pubmed.ncbi.nlm.nih.gov

Muse Cells, Nontumorigenic Pluripotent-Like Stem Cells, Have Liver Regeneration Capacity Through Specific Homing and Cell Replacement in a Mouse Model of Liver Fibrosis - PubMed

  • ️Sun Jan 01 2017

Muse Cells, Nontumorigenic Pluripotent-Like Stem Cells, Have Liver Regeneration Capacity Through Specific Homing and Cell Replacement in a Mouse Model of Liver Fibrosis

Masahiro Iseki et al. Cell Transplant. 2017.

Abstract

Muse cells, a novel type of nontumorigenic pluripotent-like stem cells, reside in the bone marrow, skin, and adipose tissue and are collectable as cells positive for pluripotent surface marker SSEA-3. They are able to differentiate into cells representative of all three germ layers. The capacity of intravenously injected human bone marrow-derived Muse cells to repair an immunodeficient mouse model of liver fibrosis was evaluated in this study. The cells exhibited the ability to spontaneously differentiate into hepatoblast/hepatocyte lineage cells in vitro. They demonstrated a high migration capacity toward the serum and liver section of carbon tetrachloride-treated mice in vitro. In vivo, they specifically accumulated in the liver, but not in other organs except, to a lesser extent, in the lungs at 2 weeks after intravenous injection in the liver fibrosis model. After homing, Muse cells spontaneously differentiated in vivo into HepPar-1 (71.1 ± 15.2%), human albumin (54.3 ± 8.2%), and anti-trypsin (47.9 ± 4.6%)-positive cells without fusing with host hepatocytes, and expressed mature functional markers such as human CYP1A2 and human Glc-6-Pase at 8 weeks after injection. Recovery in serum, total bilirubin, and albumin and significant attenuation of fibrosis were recognized with statistical differences between the Muse cell-transplanted group and the control groups, which received the vehicle or the same number of a non-Muse cell population of MSCs (MSCs in which Muse cells were eliminated). Thus, unlike ESCs and iPSCs, Muse cells are unique in their efficient migration and integration into the damaged liver after intravenous injection, nontumorigenicity, and spontaneous differentiation into hepatocytes, rendering induction into hepatocytes prior to transplantation unnecessary. They may repair liver fibrosis by two simple steps: expansion after collection from the bone marrow and intravenous injection. A therapeutic strategy such as this is feasible and may provide significant advancements toward liver regeneration in patients with liver disease.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.

Characterization of multilineage-differentiating stress-enduring (Muse) cells. (A) Growth curve of mesenchymal stem cells (MSCs) obtained by using the cell counting kit-8 (CCK8). Bone marrow (BM)-derived MSCs (1.3 × 103 cells, p = 7) were seeded onto 96-well plates, and the plates were incubated [day (D)]. (B) Stage-specific embryonic antigen-3+ (SSEA-3+) Muse cells (gate P3) and SSEA-3− non-Muse cells (gate P6) sorted from human BM-MSCs. (C) Quantitative polymerase chain reaction (qPCR) for octamer-binding transcription factor 4 (OCT4), sex-determining region Y-box 2 (SOX2), and Nanog. *p < 0.05, **p < 0.01, ***p < 0.001. (D) Cells expanded from single M-cluster on gelatin-coated dish. (E) Immunocytochemistry of cells expanded from a single M-cluster on gelatin-coated culture dish expressed hepatoblast/hepatocyte markers: Δ-like protein (DLK), α-fetoprotein, cytokeratin 19, and cytokeratin 18 (red). Blue indicates nuclei staining with 4′,6-diamidino-2-phenylindole (DAPI). Scale bars: 50 μm.

Figure 2.
Figure 2.

