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Myofibroblasts control the proliferation of fetal hepatoblasts and their differentiated cholangiocytes during the hepatoblast-to-cholangiocyte transition
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Myofibroblasts control the proliferation of fetal hepatoblasts and their differentiated cholangiocytes during the hepatoblast-to-cholangiocyte transition Wei Wang, Li Wan, Zhixin Chen, Xin Jin, Dewei Li* Department of Hepatobiliary Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 October 2019 Accepted 26 November 2019 Available online xxx
Mesenchymal cells in the liver provide the microenvironment for hepatoblasts expansion and differentiation. We have previously demonstrated that myofibroblasts (MFs) promoted hepatoblasts differentiation into cholangiocytes, whereas its role in controlling the proliferation of hepatoblasts and their differentiated cholangiocytes remains elusive. Here, we investigated the role of MFs in regulating the proliferation of hepatoblasts and their differentiated cholangiocytes using an indirect coculture system. When cocultured with hepatoblasts, MFs promoted hepatoblasts differentiation into cholangiocytes and inhibited the proliferation and stemness of hepatoblasts. However, when hepatoblasts already differentiated into cholangiocytes, MFs promoted the differentiated cholangiocytes proliferation. In addition, hepatoblast proliferation genes such as hepatocyte growth factor (HGF), insulin-like growth factor-1 and 2 (IGF-1 and 2), midkine 1 (Mdk1), and pleiotrophin (Ptn) expression in MFs were down-regulated compared with their levels in fibroblasts. Our findings uncover the role of MFs in controlling the proliferation of hepatoblasts and their differentiated cholangiocytes, potentially providing a novel therapeutic strategy for cholangiocyte regeneration. © 2019 Published by Elsevier Inc.
Keywords: Myofibroblasts Hepatoblasts Cholangiocytes Proliferation Differentiation
1. Introduction During hepatogenesis, fetal hepatoblasts, developed from foregut endodermal cells, can quickly proliferation and differentiate bipotentially into either hepatocytes or cholangiocytes [1,2]. The interaction with nonparenchymal cell, such as mesenchymal cells, stellate cells, and mesothelial cells, is important for hepatoblasts proliferation and differentiation [3e6]. Microenvironmental regulation has an important role in governing the fate of hepatoblasts in the liver development. Hepatoblasts in the periportal region differentiated into cholangiocytes [7], while hepatoblasts in the liver parenchyma specified into mature hepatocytes [8]. Several factors such as TGFb1, 2, 3 and Jagged1 secreted from the portal mesenchymal cells are important for the differentiation of hepatoblasts into cholangiocytes [9e11]. Furthermore, our previous study revealed that TGFb signaling controlled the Jagged1expression in the periportal region through the differentiation of
* Corresponding author. Department of Hepatobiliary Surgery, First Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, China. E-mail address: [email protected] (D. Li).
portal mesenchymal cells into portal myofibroblasts, which then promotes hepatoblasts differentiation into cholangiocytes [12]. However, the role of MFs in governing the proliferation of hepatoblasts and their differentiated cholangiocytes was remains unclear. Several cytokines, such as hepatocyte growth factor (HGF), insulin-like growth factor-1 and 2 (IGF-1 and 2), midkine 1 (Mdk1), and pleiotrophin (Ptn), which are known to stimulate hepatoblasts proliferation [3,5,13e17]. Mouse embryonic fibroblasts (MEFs) are widely used as feeder cells in coculture with hepatoblasts to mimic the interaction between mesenchymal cells and hepatoblasts. Soluble factors derived from MEFs can support the growth of hepatoblasts and human induced pluripotent stem cell-derived hepatic progenitor cells in vitro [14e16]. Previous studies reported that mesenchymal cells transform into MFs after culture in vitro [3,16,18,19]. Furthermore, the transformation of mesenchymal cells into MFs is associated with a change in cytokine secretion, such as HGF and Ptn [3,19]. Therefore, it is likely that fibroblasts and MFs play a different role in regulating the proliferation of hepatoblasts. However, it remains elusive the role of fibroblasts and MFs in regulating the proliferation of hepatoblasts.
https://doi.org/10.1016/j.bbrc.2019.11.174 0006-291X/© 2019 Published by Elsevier Inc.
Please cite this article as: W. Wang et al., Myofibroblasts control the proliferation of fetal hepatoblasts and their differentiated cholangiocytes during the hepatoblast-to-cholangiocyte transition, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.174
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Abbreviations
a-SMA AFP ALB CK19 Dlk1 DPBS E HGF IGF KSR Mdk MEFs MFs Ptn qRT-PCR Sox9 TGFb
alpha smooth muscle actin a-fetoprotein albumin cytokeratin19 Delta-like 1 Dubecco’s phosphate buffered saline embryonic day hepatocyte growth factor insulinlike growth factor KnockOut™ Serum Replacement midkine mouse embryonic fibroblasts myofibroblasts pleiotrophin quantitative reversetranscriptase polymerase chain reaction SRY (sex determining region Y)- box 9 transforming growth factor b
Dubecco’s phosphate buffered saline (DPBS) complemented with 3% FBS, the dissociated cells were incubated with Phycoerythrin (PE)conjugated anti-Dlk1 antibody (MBL International, Japan). Dead cells were stained with 7-AAD (KeyGEN, China), and the DLK1þ cells were analyzed and sorted by flow cytometry FACS Vantage SE cell sorter (Becton Dickinson, CA). The isolated DLK1þ cells (5 103) were Transwell cocultured in gelatin-coated 24-well culture plates with mitomycin-C-treated feeder cells (1 105) grown on Transwell membranes with 3.0 mm pore size (Corning, USA). The fetal hepatoblasts’ medium was composed of 1:1 mixture of H-CFU-C medium (DMEM/Ham’s F12 medium (GIBCO) complemented with 15% KnockOut™ Serum Replacement (KSR, Invitrogen), nicotinamide (10 mM, Sigma-Aldrich), dexamethasone (107 M, Sigma-Aldrich), L-glutamine(2 mM, Sigma-Aldrich), HEPES (2.5 mM, SigmaAldrich), 1 InsulineTransferrineSelenium X (Invitrogen), penicillin/streptomycin (GIBCO), and nonessential amino acid solution (GIBCO)) and fresh DMEM complemented with 15% KSR. HGF (40 ng/ mL. Peprotech, UK) and epidermal growth factor (20 ng/mL, Peprotech, UK) were then added to fetal hepatoblasts’ medium.
