Enhanced In Vitro Maturation of Fetal Mouse Liver Cells with Oncostatin M, Nicotinamide, and Dimethyl Sulfoxide

Although cells isolated from fetal liver are one of the major sources for liver tissue engineering, it is still very difficult to induce them to fully differentiate in vitro into mature hepatocytes. We therefore investigated the effects of nicotinamide (NA), dimethyl sulfoxide (DMSO), and oncostatin M (OSM) on differentiation in terms of the expression of various liver-specific functions, because these factors have been reported to induce the emergence of possible hepatocyte progenitor cells (small hepatocytes) in adult rat hepatocyte culture or maturation of fetal mouse liver cells in culture. Fetal liver cells isolated from mouse embryos were cultured for 5 weeks in collagen-precoated plates. NA (10 mM) and DMSO (1%) remarkably enhanced the emergence of small hepatocytes, and OSM also synergistically enhanced the selective growth of small hepatocytes and inhibited the growth of blood cell populations. In the presence of these three factors, such small hepatocytes became dominant in culture, so that they covered almost 60–70% of confluence after week 2. In addition, some of them piled up over the small hepatocyte monolayer and displayed distinctively differentiated morphology, such as the emergence of binucleated cells, formation of tight gap junctions, and possible bile duct structures. Although OSM alone had very weak effects on hepatocyte functions, albumin secretion and cytochrome P450IA1/2 capacity were greatly enhanced when combined with NA or DMSO. This functional observation closely agreed with the emergence of small hepatocytes. In contrast, ammonium removal was strongly dependent on DMSO alone. DNA amount basis functions of fetal cells with three factors at week 5 were 1/7 for albumin secretion, 3 times higher for ammonium removal, and 1/10 for P450 capacity, compared with those of cultured adult mouse hepatocytes. These results show that inclusion of NA, DMSO, and OSM in the culture medium significantly enhances in vitro maturation of fetal liver cells when compared with conventional culture conditions.

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Cultivation of Fetal Liver Cells in a Three-Dimensional Poly-L-Lactic Acid Scaffold in the Presence of Oncostatin M

Cell Transplantation ◽

10.3727/000000002783985648 ◽

2002 ◽

Vol 11 (5) ◽

pp. 403-406 ◽

Cited By ~ 27

Author(s):

Jinlan Jiang ◽

Nobuhiko Kojima ◽

Taisei Kinoshita ◽

Atsushi Miyajima ◽

Weiqun Yan ◽

...

Keyword(s):

Lactic Acid ◽

Fetal Liver ◽

Three Dimensional ◽

Liver Cells ◽

Oncostatin M ◽

Albumin Secretion ◽

Plla Scaffold ◽

Fetal Liver Cells ◽

Number Of Cells

To investigate the feasibility of fetal liver cells for liver tissue engineering, the supporting function of poly-l-lactic acid (PLLA) for fetal liver cells and the effects of oncostatin M (OSM) on hepatic differentiation were studied. After preparing three-dimensional biodegradable PLLA scaffold having a well-developed open-pore structure by a gas-forming method with ammonium chloride particles as a porogen and a gas-forming reagent, fetal liver cells separated from E14.5-C57BL/6CrSlc murine embryos were inoculated in the PLLA scaffolds. Cells were cultured in Williams' E medium with or without OSM (10 ng/ml) for 30 days with a medium change every 2 days. Results showed that there were significant increases in the number of cells and in albumin secretion in PLLA culture compared with in monolayer culture on day 15. In addition, a significant increase in albumin secretion was observed in OSM-added PLLA culture compared with OSM-free culture, and there was only a slightly enhanced albumin secretion in monolayer cultures with OSM. These results suggest that PLLA may enhance the biological activity of OSM for inducing maturation of fetal liver cells. Interestingly, the number of cells in PLLA culture with OSM decreased compared with OSM-free PLLA culture at day 15. This may be because promotion of hepatic development by OSM simultaneously suppressed in vitro hematopoiesis (i.e., blood cell production). In summary, our results indicate that the three-dimensional PLLA scaffold is a good support material for the cultivation of fetal liver cells and that OSM is capable of not only terminating hematopoiesis of the fetal liver but also stimulating the maturation of hepatic parenchymal cells in vitro.

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Basis of hematopoietic defects in platelet-derived growth factor (PDGF)-B and PDGF β-receptor null mice

Blood ◽

10.1182/blood.v97.7.1990 ◽

2001 ◽

Vol 97 (7) ◽

pp. 1990-1998 ◽

Cited By ~ 48

Author(s):

Wolfgang E. Kaminski ◽

Per Lindahl ◽

Nancy L. Lin ◽

Virginia C. Broudy ◽

Jeffrey R. Crosby ◽

...