In vitro migration of multilineage-differentiating stress-enduring (Muse) cells toward the serum and liver tissue of carbon tetrachloride (CCl4)-treated animals. (A–C) In vitro migration assay. (A) Outline of the experiment. Human Muse or non-Muse cells were placed in the upper chamber of the semipermeable membrane, and either the serum or the liver tissue collected at 1, 24, and 28 h after intraperitoneal (IP) injection of CCl4, or from intact animal, was placed in the lower chamber. After 22 h of incubation, the number of migrated cells through the semipermeable membrane was counted. Muse cells migrate to the (B) serum and (C) liver tissue with higher efficiency than that observed in non-Muse cells. (D) Western blot for C-X-C chemokine receptor type 4 (CXCR4) and hepatocyte growth factor receptor (c-Met) in human Muse and non-Muse cells. β-Actin was used as control. (E) Migration of Muse and non-Muse cells toward the serum 24 h after CCl4 injection with or without the CXCR4 antagonist AMD3100. **p < 0.01, ***p < 0.001.

Figure 3.
Figure 3.

In vivo dynamics of multilineage-differentiating stress-enduring (Muse) and non-Muse cells in 24-h carbon tetrachloride (CCl4) liver damage model. (A, B) Quantitative polymerase chain reaction (qPCR) of a human-specific Alu sequence both in intact (A) and CCl4-treated mice (B) injected with human Muse and non-Muse cells at 2 weeks (BM, bone marrow). (C) H-Golgi immunostaining in the liver at 30 days of infusion. Since integration of Muse cells that expressed the H-Golgi signal (green) was heterogenous, representative pictures of high, moderate, and low integration areas were demonstrated. A minimal number of non-Muse cells was detected. Blue indicates nuclei staining with DAPI. (D) The proportion of H-Golgi+ cells relative to the total number of cells/mm2 in the liver section. ***p < 0.001. (E, F) Because the injured liver is quite heterogenous, (B), (D), and (E), showing the integration of Muse cells into the damage tissue, were selected for demonstrative purposes. Integrated human cells positive for human mitochondria (E; green) and H-Golgi (F; green) expressed human albumin (E; red) and anti-human hepatocyte antibody (HepPar-1) (F; red), respectively, in the damaged liver of the Muse cell-injected animals (30 days). Blue indicates nuclei staining with DAPI. Scale bars: 50 μm (main figures), 25 μm (magnified boxes).

Figure 4.
Figure 4.

Functional and histological evaluations in the liver fibrosis model. (A) The liver fibrosis severe combined immunodeficiency (SCID) mouse model was carried out by intraperitoneal (IP) injection of carbon tetrachloride (CCl4; 0.5 ml/kg) twice per week for up to 8 weeks. Multilineage-differentiating stress-enduring (Muse) and non-Muse cells (5 × 104 cells) were infused via the tail vein at 2, 4, and 6 weeks, and data were collected at 8 weeks [intravenous injection (IV)]. (B, C) Serum total bilirubin (B) and serum albumin (C) levels in the Muse, vehicle, and non-Muse groups at 8 weeks of infusion. (D, E) Evaluation of the liver fibrotic area by staining with (D) Sirius red and (E) Masson's trichrome staining at 8 weeks. (F–H) ELISA for matrix metalloproteinase-1 (MMP-1), MMP-2, and MMP-9 in human Muse and non-Muse cells. **p < 0.01, ***p < 0.001. Scale bars: 50 μm.

Figure 5.
Figure 5.

Differentiation of human multilineage-differentiating stress-enduring (Muse) cells in the liver fibrosis model. (A) Immunohistochemistry of H-Golgi+ cells located in the damaged liver at 8 weeks. A substantial number of H-Golgi+ cells (green) were detected around the vessels in the Muse group, while they were almost undetectable in the non-Muse group. Since integration of Muse cells was heterogenous, representative pictures of high, moderate, and low integration areas were demonstrated. Blue indicates nuclei staining with DAPI. (B) Proportion of H-Golgi+ cells relative to the total number of cells/mm2 in the liver section. ***p < 0.001. (C) Expression of anti-human hepatocyte antibody (HepPar-1; red) in H-Golgi+ cells (green), (D) human albumin (red) in H-Golgi+ cells (green), and (E) human anti-trypsin (red) in human mitochondria+ cells (green). Scale bars: 50 μm (main figures), 25 μm (magnified boxes).