2.4. Immunostaining
In this paper, we indirect cocultured hepatoblasts with MFs to investigate the role of MFs in controlling the proliferation of hepatoblasts and their differentiated cholangiocytes. When cocultured with hepatoblasts, MFs induced hepatoblasts differentiation into cholangiocytes and suppressed hepatoblasts proliferation. In contrast, when hepatoblasts differentiated into cholangiocytes, MFs promoted the proliferation of the differentiated cholangiocytes. These results identify a precise role of MFs in the proliferation of hepatoblasts and their differentiated cholangiocytes during the hepatoblast-to-cholangiocyte transition. 2. Materials and methods 2.1. Animals C57BL/6 mice were obtained from Chongqing Medical University. All animal experiments were approved by the Ethics Committee of Animal experiments of Chongqing Medical University.
Cultured cells were fixed using 4% paraformaldehyde for 15 min. After blocking, the cells incubated overnight with the diluted primary antibodies (Table S1) at 4 C. Then the cells incubated with corresponding second antibodies follow by staining Cell nuclei with DAPI. Thereafter, the cell samples were photographed using a fluorescence microscope (Zeiss, Germany).
2.5. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis Total RNA was extracted from cultured cells using an RNeasy Micro Kit (Qiagen, The Netherlands). First-strand cDNA was synthesized using a PrimeScript™ RT reagent kit with gDNA Eraser (TaKaRa). qRT-PCR was performed using the SYBR-® Premix Ex Taq™ II kit (TaKaRa) with a CFX96 Gradient Real-Time PCR detection system (Bio-Rad Laboratories, Hercules, CA). The relative genes expression was normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primers used in PCR are listed in Table S2.
2.2. Preparation of fibroblasts and MFs MEFs were isolated as described previously [12,20]. Briefly, Anatomy of Embryonic day (E) 14.5 mice embryos, and removal of internal organs and the head. The torso was then minced and digested with 0.125% trypsineEDTA (Sigma-Aldrich, USA) for 20 min. After neutralized with 10% FBS, the cells were cultured in a High-Glucose DMEM (Sigma Aldridge, USA), complemented with 10% FBS (GIBCO, Grand Island, NY). Passage 2 MEFs were treated with TGFb1 (5 ng/ml, R&D Systems, MN), TGFb1 (5 ng/ml) and TGFbRI inhibitor SB525334 (10 mM, Selleck Chemicals, TX), TGFbRI inhibitor SB525334 (10 mM), and DMSO for 72 h. MEFs treatment with TGFb1 or TGFbRI inhibitor SB525334 were used as MFs or control for subsequent coculture experiments. Then treating the feeder cells with mitomycin C (10 mg/ml, Cayman Chemical, MI) to halt cell proliferation.
2.6. Expression of cell surface molecules after coculture After 6 days of coculture with MFs or fibroblasts, hepatoblasts were detached from the plate and isolated by Accutase treatment. The cells were washed with DPBS supplemented with 3% FBS and incubated with PE-conjugated anti-Dlk and APC-conjugated antiCD133 antibodies (MBL International, Japan) against cell surface markers for 30 min at 4 C. Isotype control cells were stained with PE-conjugated anti-Rat IgG1 and APC-conjugated anti-Rat IgG1 antibodies (MBL International, Japan). After washing with DPBS/3% FBS, cells were analyzed using a flow cytometry FACS Vantage SE cell sorter (Becton Dickinson, CA).
2.7. Statistical analysis 2.3. Isolation and indirect coculture of fetal hepatoblasts Delta-like 1 (Dlk1) positive cells were isolated from E14.5 liver as previously described [12]. Briefly, the embryonic liver tissues were dissociated with Accutase (Invitrogen, USA). After washing with
The numerical data are presented as the means ± SD from at least three independent experiments and analyzed using two sample t-tests. P-values <0.05 were considered statistically significant.
Please cite this article as: W. Wang et al., Myofibroblasts control the proliferation of fetal hepatoblasts and their differentiated cholangiocytes during the hepatoblast-to-cholangiocyte transition, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.174
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3. Results 3.1. MFs induced by TGFb signaling inhibit the proliferation of hepatoblasts Recently studies have shown that MEF support the proliferation of the fetal hepatoblasts [15,16]. However, whether fibroblasts and MFs paly a different roles in regulating the proliferation of hepatoblasts is still unknown. Thus, the role of fibroblasts and MFs in regulating the proliferation of hepatoblasts was investigated by an indirect coculture system. As our previous study described [12], we choose recombinant TGFb1 treatment group or SB525334 treatment group as MFs or fibroblasts. The role of MFs in regulating proliferation ability of hepatoblasts were assessed by cell density, cell number and Ki67 expression. After 6 days of coculture, the cell density of hepatoblasts in MFs coculture group was significantly lower than that in fibroblasts coculture group (Fig. 1A). Next, the number of total cells, adherent cells and suspended cells were analyzed. The number of total cells was significantly lower in MFs coculture group than in fibroblasts coculture group (P < 0.05, Fig. 1B). Then, we further compared the number of adherent cells and suspended cells. Interestingly, the number of suspended cells was significantly lower in MFs coculture group than in fibroblasts coculture group (P < 0.05), but the number of adherent cells in MFs coculture group had no significant difference from the control (P＞ 0.05, Fig. 1B). Furthermore, the qPCR results demonstrated that the total cells in MFs coculture group expressed lower Ki67 and afetoprotein (AFP) than did those in fibroblasts coculture group (p < 0.05, Fig. 1C). Altogether, these data indicate that MFs inhibit the proliferation of hepatoblasts.
