Self‐Renewal Capability of Hepatocytic Parental Progenitor Cells Derived From Adult Rat Liver Is Maintained Long Term When Cultured on Laminin 111 in Serum‐Free Medium

Abstract

In this study, we investigated how the ability of hepatocytic parental progenitor cells (HPPCs) to self-renew can be maintained and how laminin (LN) isoforms play an important role in their self-renewal and maturation. Hepatocytes isolated from adult rat livers were cultured on hyaluronic acid to form colonies consisting of CD44 + small hepatocytes, which could be passaged on dishes coated with Matrigel. When second-passage cells were plated on Matrigel, LN111, or LN511, HPPCs appeared on Matrigel and LN111 but not on LN511. We identified two types of cells among the second-passage cells: Small, round cells and large, flat ones were observed on Matrigel, whereas the former and latter ones were specifically attached on LN111 and LN511, respectively. We hypothesized that small and round cells are the origin of HPPC colonies, and the binding to LN111 could be key to maintaining their self-renewal capability. Among the integrins involved in LN binding, integrins α3 and β1 were expressed in colonies on LN111 more than in those on LN511, whereas β4 was more strongly expressed in colonies on LN511. Integrin α3 high α6β1 high cells could form HPPC colonies on LN111 but not on LN511, whereas integrin α6β1 low cells could not on either LN111 or LN511. In addition, neutralizing anti-integrin β1 and anti-LN111 antibodies inhibited the passaged cells' ability to attach and form colonies on LN111 by HPPCs. Matrigel overlay induced second-passage cells growing on LN111 to increase their expression of hepatic functional genes and to form 3-dimensional colonies with bile canalicular networks, whereas such a shift was poorly induced when they were grown onLN511. Conclusion: These results suggest that the self-renewal capability of HPPCs depends on LN111 through integrin β1 signaling. (Hepatology Communications 2020;4:21-37). W hole or segmental liver transplantation is widely chosen as the last option to save patients suffering from severe liver diseases, yet a persistent shortage of donor organs prevents most patients from receiving the benefits of transplantation. Thus, cell-based therapies, such as

CINC-2 and miR-199a-5p in EVs secreted by transplanted Thy1+ cells activate hepatocytic progenitor cell growth in rat liver regeneration

“…The isolation and subculture methods have been described previously [ 21 , 27 , 28 , 30 , 31 ]. Briefly, liver cells were isolated from DPPIV + rats using the two-step collagenase perfusion method [ 17 ], following which the cell suspension was centrifuged at 50× g for 1 min.…”

“…Furthermore, SHs were plated on hyaluronic acid (Sigma-Aldrich Co., St. Louis, MO)-coated dishes (1 mg/35-mm dish). SHs were cultured for 10 days and then subcultured on Matrigel-coated 12-well plates as previously described [ 31 , 32 ]. The cells were cultured in DMEM/F12 medium (Sigma-Aldrich) supplemented with 20 mM HEPES (Dojindo Chemical Laboratories, Kumamoto, Japan), 25 mM NaHCO 3 (Kanto Chemical Co. Inc., Tokyo, Japan), 30 mg/L l -proline (Sigma-Aldrich), 0.1% bovine serum albumin (Serologicals Proteins Inc., Kankakee, IL), 10 mM nicotinamide (Sigma-Aldrich), 1 mM ascorbic acid 2-phosphate (Fujifilm Wako Pure Chem., Osaka, Japan), 10 ng/mL epidermal growth factor (BD Biosciences, Bedford, MA), ITS-X (BD Biosciences), 10 −7 M dexamethasone, and antibiotics.…”

CINC-2 and miR-199a-5p in exosomes secreted by transplanted Thy1+ cells activate hepatocytic progenitor cell growth in rat liver regeneration

“…The transfections were performed using the XMIRXpress vector (SBI System Biosciences) according to the manufacturer's instructions [26,29]. Brie y, 293TN cells (3 × 10 6 cells) were plated on 75-cm 2 culture asks.…”

“…The detail of the isolation and subculture methods have been reported [20,26,28,29]. Brie y, cells were isolated from DPPIV + rats using the two-step collagenase-perfusion method [16].…”

“…The number of viable cells was counted using the trypan blue exclusion test, and 1 × 10 5 cells/mL were plated on hyaluronic acid-coated dishes (Sigma-Aldrich Co., St. Louis, MO, 1 mg/35-mm dish). SHs were cultured for 10 d and then subcultured on Matrigel-coated 12well plates as described [28,29]. The cells were cultured in DMEM/F12 medium (Sigma-Aldrich) supplemented with 20 mM HEPES (Dojindo Chemical Laboratories, Kumamoto, Japan), 25 mM NaHCO 3 (Kanto Chemical Co. Inc., Tokyo, Japan), 30 mg/L l-proline (Sigma-Aldrich), 0.1% bovine serum albumin (Serologicals Proteins Inc., Kankakee, IL), 10 mM nicotinamide (Sigma-Aldrich), 1 mM ascorbic acid 2phosphate (Fuji lm Wako Pure Chem., Osaka, Japan), 10 ng/mL epidermal growth factor (EGF, BD Biosciences, Bedford, MA), ITS-X (BD Biosciences), 10 − 7 M dexamethasone, and antibiotics.…”