Figure 6.
Figure 6.

Fluorescence in situ hybridization (FISH) and functional analyses of the multilineage-differentiating stress-enduring (Muse) group (8 weeks). (A) Upper row shows FISH, and lower row shows immunohistochemistry for H-Golgi (green)/anti-human hepatocyte antibody (HepPar-1; red) in the neighboring section. In FISH, green indicates the mouse chromosome, red indicates the human chromosome. As a consequence, *1 circle is considered the mouse cell, *2 and *3 circles are the human cells without fusion with mouse cells, and *4 circle is the human–mouse fused cells. Since sections were made at 8- to 10-μm thickness, the neighboring section do not exhibit exactly the same location of nucleoli or shape of cytoplasm. However, *1, *2, and *3 cells in FISH can be projected to corresponding cells in immunohistochemistry. The *4 cell was not projected to immunohistochemistry. The *1 cell is negative for H-Golgi. The *2 and *3 cells are double positive for H-Golgi+ and HepPar-1, thus H-Golgi+ human Muse cells are suggested to differentiate into hepatocyte marker HepPar-1+ cells without fusing with mouse hepatocytes. Blue indicates nuclei staining with DAPI. Scale bars: 25 μm. (B) Reverse transcription polymerase chain reaction (RT-PCR) of human-specific albumin, human cytochrome P450, family 1, subfamily A, polypeptide2 (CYP1A2), human glucose-6-phosphatase (Glc-6-Pase), and human and mouse β-actin. The human liver was used as a positive control, and the liver of the vehicle group was used as a negative control.

Figure 7.
Figure 7.

Differentiation of human multilineage-differentiating stress-enduring (Muse) cells into hepatocyte lineage cells by coculture with apoptotic hepatocytes. Expression of human-specific sex-determining region Y-box 17 (Sox17), human cytokeratin 18 (CK18), human prospero homeobox protein 1 (Prox1), and human a-fetoprotein (AFP) in quantitative polymerase chain reaction (qPCR). Since primers were specific for human, mouse fetus liver was consistently negative for all markers while human fetus liver was positive. Human Muse cells, originally negative for all the markers, became positive for CK18 at day 3, Sox17 and AFP at 1 week, and Prox1 at 2 weeks only when cocultured with etoposide-treated apoptotic mouse Hepa-1-6. On the other hand, supply of conditioned medium from intact or apoptotic mouse Hepa-1-6 and coculture with intact mouse Hepa-1-6 did not induce human-specific marker expressions.

Similar articles

Cited by

References

    1. Lee WM, Squires RH Jr, Nyberg SL, Doo E, Hoofnagle JH,. Acute liver failure: Summary of a workshop. Hepatology 2008; 47(4): 1401–15. - PMC - PubMed
    1. Tsochatzis EA, Bosch J, Burroughs AK. Liver cirrhosis. Lancet 2014; 383(9930): 1749–61. - PubMed
    1. Chang HM, Liao YW, Chiang CH, Chen YJ, Lai YH, Chang YL, Chen HL, Jeng SY, Hsieh JH, Peng CH, Li HY, Chien Y, Chen SY, Chen LK, Huo TI. Improvement of carbon tetrachloride-induced acute hepatic failure by transplantation of induced pluripotent stem cells without reprogramming factor c-Myc. Int J Mol Sci. 2012; 13(3): 3598–617. - PMC - PubMed
    1. Esrefoglu M. Role of stem cells in repair of liver injury: Experimental and clinical benefit of transferred stem cells on liver failure. World J Gastroenterol. 2013; 19(40): 6757–73. - PMC - PubMed
    1. Herrera MB, Fonsato V, Bruno S, Grange C, Gilbo N, Romagnoli R, Tetta C, Camussi G. Human liver stem cells improve liver injury in a model of fulminant liver failure. Hepatology 2013; 57(1): 311–9. - PubMed

MeSH terms

Substances