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3.2. MFs promote hepatoblasts differentiation and reduce the stemness of hepatoblasts Our previous studies found that MFs can drive the differentiation of hepatoblasts into cholangiocytes [12,21]. The immunostaining data showed that the adherent cells in both group expression of CK19 but almost not expression of ALB, indicated it differentiated into CK19þ ALB- cholangiocytes. Moreover, the expression of CK19 in MFs coculture group was more strong than that in control group, suggesting that MFs promote the maturation of differentiated cholangiocytes (Fig. 2B). HPCs with self-renewing potential can be highly proliferative and can form cell colonies [22e24]. Due to MFs promote the differentiation of hepatoblasts into mature cholangiocytes, we hypothesized that MFs reduce the stemness of hepatoblasts. Indeed, hepatoblasts were yielded cell colonies in fibroblasts coculture group (Fig. 2A arrows), while none of cell colonies was observed in MFs coculture group. Furthermore, we examined the expression of hepatoblast cell surface markers. Dlk1 and CD133 are hepatoblast cell surface markers derived from fetal livers [23,25,26]. The expression of Dlk1 significantly decreased in both groups (Fig. 2C). As expected, the expression of CD133 in the MFs group was lower than that in the fibroblasts group (Fig. 2C). These results suggest that MFs reduce the stemness of hepatoblasts. 3.3. MFs promoted proliferation of differentiated cholangiocytes Given that MFs reduced the proliferation of hepatoblasts, it is likely that less numbers of hepatoblasts adherent to the culture plates. Interestingly, the number of adherent cells in MFs coculture
Fig. 1. The proliferative activity of Hepatoblasts was reduced in coculture with MFs. (A) After 6 days of coculture, the density of the hepatoblasts in both groups was observed and captured with a microscope. (B) The numbers of total cells, adherent cells and suspended cells in both groups were counted. (C) qRT-PCR was performed to analyze the mRNA levels of AFP and Ki 67 in total cells. Scale bar ¼ 200 mm *P < 0.05.
Please cite this article as: W. Wang et al., Myofibroblasts control the proliferation of fetal hepatoblasts and their differentiated cholangiocytes during the hepatoblast-to-cholangiocyte transition, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.174
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Fig. 2. Regulation of stemness and differentiation of hepatoblasts cocultured with MFs. (A) Sorted Dlk-1þ cells were cocultured with mitomycin C-treated MFs or fibroblasts for 6 days. The morphological characteristics of the hepatoblasts in both groups were observed and captured with a microscope. (B) After 6 days of coculture, the expression of albumin (red) and CK19 (green) in hepatoblasts was detected. (C) Expression of cell surface molecules were analyzed after 6 days of coculture. Nuclei were counterstained with DAPI (blue). Scale bar ¼ 200 mm.
group did not vary significantly from the control (Fig. 3B). This raises the possibility that MFs promoted differentiated cholangiocytes proliferation. To validate the support role of MFs in controlling the proliferation of differentiated cholangiocytes, the proliferative ability of differentiated cholangiocytes was assessed by immunostaining for Ki-67. As expected, the percentage of Ki-67positive cells in the MFs coculture group was significantly increased from 53.47 ± 4.09% to 73.80 ± 4.94% compared with the control group (Fig. 3 A, B). Moreover, qRT-PCR data also demonstrated that the expression of Ki67 was significantly higher in MFs coculture group than in control group (Fig. 3C). Altogether, these results indicate that MFs promote the proliferation of differentiated cholangiocytes.
3.4. MFs expression of less growth factors for hepatoblasts To investigate the molecular mechanism of MFs in regulating the hepatoblasts proliferation, the expression levels of soluble factors derived from MFs and fibroblasts were analyzed. TGFb1, HGF, IGF1, IGF2, Ptn and Mdk1 were known to involve in the hepatoblasts proliferation [11,14,24,27e30]. The expression levels of HGF, IGF1, IGF2, Ptn and Mdk1 mRNAs, the key cytokines that induces hepatoblasts proliferation, were significantly lower in MFs than those in fibroblasts (Fig. 4). However, the expression levels of TGFb1, the key cytokine that inhibits hepatoblast proliferation [24], were significantly upregulated in MFs (Fig. 4). These results indicate that MFs may reduce hepatoblasts proliferation by down-
Please cite this article as: W. Wang et al., Myofibroblasts control the proliferation of fetal hepatoblasts and their differentiated cholangiocytes during the hepatoblast-to-cholangiocyte transition, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.174
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Fig. 3. MFs promote the proliferation of differentiated cholangiocytes. (A)After 6 days of coculture, the expression of Ki67 (green) in differentiated cholangiocytes was detected. Nuclei were counterstained with DAPI (blue). (B) The statistical data of the percentage of Ki-67-positive cells (n ¼ 4) in both groups were calculated. (C) qRT-PCR was performed to analyze mRNA levels of Ki67 in differentiated cholangiocytes. Scale bar ¼ 2 00 mm *P < 0.05.
