Enrichment of Hepatocytes Differentiated from Mouse Embryonic Stem Cells as a Transplantable Source

Kumashiro, Yuji; Asahina, Kinji; Ozeki, Rie; Shimizu-Saito, Keiko; Tanaka, Yujiro; Kida, Yujiro; Inoue, Kazuo; Kaneko, Michinari; Sato, Toshio; Teramoto, Koji; Arii, Shigeki; Teraoka, Hirobumi

doi:10.1097/01.tp.0000153637.44069.c6

Cited by 59 publications

(56 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although several studies have shown that ES cell-derived hepatocytes incorporate into recipient liver upon transplantation 7,10,12,26 the repopulation efficiency was very low, not exceeding 0.1%. 12,26 In our study, ES cell-derived hepatocytes replaced from 1% to 20% of the parenchyma in randomly selected areas of the liver in MUPuPA/SCID mice.…”

Section: Discussionmentioning

confidence: 99%

“…[1][2][3][4][5] Clinical application of ES cells requires a simple and efficient protocol for directing their differentiation into a specific cell type. Several studies have reported the induction of ES cell differentiation into hepatocytes both in vitro [6][7][8][9] and in vivo. 10 The in vitro approaches involve the formation of embryoid bodies (EBs) to re-create the inductive microenvironment required for liver organogenesis 11,12 or treatment with specific growth factors and cytokines critical for hepatocyte differentiation such as hepatocyte growth factor, fibroblast growth factor, and oncostatin.…”

Section: -1486)mentioning

confidence: 99%

See 1 more Smart Citation

Hepatic precursors derived from murine embryonic stem cells contribute to regeneration of injured liver

et al. 2006

View full text Add to dashboard Cite

We established an efficient system for differentiation, expansion and isolation of hepatic progenitor cells from mouse embryonic stem (ES) cells and evaluated their capacity to repopulate injured liver. Using mouse ES cells transfected with the green fluorescent protein (GFP) reporter gene regulated by albumin (ALB) enhancer/promoter, we found that a serum-free chemically defined medium supports formation of embryoid bodies (EBs) and differentiation of hepatic lineage cells in the absence of exogenous growth factors or feeder cell layers. The first GFP ؉ cells expressing ALB were detected in close proximity to "beating" myocytes after 7 days of EB cultures. GFP ؉ cells increased in number, acquired hepatocyte-like morphology and hepatocytespecific markers (i.e., ALB, AAT, TO, and G6P), and by 28 days represented more than 30% of cells isolated from EB outgrowths. The FACS-purified GFP ؉ cells developed into functional hepatocytes without evidence of cell fusion and participated in the repairing of diseased liver when transplanted into MUP-uPA/SCID mice. The ES cell-derived hepatocytes were responsive to normal growth regulation and proliferated at the same rate as the host hepatocytes after an additional growth stimulus from CCl 4 -induced liver injury. The transplanted GFP ؉ cells also differentiated into biliary epithelial cells. In conclusion, a highly enriched population of committed hepatocyte precursors can be generated from ES cells in vitro for effective cell replacement therapy. Supplementary material for this article can be found on the HEPATOLOGY website H epatocyte transplantation is an effective treatment for liver failure and/or end-stage liver disease. However, the shortage of donor organs and the difficulties of cyropreservation and long-term culturing of mature hepatocytes have limited the clinical application of cell-based therapy. Recently, the use of embryonic stem (ES) cells has attracted considerable interest for cell replacement therapy because of their capacity to proliferate indefinitely in vitro while retaining the potential to differentiate into all types of cells including hepatocytes. [1][2][3][4][5] Clinical application of ES cells requires a simple and efficient protocol for directing their differentiation into a specific cell type. Several studies have reported the induction of ES cell differentiation into hepatocytes both in vitro 6-9 and in vivo. 10 The in vitro approaches involve the formation of embryoid bodies (EBs) to re-create the inductive microenvironment required for liver organogenesis 11,12 or treatment with specific growth factors and cytokines critical for hepatocyte differentiation such as hepatocyte growth factor, fibroblast growth factor, and oncostatin. 1,13 Also, the introduction of genes promoting endodermal differentiation into ES cells, 14 modification of culture microenvironment by supplementation with extracellular matrix proteins or coculture with other cell types, 15,16 is used to direct ES cells toward a hepatocytic lineage. Nevertheless, these strategie...