Keyword(s):

Growth Factor ◽

Fetal Liver ◽

Liver Cells ◽

Platelet Derived Growth Factor ◽

Wild Type ◽

Liver Growth ◽

Hematologic Disorders ◽

Fetal Liver Cells ◽

Β Receptor

Abstract Platelet-derived growth factor (PDGF)-B and PDGF β-receptor (PDGFRβ) deficiency in mice is embryonic lethal and results in cardiovascular, renal, placental, and hematologic disorders. The hematologic disorders are described, and a correlation with hepatic hypocellularity is demonstrated. To explore possible causes, the colony-forming activity of fetal liver cells in vitro was assessed, and hematopoietic chimeras were demonstrated by the transplantation of mutant fetal liver cells into lethally irradiated recipients. It was found that mutant colony formation is equivalent to that of wild-type controls. Hematopoietic chimeras reconstituted with PDGF-B−/−, PDGFRβ−/−, or wild-type fetal liver cells show complete engraftment (greater than 98%) with donor granulocytes, monocytes, B cells, and T cells and display none of the cardiovascular or hematologic abnormalities seen in mutants. In mouse embryos, PDGF-B is expressed by vascular endothelial cells and megakaryocytes. After birth, expression is seen in macrophages and neurons. This study demonstrates that hematopoietic PDGF-B or PDGFRβ expression is not required for hematopoiesis or integrity of the cardiovascular system. It is argued that metabolic stress arising from mutant defects in the placenta, heart, or blood vessels may lead to impaired liver growth and decreased production of blood cells. The chimera models in this study will serve as valuable tools to test the role of PDGF in inflammatory and immune responses.

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Hemoglobin switching in sheep: characteristics of BFU-E-derived colonies from fetal liver

Blood ◽

10.1182/blood.v56.3.495.bloodjournal563495 ◽

1980 ◽

Vol 56 (3) ◽

pp. 495-500

Author(s):

JE Barker

Keyword(s):

Stem Cells ◽

Fetal Liver ◽

Fetal Hemoglobin ◽

Liver Cells ◽

S Phase ◽

Hemoglobin Switching ◽

Fetal Liver Cells ◽

Beta 2 ◽

Number Of Cells

Two types of erythroid colonies were generated in vitro from sheep fetal liver cells. The first type consisted of single colonies of 8–256 cells that were well hemoglobinized by 4 days; these are thought to originate from CFU-E. The second type consisted of macroscopic colonies composed of several subcolonies that matured between days 3 and 8 in vitro. At maturity, each contained 256 to > 1000 cells that formed a discrete macroscopic cluster. The macroscopic colonies, not previously described in sheep, are thought to be derived from BFU-E. The characteristics of sheep BFU-E were defined and the production of fetal hemoglobin (HbF, alpha 1, gamma 2) and HbC (alpha 2 beta 2) was compared in colonies derived from CFU-E or BFU-E. Bursts developed at erythropoietin (epo) concentrations as low as 0.1 U/ml, although the number observed increased with epo concentration up to 10 U/ml. The number of bursts observed was approximately proportional to the number of cells plated. As shown by thymidine suicide, approximately 50% of both the BFU e and CFU-E were in S-phase when obtained from the fetus. BFU-E were smaller and partially separable from CFU-E after sedimentation at unit gravity. The beta c/gamma synthetic ratio in colonies derived from BFU-E was greater than in CFU-E-derived colonies. These data suggest that the capacity for generation of erythroblasts making HbC is greater in the earlier or more primitive erythroid stem cells in fetal liver.

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Disruption of palladin leads to defects in definitive erythropoiesis by interfering with erythroblastic island formation in mouse fetal liver

Blood ◽

10.1182/blood-2007-01-068528 ◽

2007 ◽

Vol 110 (3) ◽

pp. 870-876 ◽

Cited By ~ 28

Author(s):

Xue-Song Liu ◽

Xi-Hua Li ◽

Yi Wang ◽

Run-Zhe Shu ◽

Long Wang ◽

...

Keyword(s):