Fig. 4. Expression of soluble factors related to hepatoblast proliferation in MFs and fibroblasts. The expression of TGFb1, HGF, IGF1, IGF2, Ptn and Mdk1 mRNA in MFs and fibroblasts was detected by qRT-PCR. *P < 0.05.
regulating the expression of HGF, IGF1, IGF2, Ptn and Mdk1, and upregulating the expression of TGFb1. 4. Discussion In this study, we sought to reveal the role of MFs in regulating the proliferation of hepatoblasts and their differentiated cholangiocytes during the hepatoblast-to-cholangiocyte transition. In vitro coculture analysis revealed that MFs reduced the proliferation and stemness of hepatoblasts. In contrast, when hepatoblasts differentiate into cholangiocytes, the number of Ki67-positive cholangiocytes in MFs coculture group was higher than that in fibroblasts coculture group, indicating that MFs promote the proliferation of differentiated cholangiocytes. Moreover, MFs expression
of less growth factors, such as HGF, IGF1, IGF2, Ptn and Mdk1, which are important for hepatoblasts proliferation. A recent study reported that prenatal or postnatal murine liver stromal cells as feeder cells played a different role in regulating the proliferation of hepatoblasts [31]. MEFs are widely used as feeder cells in coculture with hepatoblasts [14e16]. In addition, MEFs transform into MFs after culture in vitro [12]. However, whether active or inactive state of MEFs play a different role of in regulating the proliferation of hepatoblasts remains poorly understood. In this study, two types of feeder cells (namely, fibroblasts and MFs) derived from MEFs were used to indirectly coculture with hepatoblasts. After 6 days coculture, the total number of hepatoblasts in the MFs coculture group was significantly decreased compared with the control. Furthermore, the qRT-PCR results also demonstrated that Ki67 transcripts in MFs coculture group was significantly lower than that in fibroblasts coculture group. These results suggest MFs inhibited hepatoblasts proliferation compared with fibroblasts. Self-renewing hepatoblasts can form cell colonies in vitro culture [23,32]. Hepatoblasts proliferated quickly and formed cell colonies on day 1 and day 2. However, these cell colonies scattered in the following days [33]. Nevertheless, Cell colonies remain observed in fibroblasts coculture group after 6 days coculture, indicating that these cells possess stem cell properties. In contrast, none of cell colonies observed in MFs coculture group, indicating that MFs reduce the stemness of hepatoblasts. Dlk1 and CD133 are hepatoblast cell surface markers derived from fetal livers [23,25,26]. Consistent with a previous study [23], the expression of Dlk1 significantly decreased in both groups after culture in vitro. However, the expression of CD133 in the MFs coculture group was lower than in the fibroblasts coculture group. These results
Please cite this article as: W. Wang et al., Myofibroblasts control the proliferation of fetal hepatoblasts and their differentiated cholangiocytes during the hepatoblast-to-cholangiocyte transition, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.174
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suggested that MFs inhibit the stemness of hepatoblasts. HGF, IGF1, IGF2, Ptn and Mdk1 produced by nonparenchymal cells promote hepatoblasts proliferation [3,5,13e17]. TGFb1 were known to inhibit hepatoblasts proliferation [24]. Importantly, when fibroblasts were activated by TGFb1, the expression of HGF, IGF1, IGF2, Ptn and Mdk1 significantly decreased, but the expression of TGFb1 significantly increased. Similarly, previous studies have reported that MFs transform form fetal liver mesenchymal cells exhibited less growth factors expression [3,19]. Therefore, it is likely that the MFs inhibited the proliferation of hepatoblasts by down-regulating the expression of HGF, IGF1, IGF2, Ptn and Mdk1 and up-regulating the expression of TGFb1. Whether MFs play a diverse role in regulating the proliferation of hepatoblasts and their differentiated cholangiocytes remains unknown. A recent study provided evidence that mature cholangiocytes exhibit higher proliferation rates compared with immature cholangiocytes [34]. Our results showed that MFs induced the maturation of the differentiated cholangiocytes. Therefore, MFs is likely not only drive the differentiation of hepatoblasts to mature cholangiocytes but also promote the proliferation of differentiated cholangiocytes. Indeed, the percentage of Ki67þ cells significantly increased in MFs coculture group compared with those in the controls, suggested MFs can promote the proliferation of differentiated cholangiocytes. Interestingly, there was no significant difference in the number of adherent cells in two groups. It can be speculated that MFs reduced the proliferation of hepatoblasts, thus less numbers of hepatoblasts gave rise to cholangiocytes. It will be important to determine MFs act solely to expand the differentiated cholangiocytes population. Previous studies reported that TGFb 1 and Jagged1 promote the maturation of hepatoblasts toward cholangiocytes [9e11,28]. Our previous study found that TGFb signaling upregulated Jagged1 expression in fibroblasts [12]. The results showed that TGFb1 expression was significantly increased in MFs. Thus, it is likely that MFs promote differentiated cholangiocytes maturation through the upregulation of TGFb1 and Jagged1. However, the molecular mechanisms promoted the proliferation of differentiated cholangiocytes are unknown and await further investigation. In summary, our data demonstrate that MFs reduced the proliferation and stemness of hepatoblasts with increased biliary differentiation, but promote the proliferation of their differentiated cholangiocytes. Our observations elucidate the role of MFs in regulating the proliferation of hepatoblasts and their differentiated cholangiocytes, and these findings might ultimately contribute to cholangiocyte regeneration and chronic liver disease therapies. Declaration of competing interest The authors declare that they have no competing interests. Acknowledgements We would like to thank the support of research grants from the General Program of National Natural Science Foundation of China (Grant No.81470898). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.174. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.174.
