Identification Of A Common Precursor For Hepatic And Hematopoietic Lineage & Regulation Of Epimorphin On Differentiation Of Hepatic Stem/Progenitor Cells

Fetal liver is a good model in which the differentiation and proliferation of stem cells is modulated. Some different germ layer derived cells including mesoderm -originated hematopoietic cells, mesenchymal cells and endoderm-originated epithelial cells can develop into maturation reciprocally in fetal liver. The academic dissertation can be divided into two parts. In part I, we are trying to set up a new clonal model to facilitate the researches on developmental relationship between hematopoietic and hepatic lineage. In part II, we explore the mechanism of epimorphin (EPM) in differentiation of hepatic stem cells. EPM, a mesenchymal associated protein, is not expressed on ductal plate of fetal liver until hematopoiesis is terminated and hepatic maturation is started in fetal liver.Part I. Identification of a Common Precursor for Hepatic and Hematopoietic LineageFetal liver is a major organ for hematopoietic and hepatic development during ontogenesis. Hematopoietic and hepatic system are intertwined and regulated reciprocally in each stage of fetal liver development. As fetal liver is a good model in which the differentiation and proliferation of stem cells is modulated, it will help to understand a series of key questions such as hematopoiesis origin, hepatic development, multipotential differentiation of stem cells and underlying mechanisms by the way of a systematic study of fetal liver development. It can be expected to benefit from the above studies in the fields of progression and therapeutic strategy in liver and blood diseases. A hypothesis of a common precursor for hematopoietic and hepatic lineage in earlier developmental stage of fetal liver is presented previously. Whether the presentation is right or not will be a founding valuable to hematopoiesis origin and hepatic development. Support for this concept has been provided at some extent, by confirming the existence of the subpopulation with biopotential differentiation capacity for hematopoietic and hepatic lineage in bone marrow or other hematopoietic site. The previous study in our lab also demonstrated that a subset of umbilical cord bloodβ2m-/c-Met+ cells can differentiate into both hepatocyte-like cells and hematopoietic cells in vitro. However, most of the studies were subjected to the limitations in tissue sources, cell models, identification techniques. Most importantly, it is hard to exclude the possibility that individual hepatic stem/progenitor cells and hematopoietic stem/progenitor cells (HS/PCs) were contained in cell populations, which appear to retain the capacity of differentiation into both lineages due to the existence of two distinct stem cell compartments. To address the issue, a high proliferative potential colony forming cells (HPP-CFC) model of mouse fetal liver was set up. HPP-CFC is well known as the earliest multipotential precursors within the hematopoietic hierarchy that can be cultured in vitro without stromal support. In addition to hematopietic potential, HPP-CFC derived from aorta-gonadal- mesoneohros (AGM) region also has endothelial potential. Here we examined hepatic differentiation capacity of HPP-CFC derived from fetal liver at single colony level.1. The Establishment of Mouse Fetal Liver-derived HPP-CFC Model and Differentiation into Hepatic LineageSome differentiational assays based on individual HPP colonies were performed. Under the condition of combinations of hematopoietic and hepatic factors, some individual HPP colonies were induced into hematopoietic or hepatic cells, which were identified with transmission electron microscope, nested RT-PCR and immuno- fluorescence staining. The results showed that induced HPP colonies cells with a specific ultrastructure similar to hepatic epithelial cells, such as many microvilli on the cell surface, large nucleolus and many round-shaped mitochondria, and lots of glycogen granule in the cytoplasm. They also expressed hepatic markers including albumin (ALB),α-fetoprotein (AFP), and cytokeratins (CK8, CK18) at different extent of percentage at mRNA or protein level.2. Mouse Fetal Liver Cells SortingTo further explore the phenotype of the cell populations with hepatic and hematopoietic differentiation potential, we undertook cell sorting experiments. The magnetic activated cell sorting (MACS) results suggested that the fetal liver-derived HPP-CFCs were all from CD45+ cells, while CD45- cells had no capacity to form hematopoietic colonies at all. The fluorescence activated cell sorting (FACS) sorted CD49f+/Sca-1+ cells had no difference of hepatic differentiation