Grant amount：\4680000 （ Direct Cost: \3600000 、 Indirect Cost：\1080000 ）

We established a method to identify and separate liver stem cells from CD45-, TER119-, CD31-, EpCAM-negative, and ICAM-1-positive cells as donor cells for hepatocyte-transplantation. From this cell population, cloned liver stem cells were established. Besides, donor cells treated with oncostatin M had improved regeneration replacement efficiency. Twenty-five studies targeting liver volume assessment were collected and reviewed to improve donor collection efficiency. When projected on a three-dimensional scatter plot as a standard liver volume calculation method, it was found that it was classified into two clusters.
The involvement of the structural variant of laminin could be on the self-renewal performance of the parental progenitor cells. When hyaluronic acid was used as a substrate, CD44-positive small hepatocytes proliferated, and the cell recovery rate and the retransplantation rate were improved under the conditions using Matrigel as a substrate.

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Grant amount：\4810000 （ Direct Cost: \3700000 、 Indirect Cost：\1110000 ）

We aimed at establishing the cell harvest method for human hepatocyte with safety and technical issues. We reported an effective method for donor cell harvests with video presentation. We also elucidate clinical values of lactate levels during the cell harvesting. We developed a standard for evaluating postoperative complications. We investigate a cell harvest method regarding anatomical location of the donor cells. BCAA plays an important role to maintain nutritional balance. We also aimed at establishing a banking hepatic stem cells and investigate cell preparation and cell nature. In the research period, we success to separate and culture hepatic stem cells. We tried to protect the method by a patent. Finally, we found an antigen to identify hepatic stem cells and select specific cells for regenerative therapy.

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Grant amount：\3770000 （ Direct Cost: \2900000 、 Indirect Cost：\870000 ）

We performed the experiments to establish hepatocytic progenitor cell lines that can be subcultured. CD44-positive small hepatocytes (CD44-SHs) sorted from SH colonies were cultured in serum-free medium on Matrigel-coated dishes. CD44-SHs could be subcultured at least 4 times and maintain the growth ability for more than 4 months. The cells showed expression of hepatocyte-specific genes and proteins such as albumin and HNF4α in a whole period. CD44-SHs morphologically and functionally demonstrated typical characteristics as hepatocytes. When the cells were transplanted into NOG mouse livers, they could be engrafted and differentiate into hepatocytes. On the other hand, mouse SHs derived from a healthy adult mouse could be lined, which possessed hepatocytic characteristics. Although we succeeded in performing one passage of human SHs, we have not yet succeeded the sequential passages of human SHs. We try to establish human SH lines for clinical application in near future.

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Authorship：Principal investigator  Grant type：Competitive

Grant amount：\4940000 （ Direct Cost: \3800000 、 Indirect Cost：\1140000 ）

In retrorsine (Ret)/70% partial hepatectomy (PH) model rat livers small hepatocyte-like progenitor cells (SHPCs) transiently appear to form clusters. However, the molecular mechanisms regulating their proliferation remain unknown. We found that transplantation of Thy1+ cells isolated from galactosamine-treated rat livers transiently increased the liver mass by expanding SHPCs in Ret/PH model livers. In this study, we examined the mechanism of the enhanced liver regeneration. Extracellular vesicles secreted from Thy1+ cells induced interleukin 17 b (IL17b) and IL25 expression in sinusoidal endothelial cells and Kupffer cells, respectively, whereas they induced IL17 receptor b (IL17rb) in SHPCs. In conclusion, Thy1+ cells transplantation enhanced the growth of intrinsic hepatic progenitor cells via IL17RB signals.

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Grant amount：\18330000 （ Direct Cost: \14100000 、 Indirect Cost：\4230000 ）

We showed that a small part of Thy1-positive cells isolated from galactosamine-treated rat livers were hepatic stem/progenitors and could differentiate into hepatocytes by HGF/FGF via CD44-positive ones. However, it was hard for the progenitors to fully differentiate into mature hepatocytes. Transplantation of the Thy1-positive cells or rat bone marrow-derived mesenchymal cells stimulated the growth of resident hepatocytic progenitors in recipient rat livers by IL17B and IL25 that were secreted by sinusoidal endothelial and Kupffer cells, respectively. Laminin alpha 1 chains are necessary to form bile ducts in fetal mouse livers and expression of laminin alpha 5 chains is critical to differentiate into mature cholangiocytes. Plasticity of cholangiocytes to hepatocytes was rapidly lost after birth and Grhl2 expression is crucial to maturation of the cells.

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Grant amount：\3640000 （ Direct Cost: \2800000 、 Indirect Cost：\840000 ）

We have developed devices made of silicon for the co-culture of rat small hepatocytes (SHs) and biliary epithelial cells (BECs). Cells could proliferate and survive on a silicon-device that oxygen freely passes through. We coated channels of the silicon-device with various ECM to examine their effects on morphogenesis. However, we have not found an optimal condition for that bile ducts (BDs) reconstructed by BECs connect to bile canaliculi of hepatic organoids reconstructed by matured SHs. By using microfluidic platform, we analyzed angiogenesis in 3D cultures in which collagen gel separated rat mature hepatocytes and endothelial cells (ECs), and found that ECs formed vascular sprouts in collagen gels. Laminins 111 and 521 were necessary for hepatic progenitor cells to differentiate into BECs and to form mature duct structures, respectively. A transcription factor Grhl2 enhanced the morphogenesis of BDs by regulating the expression and the location of claudins 3 and 4.