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Please cite this article as: W. Wang et al., Myofibroblasts control the proliferation of fetal hepatoblasts and their differentiated cholangiocytes during the hepatoblast-to-cholangiocyte transition, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.174
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Myofibroblasts control the proliferation of fetal hepatoblasts and their differentiated cholangiocytes during the hepatoblast-to-cholangiocyte transition Wei Wang, Li Wan, Zhixin Chen, Xin Jin, Dewei Li* Department of Hepatobiliary Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 October 2019 Accepted 26 November 2019 Available online xxx
Mesenchymal cells in the liver provide the microenvironment for hepatoblasts expansion and differentiation. We have previously demonstrated that myofibroblasts (MFs) promoted hepatoblasts differentiation into cholangiocytes, whereas its role in controlling the proliferation of hepatoblasts and their differentiated cholangiocytes remains elusive. Here, we investigated the role of MFs in regulating the proliferation of hepatoblasts and their differentiated cholangiocytes using an indirect coculture system. When cocultured with hepatoblasts, MFs promoted hepatoblasts differentiation into cholangiocytes and inhibited the proliferation and stemness of hepatoblasts. However, when hepatoblasts already differentiated into cholangiocytes, MFs promoted the differentiated cholangiocytes proliferation. In addition, hepatoblast proliferation genes such as hepatocyte growth factor (HGF), insulin-like growth factor-1 and 2 (IGF-1 and 2), midkine 1 (Mdk1), and pleiotrophin (Ptn) expression in MFs were down-regulated compared with their levels in fibroblasts. Our findings uncover the role of MFs in controlling the proliferation of hepatoblasts and their differentiated cholangiocytes, potentially providing a novel therapeutic strategy for cholangiocyte regeneration. © 2019 Published by Elsevier Inc.
Keywords: Myofibroblasts Hepatoblasts Cholangiocytes Proliferation Differentiation
1. Introduction During hepatogenesis, fetal hepatoblasts, developed from foregut endodermal cells, can quickly proliferation and differentiate bipotentially into either hepatocytes or cholangiocytes [1,2]. The interaction with nonparenchymal cell, such as mesenchymal cells, stellate cells, and mesothelial cells, is important for hepatoblasts proliferation and differentiation [3e6]. Microenvironmental regulation has an important role in governing the fate of hepatoblasts in the liver development. Hepatoblasts in the periportal region differentiated into cholangiocytes [7], while hepatoblasts in the liver parenchyma specified into mature hepatocytes [8]. Several factors such as TGFb1, 2, 3 and Jagged1 secreted from the portal mesenchymal cells are important for the differentiation of hepatoblasts into cholangiocytes [9e11]. Furthermore, our previous study revealed that TGFb signaling controlled the Jagged1expression in the periportal region through the differentiation of
* Corresponding author. Department of Hepatobiliary Surgery, First Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, China. E-mail address: [email protected] (D. Li).
portal mesenchymal cells into portal myofibroblasts, which then promotes hepatoblasts differentiation into cholangiocytes [12]. However, the role of MFs in governing the proliferation of hepatoblasts and their differentiated cholangiocytes was remains unclear. Several cytokines, such as hepatocyte growth factor (HGF), insulin-like growth factor-1 and 2 (IGF-1 and 2), midkine 1 (Mdk1), and pleiotrophin (Ptn), which are known to stimulate hepatoblasts proliferation [3,5,13e17]. Mouse embryonic fibroblasts (MEFs) are widely used as feeder cells in coculture with hepatoblasts to mimic the interaction between mesenchymal cells and hepatoblasts. Soluble factors derived from MEFs can support the growth of hepatoblasts and human induced pluripotent stem cell-derived hepatic progenitor cells in vitro [14e16]. Previous studies reported that mesenchymal cells transform into MFs after culture in vitro [3,16,18,19]. Furthermore, the transformation of mesenchymal cells into MFs is associated with a change in cytokine secretion, such as HGF and Ptn [3,19]. Therefore, it is likely that fibroblasts and MFs play a different role in regulating the proliferation of hepatoblasts. However, it remains elusive the role of fibroblasts and MFs in regulating the proliferation of hepatoblasts.
https://doi.org/10.1016/j.bbrc.2019.11.174 0006-291X/© 2019 Published by Elsevier Inc.
Please cite this article as: W. Wang et al., Myofibroblasts control the proliferation of fetal hepatoblasts and their differentiated cholangiocytes during the hepatoblast-to-cholangiocyte transition, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.174
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Abbreviations
a-SMA AFP ALB CK19 Dlk1 DPBS E HGF IGF KSR Mdk MEFs MFs Ptn qRT-PCR Sox9 TGFb
alpha smooth muscle actin a-fetoprotein albumin cytokeratin19 Delta-like 1 Dubecco’s phosphate buffered saline embryonic day hepatocyte growth factor insulinlike growth factor KnockOut™ Serum Replacement midkine mouse embryonic fibroblasts myofibroblasts pleiotrophin quantitative reversetranscriptase polymerase chain reaction SRY (sex determining region Y)- box 9 transforming growth factor b
Dubecco’s phosphate buffered saline (DPBS) complemented with 3% FBS, the dissociated cells were incubated with Phycoerythrin (PE)conjugated anti-Dlk1 antibody (MBL International, Japan). Dead cells were stained with 7-AAD (KeyGEN, China), and the DLK1þ cells were analyzed and sorted by flow cytometry FACS Vantage SE cell sorter (Becton Dickinson, CA). The isolated DLK1þ cells (5 103) were Transwell cocultured in gelatin-coated 24-well culture plates with mitomycin-C-treated feeder cells (1 105) grown on Transwell membranes with 3.0 mm pore size (Corning, USA). The fetal hepatoblasts’ medium was composed of 1:1 mixture of H-CFU-C medium (DMEM/Ham’s F12 medium (GIBCO) complemented with 15% KnockOut™ Serum Replacement (KSR, Invitrogen), nicotinamide (10 mM, Sigma-Aldrich), dexamethasone (107 M, Sigma-Aldrich), L-glutamine(2 mM, Sigma-Aldrich), HEPES (2.5 mM, SigmaAldrich), 1 InsulineTransferrineSelenium X (Invitrogen), penicillin/streptomycin (GIBCO), and nonessential amino acid solution (GIBCO)) and fresh DMEM complemented with 15% KSR. HGF (40 ng/ mL. Peprotech, UK) and epidermal growth factor (20 ng/mL, Peprotech, UK) were then added to fetal hepatoblasts’ medium.
