, radiofrequency ablation, and percutaneous ethanol injection. We present a review paper on CHC highlighting the risk factors, origin, histological classification and therapeutic modalities.

Thy-1 (CD90)-Positive Hepatic Progenitor Cells, Hepatoctyes, and Non-parenchymal Liver Cells Isolated from Human Livers.

In response to liver injury, hepatic cells, especially hepatocytes, can rapidly proliferate to repair liver damage. Additionally, it was shown that under certain circumstances liver resident cells with progenitor capabilities are involved in liver cell proliferation and differentiation. These hepatic progenitor cells (HPCs), known as oval cells in rodents, are derived from the canals of Hering, which are located in the periportal region of the liver. Regarding to different cell niches, which were defined for human HPCs, several markers have been used to identify these cells such as CD34, c-kit, OV-6, and Thy-1 (CD90). The latter was shown to be expressed on HPCs in human liver tissue with histological signs of regeneration. In this chapter we describe a detailed method for the isolation of Thy-1 positive cells from human resected liver tissue. Based on a procedure for isolating primary human hepatocytes and non-parenchymal cells (NPCs) we expanded this protocol to additional enzymatic dissociation, filtration, and centrifugation steps. This results in a bile duct cell enriched fraction of NPCs from which Thy-1 (CD90) positive cells were purified by Thy-1 positivity selection using MACS technique. Bipotential progenitor cells from human liver resections can be isolated using Thy-1 and was shown to be a suitable tool for the enrichment of liver resident progenitor cells for xenotransplantation.

ITRAQ-Based Membrane Proteomics Reveals Plasma Membrane Proteins Change During HepaRG Cell Differentiation.

HepaRG cell, a stabilized bipotent liver progenitor cell line, exhibits hepatocyte functions only after differentiation. However, the mechanism of transition from nondifferentiated to differentiated states, accompanied by proliferation migration and differentiation, remains poorly understood, particularly those proteins residing in the plasma membrane. In this study, the membrane protein expression change of HepaRG cell during differentiation were systematically analyzed using an iTRAQ labeled quantitative membrane proteomics approach. A total of 70 membrane proteins were identified to be differentially expressed among 849 quantified membrane proteins. Function and disease clustering analysis proved that 11 of these proteins are involved in proliferation, migration, and differentiation. Two key factors (MMP-14 and OCLN) were validated by qRT-PCR and Western blot. Blockade of MMP-14 further demonstrated its important function during tumor cell migration. The data sets have been uploaded to ProteomeXchange with the identifier PXD004752.

Conversion of Terminally Committed Hepatocytes to Culturable Bipotent Progenitor Cells with Regenerative Capacity

A challenge for advancing approaches to liver regeneration is loss of functional differentiation capacity when hepatocyte progenitors are maintained in culture. Recent lineage-tracing studies have shown that mature hepatocytes (MHs) convert to an immature state during chronic liver injury, and we investigated whether this conversion could be recapitulated invitro and whether such converted cells could represent a source of expandable hepatocytes. We report that a cocktail of small molecules, Y-27632, A-83-01, and CHIR99021, can convert rat and mouse MHs invitro into proliferative bipotent cells, which we term chemically induced liver progenitors (CLiPs). CLiPs can differentiate into both MHs and biliary epithelial cells that can form functional ductal structures. CLiPs in long-term culture did not lose their proliferative capacity or their hepatic differentiation ability, and rat CLiPs were shown to extensively repopulate chronically injured liver tissue. Thus, our study advances the goals of liver regenerative medicine.