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Authorship：Principal investigator  Grant type：Competitive

Grant amount：\4420000 （ Direct Cost: \3400000 、 Indirect Cost：\1020000 ）

Oval cells and small hepatocytes (SHs) are known to be hepatic stem and progenitor cells, respectively. We have used Thy1 and CD44 as markers of stem and progenitor cells, respectively. In the present study we examined the differentiation capacity of Thy1+ cells and their contribution to liver regeneration. (1) Thy1+ cells could differentiate to the CD44+ SHs through Thy1+/CD44+ cells. Thy1+/CD44+ cells could also differentiate into bile duct cells in collagen sandwich culture. (2) Although transplanted Thy1+ cells could form only a small number of foci, , they could increase the number and the size of resident small hepatocyte-like progenitor cells (SHPCs). (3) The proliferation of SHs isolated was enhanced when the cells were indirectly co-cultured with Thy1+ cells. These results suggest that growth of hepatic progenitor cells may be enhanced by factors secreted by Thy1+ cells.

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Authorship：Principal investigator  Grant type：Competitive

Grant amount：\3900000 （ Direct Cost: \3000000 、 Indirect Cost：\900000 ）

Oval cells and small hepatocytes(SHs) are known as hepatic stem and progenitor cells, and Thy1 and CD44 are used as a marker, respectively. Although oval cells are believed to differentiate into mature hepatocytes(MHs) through SHs, the details of their differentiation process are not well understood. In the present experiment we used Thy1^+ oval and CD44^+cells isolated from D-galactosamine-treated rat livers. (1) EGF, basic FGF, or HGF could trigger the hepatic differentiation of sorted Thy1^+ cells to form epithelial cell colonies. (2) Thy1^+oval and CD44^+cells are able to differentiate into hepatocytes, the degree of hepatic maturation of the induced hepatocytes may not be equal to that of normal resident ones. (3) Thy1^+, CD44^+cells and MHs were transplanted into livers treated with retrorsine following partial hepatectomy. Both stem and progenitor cells could differentiate into hepatocytes in host livers. Repopulation efficiency and the survival period of Thy1^+, CD44^+ cells in the long term was very low. The short life of the cells may be due to their cellular senescence.

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Grant amount：\17420000 （ Direct Cost: \13400000 、 Indirect Cost：\4020000 ）

We aimed to develop the methods of producing a large number of small hepatocytes(SHs), hepatic progenitor cells, and to establish the methods of hepatic organoid formation in vitro. In addition, we examined how cells and tissues could be transplanted into liver. Unlike mature hepatocytes the growth of rat SHs was regulated by Follistatin/Activin system. Although oval cells, hepatic stem cells, differentiated into hepatocytes via SHs, they could not completely mature. The transplanted hepatic stem/progenitor cells could not survive for a long term in the recipient liver and disappeared by cellular senescence. Thus, the role of hepatic stem/progenitor cells in severe liver failure may be to compensate for the lost hepatic functions transiently. To use the model of hepatic sinusoids constituting rat SHs, stellate cells, and endothelial cells, we clarified that quiescent stellate cells might regulate the capillary formation of endothelial cells and maturation of SHs.

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Authorship：Principal investigator  Grant type：Competitive

Grant amount：\3780000 （ Direct Cost: \3300000 、 Indirect Cost：\480000 ）

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Grant amount：\16900000 （ Direct Cost: \15400000 、 Indirect Cost：\1500000 ）

We have reported that CD44 is specifically expressed in rat small hepatocytes and that CD44^+ hepatocytes transiently appear in injured rat livers. Using a specific antibody, we sorted CD44^+ cells from galactosamine-treated rat livers and cultured them. The phenotypes and gene expression of the cells were similar to those of cultured SHs and the ^+ cells could differentiate into hepatocytes (J Hepatol, 2006). In addition, some Thy1^+ cells that appeared in the galactosamine-treated livers could differentiate into + SHs and mature into hepatocytes. When the Thyl+ and CD44^+ cells were transplanted into livers treated with retrorsine/PH, they could survive and proliferate to form hepatic foci in the recipient livers. Furthermore, when cultured SHs were transplanted into congenic rat livers treated with radiation/PH, the cells migrated into hepatic trabecules and the liver was partially repopulated by the donor cells (Liver Transplant, 2006). Rat SHs cultured in hyaluronic acid (HA ; ligand for CD44)-coated dishes and serum-free medium could selectively proliferate with hepatic functions (Nature Protocols, 2007) and this method could be applied for the isolation of human SHs from normal liver tissues, which were surgically resected from patients with informed consent. The number of human SHs increased about 100-fold within 3 weeks and the cells showed hepatic characteristics such as albumin secretion (Cell Transplant, 2008). Furthermore, we found that the proliferation of rat SHs was enhanced by follistatin produced by SHs to inhibit the action of activin A, which is secreted by mature hepatocytes. On the other hand, we succeeded in reconstructing functional hepatic tissues by stacking up two-dimensional tissues composed of rat SHs (FASEB J, 2006). In addition, we could culture normal rat biliary epithelial cells for a long term and reconstruct functional bile ductules between collagen gels (Am J Pathol, 2008).

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