potential compared with whole fetal liver cells. One possibility is that CD49f+/Sca-1+ cells can not enrich the hypothesized common precursors. Alternatively, these cell populations have a predominant hematopoietic response to the effects of these combined factors in methylcellulose medium.3. Identification of the Clonality of Mouse Fetal Liver-derived HPP-CFCThe clonality of HPP-CFC was then confirmed by cell mixing assay with GFP and Sry marker. GFP or Sry positive-HPP colonies were examined by nested RT-PCR combined with fluorescence microscope. The experimental results showing that our single clonal model is highly reliable.It was surprisingly that these induced clonal cells also expressed mesenchymal markerα-SMA (α-smooth muscle actin) and primary endothelial cell marker Flk-1(fetal liver kianse-1), suggesting that these CK8+/α-SMA+ cells may represent the epithelial-mesenchymal transition (EMT) cells with multipotential capacity, which may originate from mesendoderm in developing fetal liver.Taken together, the HPP-CFC may represent a novel clonal model of hypothesized common precursors for hepatic and hematopoietic lineage in the mouse feta liver and will shed light on the associations underlying the hepatic and hematopoietic development, although the model still needs further improvement.Part II. Regulation of Epimorphin (EPM) on Differentiation of Hepatic Stem/Progenitor CellsWe initially verified the concept of a common precursor for hepatic and hematopoietic lineage in the fetal liver microenvironment. In fact, fetal liver is undergoing an developmental process characterized by changes in the microenvironment from hematopoietic supportive functions to enhancing hepatic maturation in the late stage of embryonic development. EPM can be not detected on ductal plate of fetal liver until hematopoiesis is terminated and bilayered ductal plate is profoundly remodeled into focal dilations that are developed into bile ducts eventually. The expression of EPM is reported to be correlated with the up-regulation of its receptor,αvβ1 integrin in hepatoblasts around E17.5 in fetal liver. We then asked whether EPM was possibly involved in the specific differentiation of the common precursors we studied into hepatic lineage.The current studies show that EPM can direct the key processes of tubulogenesis in many epithelial organs including lung, mammary gland, pancreas, gallbladder, hair follicles, intestine, skin, kidney and sexual gland. Some reports suggested that the expression of EPM is enhanced in the late stage of liver regeneration and EPM is involved in the differentiation of hepatic stem-like cells in vitro. We then conclude that EPM may play an essential role in bile ducts morphogenesis and the differentiation of hepatic stem cells into biliary lineage. It will not only help to understand the process in specification of the developing epithelial cell in fetal liver, but also help to direct the studies in liver tissue engineering in vitro.Construction of liver units for tissue engineering may be emerging as a new trend in the field of stem cells based-liver tissue engineering in the future. So far, much attention has been paid during the past years to the induction of hepatocyte and endothelial-like cells from stem cells, but little is known about differentiation and morphogenesis of the biliary tract. It may be due to the difficulties in induction and maintenance the polarity of cellular organization and distribution in bile duct structures in addition to the plasma membrane polarity as well as in hepatocytes. Although the percentage of biliary epithelial cells (BEC) is little in total of liver mass, BEC-delineated intrahepatic bile ducts (IHBD) is essential in coordination of liver metabolism function by transitting and modifying biles produced by hepatocytes to gallbladder.We will focus on the role and underlying mechanisms of EPM in bile ducts morphogenesis and differentiation of hepatic stem cells into biliary lineage in CCl4 injured liver model and WB-F344 stem cell lines in vitro.1. I-EPM Induced Bile Duct-like Structures of WB Cells in vitroTo confirm the involvement of EPM in morphogenesis and function maintenance of bile ducts, we carried out the spatial co-localization study of EPM and bile ducts. Our findings showed a strong and previously undocumented expression of EPM in the mesenchyme around the bile ducts in CCl4-treated and normal adult liver. These results suggested that EPM may be involved in maintaining normal morphogenesis and/or regeneration of IHBD in vivo.Considering that EPM may be involved in maintaining normal morphogenesis and/or regeneration of IHBD in vivo, we will further investigate the role of EPM in differentiation