Fetal Liver ◽

Liver Cells ◽

Normal Function ◽

Yolk Sac ◽

Neural Tube Closure ◽

Island Formation ◽

Erythroblastic Island ◽

Fetal Liver Cells ◽

Definitive Erythropoiesis

Abstract Palladin was originally found up-regulated with NB4 cell differentiation induced by all-trans retinoic acid. Disruption of palladin results in neural tube closure defects, liver herniation, and embryonic lethality. Here we further report that Palld−/− embryos exhibit a significant defect in erythropoiesis characterized by a dramatic reduction in definitive erythrocytes derived from fetal liver but not primitive erythrocytes from yolk sac. The reduction of erythrocytes is accompanied by increased apoptosis of erythroblasts and partial blockage of erythroid differentiation. However, colony-forming assay shows no differences between wild-type (wt) and mutant fetal liver or yolk sac in the number and size of colonies tested. In addition, Palld−/− fetal liver cells can reconstitute hematopoiesis in lethally irradiated mice. These data strongly suggest that deficient erythropoiesis in Palld−/− fetal liver is mainly due to a compromised erythropoietic microenvironment. As expected, erythroblastic island in Palld−/− fetal liver was found disorganized. Palld−/− fetal liver cells fail to form erythroblastic island in vitro. Interestingly, wt macrophages can form such units with either wt or mutant erythroblasts, while mutant macrophages lose their ability to bind wt or mutant erythroblasts. These data demonstrate that palladin is crucial for definitive erythropoiesis and erythroblastic island formation and, especially, required for normal function of macrophages in fetal liver.

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Stimulating effects of fibroblast growth factors on hepatic function of fetal liver cells synergistically with oncostatin M in three-dimensional culture

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2008.10.004 ◽

2009 ◽

Vol 107 (3) ◽

pp. 307-311 ◽

Cited By ~ 1

Author(s):

Kazunori Minagawa ◽

Toshie Koyama ◽

Hirotoshi Miyoshi

Keyword(s):

Growth Factors ◽

Fetal Liver ◽

Three Dimensional ◽

Fibroblast Growth Factors ◽

Hepatic Function ◽

Liver Cells ◽

Oncostatin M ◽

Fibroblast Growth ◽

Fetal Liver Cells ◽

Three Dimensional Culture

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Differentiation of erythroid progenitor (CFU-E) cells from mouse fetal liver cells and murine erythroleukemia (TSA8) cells without proliferation.

Molecular and Cellular Biology ◽

10.1128/mcb.8.6.2604 ◽

1988 ◽

Vol 8 (6) ◽

pp. 2604-2609 ◽

Cited By ~ 9

Author(s):

T Noguchi ◽

H Fukumoto ◽

Y Mishina ◽

M Obinata

Keyword(s):

Colony Formation ◽

Fetal Liver ◽

Liver Cells ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Proliferation And Differentiation ◽

Erythroleukemia Cell ◽

Fetal Liver Cells ◽

Murine Erythroleukemia

Erythropoietin (epo) appears to play a significant role in influencing the proliferation and differentiation of erythroid progenitor (CFU-E) cells. To determine the mechanism of action of epo, the effect of drugs on the in vitro colony formation of CFU-E cells induced from a novel murine erythroleukemia cell line, TSA8, was examined. While cytosine arabinoside inhibited colony formation and terminal differentiation of the CFU-E cells responding to epo, herbimycin, which is a drug that inhibits src-related phosphorylation, inhibited colony formation only. The same effect of herbimycin was observed with normal CFU-E cells from mouse fetal liver cells. These results suggest that epo induces two signals, one for proliferation and the other for differentiation, and that the two signals are not linked in erythroid progenitor cells.

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Interleukin-4 inhibits the expression of Kit and tryptase during stem cell factor-dependent development of human mast cells from fetal liver cells

Blood ◽

10.1182/blood.v84.5.1519.1519 ◽

1994 ◽

Vol 84 (5) ◽

pp. 1519-1527 ◽

Cited By ~ 49

Author(s):

G Nilsson ◽

U Miettinen ◽

T Ishizaka ◽

LK Ashman ◽

AM Irani ◽

...

Keyword(s):

Mast Cell ◽

Stem Cell ◽

Mast Cells ◽

Stem Cell Factor ◽

Fetal Liver ◽

Liver Cells ◽

Interleukin 4 ◽

Human Mast Cells ◽

Fetal Liver Cells

Abstract Although interleukin-4 (IL-4) in mice is known to augment the proliferation of mast cells and to modulate the expression of certain mast cell protease transcripts, its effect on human mast cells is less well understood. The current study examined the effects of recombinant human IL-4 (rhuIL-4) on stem cell factor (SCF)-dependent fetal liver- derived human mast cells in liquid culture. In no case did rhuIL-4 augment proliferation of mast cells. rhuIL-4 selectively inhibited certain aspects of the development of mast cells in cultures of fetal liver cells with rhuSCF. These include lower numbers and percentages of cells expressing tryptase and surface Kit, smaller cells, and lower contents of cells for tryptase, histamine, and Kit. Development of metachromasia was not attenuated. The downregulation of Kit, the surface receptor for SCF, is probably a critical factor, because cells lacking this molecule would not be able to respond to SCF. In contrast to mast cell progenitors, mast cells already developed in vitro from fetal liver cells are relatively resistant to rhuIL-4, but are still dependent for survival on the presence of rhuSCF.

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