2.4. Immunostaining
In this paper, we indirect cocultured hepatoblasts with MFs to investigate the role of MFs in controlling the proliferation of hepatoblasts and their differentiated cholangiocytes. When cocultured with hepatoblasts, MFs induced hepatoblasts differentiation into cholangiocytes and suppressed hepatoblasts proliferation. In contrast, when hepatoblasts differentiated into cholangiocytes, MFs promoted the proliferation of the differentiated cholangiocytes. These results identify a precise role of MFs in the proliferation of hepatoblasts and their differentiated cholangiocytes during the hepatoblast-to-cholangiocyte transition. 2. Materials and methods 2.1. Animals C57BL/6 mice were obtained from Chongqing Medical University. All animal experiments were approved by the Ethics Committee of Animal experiments of Chongqing Medical University.
Cultured cells were fixed using 4% paraformaldehyde for 15 min. After blocking, the cells incubated overnight with the diluted primary antibodies (Table S1) at 4 C. Then the cells incubated with corresponding second antibodies follow by staining Cell nuclei with DAPI. Thereafter, the cell samples were photographed using a fluorescence microscope (Zeiss, Germany).
2.5. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis Total RNA was extracted from cultured cells using an RNeasy Micro Kit (Qiagen, The Netherlands). First-strand cDNA was synthesized using a PrimeScript™ RT reagent kit with gDNA Eraser (TaKaRa). qRT-PCR was performed using the SYBR-® Premix Ex Taq™ II kit (TaKaRa) with a CFX96 Gradient Real-Time PCR detection system (Bio-Rad Laboratories, Hercules, CA). The relative genes expression was normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primers used in PCR are listed in Table S2.
2.2. Preparation of fibroblasts and MFs MEFs were isolated as described previously [12,20]. Briefly, Anatomy of Embryonic day (E) 14.5 mice embryos, and removal of internal organs and the head. The torso was then minced and digested with 0.125% trypsineEDTA (Sigma-Aldrich, USA) for 20 min. After neutralized with 10% FBS, the cells were cultured in a High-Glucose DMEM (Sigma Aldridge, USA), complemented with 10% FBS (GIBCO, Grand Island, NY). Passage 2 MEFs were treated with TGFb1 (5 ng/ml, R&D Systems, MN), TGFb1 (5 ng/ml) and TGFbRI inhibitor SB525334 (10 mM, Selleck Chemicals, TX), TGFbRI inhibitor SB525334 (10 mM), and DMSO for 72 h. MEFs treatment with TGFb1 or TGFbRI inhibitor SB525334 were used as MFs or control for subsequent coculture experiments. Then treating the feeder cells with mitomycin C (10 mg/ml, Cayman Chemical, MI) to halt cell proliferation.
2.6. Expression of cell surface molecules after coculture After 6 days of coculture with MFs or fibroblasts, hepatoblasts were detached from the plate and isolated by Accutase treatment. The cells were washed with DPBS supplemented with 3% FBS and incubated with PE-conjugated anti-Dlk and APC-conjugated antiCD133 antibodies (MBL International, Japan) against cell surface markers for 30 min at 4 C. Isotype control cells were stained with PE-conjugated anti-Rat IgG1 and APC-conjugated anti-Rat IgG1 antibodies (MBL International, Japan). After washing with DPBS/3% FBS, cells were analyzed using a flow cytometry FACS Vantage SE cell sorter (Becton Dickinson, CA).
2.7. Statistical analysis 2.3. Isolation and indirect coculture of fetal hepatoblasts Delta-like 1 (Dlk1) positive cells were isolated from E14.5 liver as previously described [12]. Briefly, the embryonic liver tissues were dissociated with Accutase (Invitrogen, USA). After washing with
The numerical data are presented as the means ± SD from at least three independent experiments and analyzed using two sample t-tests. P-values <0.05 were considered statistically significant.
Please cite this article as: W. Wang et al., Myofibroblasts control the proliferation of fetal hepatoblasts and their differentiated cholangiocytes during the hepatoblast-to-cholangiocyte transition, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.174
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3. Results 3.1. MFs induced by TGFb signaling inhibit the proliferation of hepatoblasts Recently studies have shown that MEF support the proliferation of the fetal hepatoblasts [15,16]. However, whether fibroblasts and MFs paly a different roles in regulating the proliferation of hepatoblasts is still unknown. Thus, the role of fibroblasts and MFs in regulating the proliferation of hepatoblasts was investigated by an indirect coculture system. As our previous study described [12], we choose recombinant TGFb1 treatment group or SB525334 treatment group as MFs or fibroblasts. The role of MFs in regulating proliferation ability of hepatoblasts were assessed by cell density, cell number and Ki67 expression. After 6 days of coculture, the cell density of hepatoblasts in MFs coculture group was significantly lower than that in fibroblasts coculture group (Fig. 1A). Next, the number of total cells, adherent cells and suspended cells were analyzed. The number of total cells was significantly lower in MFs coculture group than in fibroblasts coculture group (P < 0.05, Fig. 1B). Then, we further compared the number of adherent cells and suspended cells. Interestingly, the number of suspended cells was significantly lower in MFs coculture group than in fibroblasts coculture group (P < 0.05), but the number of adherent cells in MFs coculture group had no significant difference from the control (P＞ 0.05, Fig. 1B). Furthermore, the qPCR results demonstrated that the total cells in MFs coculture group expressed lower Ki67 and afetoprotein (AFP) than did those in fibroblasts coculture group (p < 0.05, Fig. 1C). Altogether, these data indicate that MFs inhibit the proliferation of hepatoblasts.