show abstract

Section: Discussionmentioning

confidence: 99%

Section: -1486)mentioning

confidence: 99%

Hepatic precursors derived from murine embryonic stem cells contribute to regeneration of injured liver

et al. 2006

View full text Add to dashboard Cite

show abstract

“…Because of their simplicity, efficiency, rapidness, and excellent yields they have also become very popular for the separation of cells of various sizes. The Ficoll gradient yielded a twofold more mononuclear cell separation from bone marrow samples compared to Percoll [Cheng et al, 2003] and a two-layer Percoll gradient gave good separation of mESC-derived hepatocytes [Kumashiro et al, 2005]. Because Percoll may not be very safe for clinical application in the human as it is a PVP-coated silica preparation, Puresperm (a silane-coated silica preparation) yielded good separation of motile sperm from dead sperm and cell debris when a three-layer gradient was used [Chen and Bongso, 1999].…”

Section: Approaches To Eliminate Rogue Undifferentiated Hescs and Tummentioning

confidence: 99%

Taking stem cells to the clinic: Major challenges

Bongso

Fong

Gauthaman

2008

J of Cellular Biochemistry

167

111

View full text Add to dashboard Cite

Stem cell therapy offers tremendous promise in the treatment of many incurable diseases. A variety of stem cell types are being studied but human embryonic stem cells (hESCs) appear to be the most versatile as they are pluripotent and can theoretically differentiate into all the tissues of the human body via the three primordial germ layers and the male and female germ lines. Currently, hESCs have been successfully converted in vitro into functional insulin secreting islets, cardiomyocytes, and neuronal cells and transfer of such cells into diabetic, ischaemic, and parkinsonian animal models respectively have shown successful engraftment. However, hESC-derived tissue application in the human is fraught with the problems of ethics, immunorejection, tumorigenesis from rogue undifferentiated hESCs, and inadequate cell numbers because of long population doubling times in hESCs. Human mesenchymal stem cells (hMSC) though not tumorigenic, also have their limitations of multipotency, immunorejection, and are currently confined to autologous transplantation with the genuine benefits in allogeneic settings not conclusively shown in large controlled human trials. Human Wharton's jelly stem cells (WJSC) from the umbilical cord matrix which are of epiblast origin and containing both hESC and hMSC markers appear to be less troublesome in not being an ethically controversial source, widely multipotent, not tumorigenic, maintain ''stemness'' for several serial passages and because of short population doubling time can be scaled up in large numbers. This report describes in detail the hurdles all these stem cell types have to overcome before stem cell-based therapy becomes a genuine reality.

show abstract

“…that ES cells can be differentiated into hepatocyte-like cells [1][2][3][4][5][6] . ES cell-derived hepatocytes would be useful as a source of material for cell transplantation, bioartificial liver (BAL) support devices, studies on hepatocyte biology and hepatitis viruses and for drug testing.…”

Section: Protocols Have Been Developed To Differentiate and Enrich Vamentioning

confidence: 99%

Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines

et al. 2007

View full text Add to dashboard Cite

This protocol describes a co-culture system for the in vitro differentiation of mouse embryonic stem cells into hepatocyte-like cells. Differentiation involves four steps: (i) formation of embryoid bodies (EB), (ii) induction of definitive endoderm from 2-d-old EBs, (iii) induction of hepatic progenitor cells and (iv) maturation into hepatocyte-like cells. Differentiation is completed by 16 d of culture. EBs are formed, and cells can be induced to differentiate into definitive endoderm by culture in Activin A and fibroblast growth factor 2 (FGF-2). Hepatic differentiation and maturation of cells is accomplished by withdrawal of Activin A and FGF-2 and by exposure to liver nonparenchymal cell-derived growth factors, a deleted variant of hepatocyte growth factor (dHGF) and dexamethasone. Approximately 70% of differentiated embryonic stem (ES) cells express albumin and can be recovered by albumin promoter-based cell sorting. The sorted cells produce albumin in culture and metabolize ammonia, lidocaine and diazepam at approximately two-thirds the rate of primary mouse hepatocytes.

show abstract

Enrichment of Hepatocytes Differentiated from Mouse Embryonic Stem Cells as a Transplantable Source

Abstract: Functional hepatocytes can be differentiated from mouse ES cells by way of EB formation. The elimination of undifferentiated cells from the EBs provides transplantable cells for liver failure without tumorigenicity.

Cited by 59 publications

References 43 publications

Hepatic precursors derived from murine embryonic stem cells contribute to regeneration of injured liver

Hepatic precursors derived from murine embryonic stem cells contribute to regeneration of injured liver

Taking stem cells to the clinic: Major challenges

Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines

Contact Info

Product

Resources

About