Efficient large-scale generation of functional hepatocytes from mouse embryonic stem cells grown in a rotating bioreactor with exogenous growth factors and hormones

Abstract

Introduction: Embryonic 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.

Methods: In 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.

Results: During 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.

Conclusions: Large 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.

Figure 1

Morphologic and histologic characteristics of EBs and EB-derived cells in the rotating bioreactor. (A) A rotating bioreactor was used to create a 3D RCCS for mES cell culture. (B) A large number of homogeneous EBs was formed at day 7 in a rotating bioreactor with exogenous growth factors and hormones. (C) Image of EBs by stereomicroscopy at day 14. (D-F) Images of EBs with H&E staining at day 7 (D), 14 (E), 21 (F). EB-derived cells from day 14 and day 21, but not day 7, exhibited polyhedral contours, large nuclei, and a cordlike arrangement. (G-I) Images of EB-derived cells. When the day 21 EB-derived cells were seeded in six-well plates (G), most of the cells had a uniform pyramidal shape after 3 days (H) and 5 days (I) of culture. Scale bar, 100 μm.

Figure 2

Liver-specific gene expression of EBs. The expression of the liver-specific genes in the EBs was analyzed by RT-PCR (A, B) and WB analysis (C, D). (A, C) The expression of the hepatic genes (A) and proteins (C) in the EBs at different time point of culture. (B, D) The expression of the hepatic genes (B) and proteins (D) in the mES cells from the 3D culture and the differentiated mES cells from the 2D culture. P, positive control.

Figure 3

Immunohistochemical and immunocytochemical staining of EBs from the rotating bioreactor. (A) Immunohistochemical staining was performed on sections of day-14 EBs by using anti-ALB, anti-AFP, and anti-CK18 antibodies (red). DAPI was also included for nuclear staining (blue). (B) Immunocytochemical staining for ALB, AFP, and CK18 was performed on the day 14 EB-derived cells cultured on chamber slides for 5 days. Scale bar, 100 μm.

Figure 4

ALB secretion from EB-derived cells. ALB production by day 7, 14, and 21 EB-derived cells was quantified by using an ELISA kit after 72 hours of monolayer culture. The results are expressed as the means ± SD of six experiments. *P < 0.05.

Figure 5

Cytochrome P450 activity in EBs and EB-derived cells. (A-C) PROD activity (red) was detected in day-21 EBs (A), day-21 EB-derived cells grown in monolayer culture (B), and mES cells differentiated in 2D culture (C) by CLSM. Scale bar, 100 μm. (D-F) Flow cytometric analysis showed that the number of PROD-positive day-21 EB-derived cells (D) was higher than the number of differentiated mES cells from the 2D culture (E). A significant difference in the number of PROD-positive cells was noted between the differentiated cell samples from the 3D and 2D cultures (F). *P < 0.05.

Figure 6

LDL-uptake analysis of EBs and EB-derived cells. (A-C) CLSM images indicated that the day-21 EBs (A) could incorporate DiI-Ac-LDL (red). Additionally, more day 21 EB-derived cells grown in monolayer culture incorporated DiI-Ac-LDL (B) than the mES cells differentiated in 2D culture (C). Scale bar, 100 μm. (D-F) Flow-cytometric analysis showed that the number of LDL uptake-positive cells derived from day-21 EBs (D) was higher than the number of differentiated mES cells from 2D culture exhibiting LDL uptake (E). A significant difference in the LDL uptake between the differentiated cell samples from the 3D and 2D cultures (F). *P < 0.05.

Figure 7

PAS staining of EBs and EB-derived cells. PAS staining, used to detect glycogen in the EB sections, showed that the number of positive cells increased from day 7 (A) to day 14 (B) and day 21 (C). More PAS-positive cells were present among the day 21 EB-derived cells grown in monolayer culture (D) than the differentiated mES cells from the 2D culture (E). In contrast, the undifferentiated mES cells were negative for PAS staining (F). Scale bar, 50 μm.

Figure 8

Indocyanine green staining of EBs and EB-derived cells. A low level of indocyanine green staining was found in the day 7 EB-derived cells (A), whereas an increasing number of ICG-positive cells appeared at day 14 (B) and day 21 (C). Additionally, more ICG-positive cells were present among the day 21 EB-derived cells grown in monolayer culture (D) compared with the differentiated mES cells from the 2D culture (E). The undifferentiated mES cells were negative for ICG staining (F). Scale bar, 50 μm.

Figure 9

Transplantation of EB-derived cells. (A) Day 14 EB-derived cells labeled with DiI (red) were transplanted into the spleens of nude mice under anesthesia. (B) No development of tumors, such as teratomas, was observed in the livers of the recipient mice 2 months after the cell injection. (C) One month after transplantation, fluorescence from the EB-derived cells labeled with DiI (red) was detected in frozen sections of the recipient livers. Scale bar, 200 μm.