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3.2. MFs promote hepatoblasts differentiation and reduce the stemness of hepatoblasts Our previous studies found that MFs can drive the differentiation of hepatoblasts into cholangiocytes [12,21]. The immunostaining data showed that the adherent cells in both group expression of CK19 but almost not expression of ALB, indicated it differentiated into CK19þ ALB- cholangiocytes. Moreover, the expression of CK19 in MFs coculture group was more strong than that in control group, suggesting that MFs promote the maturation of differentiated cholangiocytes (Fig. 2B). HPCs with self-renewing potential can be highly proliferative and can form cell colonies [22e24]. Due to MFs promote the differentiation of hepatoblasts into mature cholangiocytes, we hypothesized that MFs reduce the stemness of hepatoblasts. Indeed, hepatoblasts were yielded cell colonies in fibroblasts coculture group (Fig. 2A arrows), while none of cell colonies was observed in MFs coculture group. Furthermore, we examined the expression of hepatoblast cell surface markers. Dlk1 and CD133 are hepatoblast cell surface markers derived from fetal livers [23,25,26]. The expression of Dlk1 significantly decreased in both groups (Fig. 2C). As expected, the expression of CD133 in the MFs group was lower than that in the fibroblasts group (Fig. 2C). These results suggest that MFs reduce the stemness of hepatoblasts. 3.3. MFs promoted proliferation of differentiated cholangiocytes Given that MFs reduced the proliferation of hepatoblasts, it is likely that less numbers of hepatoblasts adherent to the culture plates. Interestingly, the number of adherent cells in MFs coculture
Fig. 1. The proliferative activity of Hepatoblasts was reduced in coculture with MFs. (A) After 6 days of coculture, the density of the hepatoblasts in both groups was observed and captured with a microscope. (B) The numbers of total cells, adherent cells and suspended cells in both groups were counted. (C) qRT-PCR was performed to analyze the mRNA levels of AFP and Ki 67 in total cells. Scale bar ¼ 200 mm *P < 0.05.
Please cite this article as: W. Wang et al., Myofibroblasts control the proliferation of fetal hepatoblasts and their differentiated cholangiocytes during the hepatoblast-to-cholangiocyte transition, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.174
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Fig. 2. Regulation of stemness and differentiation of hepatoblasts cocultured with MFs. (A) Sorted Dlk-1þ cells were cocultured with mitomycin C-treated MFs or fibroblasts for 6 days. The morphological characteristics of the hepatoblasts in both groups were observed and captured with a microscope. (B) After 6 days of coculture, the expression of albumin (red) and CK19 (green) in hepatoblasts was detected. (C) Expression of cell surface molecules were analyzed after 6 days of coculture. Nuclei were counterstained with DAPI (blue). Scale bar ¼ 200 mm.
group did not vary significantly from the control (Fig. 3B). This raises the possibility that MFs promoted differentiated cholangiocytes proliferation. To validate the support role of MFs in controlling the proliferation of differentiated cholangiocytes, the proliferative ability of differentiated cholangiocytes was assessed by immunostaining for Ki-67. As expected, the percentage of Ki-67positive cells in the MFs coculture group was significantly increased from 53.47 ± 4.09% to 73.80 ± 4.94% compared with the control group (Fig. 3 A, B). Moreover, qRT-PCR data also demonstrated that the expression of Ki67 was significantly higher in MFs coculture group than in control group (Fig. 3C). Altogether, these results indicate that MFs promote the proliferation of differentiated cholangiocytes.
3.4. MFs expression of less growth factors for hepatoblasts To investigate the molecular mechanism of MFs in regulating the hepatoblasts proliferation, the expression levels of soluble factors derived from MFs and fibroblasts were analyzed. TGFb1, HGF, IGF1, IGF2, Ptn and Mdk1 were known to involve in the hepatoblasts proliferation [11,14,24,27e30]. The expression levels of HGF, IGF1, IGF2, Ptn and Mdk1 mRNAs, the key cytokines that induces hepatoblasts proliferation, were significantly lower in MFs than those in fibroblasts (Fig. 4). However, the expression levels of TGFb1, the key cytokine that inhibits hepatoblast proliferation [24], were significantly upregulated in MFs (Fig. 4). These results indicate that MFs may reduce hepatoblasts proliferation by down-
Please cite this article as: W. Wang et al., Myofibroblasts control the proliferation of fetal hepatoblasts and their differentiated cholangiocytes during the hepatoblast-to-cholangiocyte transition, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.174
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Fig. 3. MFs promote the proliferation of differentiated cholangiocytes. (A)After 6 days of coculture, the expression of Ki67 (green) in differentiated cholangiocytes was detected. Nuclei were counterstained with DAPI (blue). (B) The statistical data of the percentage of Ki-67-positive cells (n ¼ 4) in both groups were calculated. (C) qRT-PCR was performed to analyze mRNA levels of Ki67 in differentiated cholangiocytes. Scale bar ¼ 2 00 mm *P < 0.05.