and duct formation (DF) of hepatic stem cells into biliary lineage in WB-F344 cell model in vitro.Insoluble EPM (i-EPM) had a slight inhibition effect on proliferation of WB cells. WB cells contact with i-EPM could be differentiated into duct-like structures, a specific morphogenesis which could be blocked by anti-EPM antibody and anti-EPM receptor (β1 integrin) antibody. RT-PCR and Western Blotting analysis of i-EPM-treated WB cells showed that hepatocytic markers including ALB, CK18 and TAT were not induced, hepatoblast and/or hepatocyte associated markers including AFP, HNF3α, and HNF6 were suppressed, and bile duct marker CK19 was up-regulated. Taken together, these results indicated that the duct-like structures induced by i-EPM in our system are mostly immature biliary ones, since i-EPM did not elevate the expression level of some other functional biliary genes such as GGT and Yp.2. I-EPM Guided the Mitosis Orientation (MO) of WB cellsA hypothesis that orientation of EPM presentation might in turn control the orientation of the mitotic spindle axis of 3D collagen containing-mammary epithelial cells clusters was once proposed by Hirai. Regulation of MO is essential in the self-renewal and differentiation of stem cells. Here, we will investigate the bio-mechanical role of i-EPM in the differentiation of WB cells.In micropattern model, most of the WB cells divide along the tangential direction of the interface between the cell membrane and i-EPM, which can be blocked by antiβ1-integrin antibody, anti-EPM antibody, and La-A. Guidance of MO can also be observed in the duct-like structures formed on the 2D substrata. Together, these results suggested that i-EPM has the ability to guide MO determination of the WB cells by the way of mediating focal adhesion (FA) assembly and F-actin bundles alignment, a way as important as in DF.3. MO is Secondary to SFO under the Stimulation of StressThe La-A blocking results in micropattern experiments demonstrated a potential role of SF in i-EPM guiding MO determination. To further confirm the effects of SFO on guiding MO, we established a modified system of static uniaxial stretch to study the regulation of SFO on MO. The results showed MO is secondary to SFO and had a high correlation of orientation with SFO under the stimulation of the lasting stress.4. The Role of FAs, SFO, MO Determination in i-EPM-induced Bile Duct-like Formation of WB CellsIn section I, we confirmed that i-EPM had the ability to differentiate WB cells into DF, assumed to be immature biliary ones. Meanwhile, we found an interesting phenomenon that soluble EPM (s-EPM) sharing the same active EPM domain with i-EPM had no morphological effects on the WB cells. It may be due to the differences between i-EPM and s-EPM in spatial distribution and focal adhesions formation etc., which triggereda new pathway different from biochemical signaling pathway. Our previous study demonstrated that Matigel-induced biliary differentiation of WB cells is required for RhoA activity, which plays an independent bio-mechanical role in stem cells differentiation via mediating cell shape changes and cytoskeletal arrangement. We ask whether i-EPM also promotes WB DF via a biomechanical signaling pathway.Our results demonstrated that i-EPM induced a quick assembly of focal adhesions of WB cells. F-actin microfilament bundles (i.e. stress fibers, SF) were organized into cortical bundles which predominantly arranged along the long axis of the i-EPM-treated cells. SF appeared to be randomly oriented in the cells on i-EPM free or blocked substrata, especially for s-EPM. An actin polymerization inhibitor (La-A) can disrupt the DF of WB cells.Lastly, we asked whether MO determination is necessary or sufficient in i-EPM -induced DF of WB cells. All of the factors (antiβ1-integrin antibody, anti-EPM antibody, and La-A) which could disrupt MO determination in our experiments had a suppression impact on the duct-like structure formation. However, fibronectin (FN), which was reported to have the ability to regulate MO of the cells, failed to induce DF of WB cells. Together, these results suggest that the guidance of MO is necessary but not sufficient for the i-EPM-induced DF of the WB cells.Above all, in addition to traditional biochemical signaling pathway, a new biophysical signaling pathway (EPM-β1 integrin-SFO-MO-DF) independent of biochemical signals, was revealed in i-EPM-inducedbile duct formation of WB cells. Briefly, i-EPM has the ability to guide MO determination of the WB cells along the tangential direction of cell-EPM contact surface via mediating focal adhesion assembly and F-actin bundles alignment, which may be vital to bile duct-like formation of the WB cells.