Abstract
IntroductionEmbryonic stem (ES) cells are considered a potentially advantageous source of hepatocytes for both transplantation and the development of bioartificial livers. However, the efficient large-scale generation of functional hepatocytes from ES cells remains a major challenge, especially for those methods compatible with clinical applications.MethodsIn this study, we investigated whether a large number of functional hepatocytes can be differentiated from mouse ES (mES) cells using a simulated microgravity bioreactor. mES cells were cultured in a rotating bioreactor in the presence of exogenous growth factors and hormones to form embryoid bodies (EBs), which then differentiated into hepatocytes.ResultsDuring the rotating culture, most of the EB-derived cells gradually showed the histologic characteristics of normal hepatocytes. More specifically, the expression of hepatic genes and proteins was detected at a higher level in the differentiated cells from the bioreactor culture than in cells from a static culture. On further growing, the EBs on tissue-culture plates, most of the EB-derived cells were found to display the morphologic features of hepatocytes, as well as albumin synthesis. In addition, the EB-derived cells grown in the rotating bioreactor exhibited higher levels of liver-specific functions, such as glycogen storage, cytochrome P450 activity, low-density lipoprotein, and indocyanine green uptake, than did differentiated cells grown in static culture. When the EB-derived cells from day-14 EBs and the cells’ culture supernatant were injected into nude mice, the transplanted cells were engrafted into the recipient livers.ConclusionsLarge quantities of high-quality hepatocytes can be generated from mES cells in a rotating bioreactor via EB formation. This system may be useful in the large-scale generation of hepatocytes for both cell transplantation and the development of bioartificial livers.
Highlights
Embryonic stem (ES) cells are considered a potentially advantageous source of hepatocytes for both transplantation and the development of bioartificial livers
We demonstrated that the growth and differentiation of ES cells in a biodegradable polymer scaffold within a rotating microgravity bioreactor yields organized functional hepatocytes that can be used in research on bioartificial livers and engineered liver tissue [20]
embryoid body (EB) formation and hepatic differentiation in a rotating bioreactor mouse ES (mES) cells were dissociated by using trypsin and seeded into a rotating bioreactor (Synthecon Inc., Houston, TX, USA) at a concentration of 1 × 106 cells/ml Iscove modified Dulbecco medium (IMDM), which was supplemented with 10-7 M dexamethasone
Summary
Embryonic stem (ES) cells are considered a potentially advantageous source of hepatocytes for both transplantation and the development of bioartificial livers. The efficient large-scale generation of functional hepatocytes from ES cells remains a major challenge, especially for those methods compatible with clinical applications. Emergency liver transplantation remains the most successful treatment in many cases of ALF. Both hepatocyte transplantation and bioartificial livers have been investigated as a “bridge” or alternative to liver transplantation, which are promising treatment options for ALF patients awaiting a donor liver [2,3,4]. Hepatocyte transplantation and the development of bioartificial livers entail a large quantity of high-quality hepatocytes, which requires a donor liver. An urgent need exists for an alternative and adequate supply of suitable hepatocytes for both hepatocyte transplantation and bioartificial livers [5]
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