Fig. 4. Expression of soluble factors related to hepatoblast proliferation in MFs and fibroblasts. The expression of TGFb1, HGF, IGF1, IGF2, Ptn and Mdk1 mRNA in MFs and fibroblasts was detected by qRT-PCR. *P < 0.05.
regulating the expression of HGF, IGF1, IGF2, Ptn and Mdk1, and upregulating the expression of TGFb1. 4. Discussion In this study, we sought to reveal the role of MFs in regulating the proliferation of hepatoblasts and their differentiated cholangiocytes during the hepatoblast-to-cholangiocyte transition. In vitro coculture analysis revealed that MFs reduced the proliferation and stemness of hepatoblasts. In contrast, when hepatoblasts differentiate into cholangiocytes, the number of Ki67-positive cholangiocytes in MFs coculture group was higher than that in fibroblasts coculture group, indicating that MFs promote the proliferation of differentiated cholangiocytes. Moreover, MFs expression
of less growth factors, such as HGF, IGF1, IGF2, Ptn and Mdk1, which are important for hepatoblasts proliferation. A recent study reported that prenatal or postnatal murine liver stromal cells as feeder cells played a different role in regulating the proliferation of hepatoblasts [31]. MEFs are widely used as feeder cells in coculture with hepatoblasts [14e16]. In addition, MEFs transform into MFs after culture in vitro [12]. However, whether active or inactive state of MEFs play a different role of in regulating the proliferation of hepatoblasts remains poorly understood. In this study, two types of feeder cells (namely, fibroblasts and MFs) derived from MEFs were used to indirectly coculture with hepatoblasts. After 6 days coculture, the total number of hepatoblasts in the MFs coculture group was significantly decreased compared with the control. Furthermore, the qRT-PCR results also demonstrated that Ki67 transcripts in MFs coculture group was significantly lower than that in fibroblasts coculture group. These results suggest MFs inhibited hepatoblasts proliferation compared with fibroblasts. Self-renewing hepatoblasts can form cell colonies in vitro culture [23,32]. Hepatoblasts proliferated quickly and formed cell colonies on day 1 and day 2. However, these cell colonies scattered in the following days [33]. Nevertheless, Cell colonies remain observed in fibroblasts coculture group after 6 days coculture, indicating that these cells possess stem cell properties. In contrast, none of cell colonies observed in MFs coculture group, indicating that MFs reduce the stemness of hepatoblasts. Dlk1 and CD133 are hepatoblast cell surface markers derived from fetal livers [23,25,26]. Consistent with a previous study [23], the expression of Dlk1 significantly decreased in both groups after culture in vitro. However, the expression of CD133 in the MFs coculture group was lower than in the fibroblasts coculture group. These results
Please cite this article as: W. Wang et al., Myofibroblasts control the proliferation of fetal hepatoblasts and their differentiated cholangiocytes during the hepatoblast-to-cholangiocyte transition, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.174
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suggested that MFs inhibit the stemness of hepatoblasts. HGF, IGF1, IGF2, Ptn and Mdk1 produced by nonparenchymal cells promote hepatoblasts proliferation [3,5,13e17]. TGFb1 were known to inhibit hepatoblasts proliferation [24]. Importantly, when fibroblasts were activated by TGFb1, the expression of HGF, IGF1, IGF2, Ptn and Mdk1 significantly decreased, but the expression of TGFb1 significantly increased. Similarly, previous studies have reported that MFs transform form fetal liver mesenchymal cells exhibited less growth factors expression [3,19]. Therefore, it is likely that the MFs inhibited the proliferation of hepatoblasts by down-regulating the expression of HGF, IGF1, IGF2, Ptn and Mdk1 and up-regulating the expression of TGFb1. Whether MFs play a diverse role in regulating the proliferation of hepatoblasts and their differentiated cholangiocytes remains unknown. A recent study provided evidence that mature cholangiocytes exhibit higher proliferation rates compared with immature cholangiocytes [34]. Our results showed that MFs induced the maturation of the differentiated cholangiocytes. Therefore, MFs is likely not only drive the differentiation of hepatoblasts to mature cholangiocytes but also promote the proliferation of differentiated cholangiocytes. Indeed, the percentage of Ki67þ cells significantly increased in MFs coculture group compared with those in the controls, suggested MFs can promote the proliferation of differentiated cholangiocytes. Interestingly, there was no significant difference in the number of adherent cells in two groups. It can be speculated that MFs reduced the proliferation of hepatoblasts, thus less numbers of hepatoblasts gave rise to cholangiocytes. It will be important to determine MFs act solely to expand the differentiated cholangiocytes population. Previous studies reported that TGFb 1 and Jagged1 promote the maturation of hepatoblasts toward cholangiocytes [9e11,28]. Our previous study found that TGFb signaling upregulated Jagged1 expression in fibroblasts [12]. The results showed that TGFb1 expression was significantly increased in MFs. Thus, it is likely that MFs promote differentiated cholangiocytes maturation through the upregulation of TGFb1 and Jagged1. However, the molecular mechanisms promoted the proliferation of differentiated cholangiocytes are unknown and await further investigation. In summary, our data demonstrate that MFs reduced the proliferation and stemness of hepatoblasts with increased biliary differentiation, but promote the proliferation of their differentiated cholangiocytes. Our observations elucidate the role of MFs in regulating the proliferation of hepatoblasts and their differentiated cholangiocytes, and these findings might ultimately contribute to cholangiocyte regeneration and chronic liver disease therapies. Declaration of competing interest The authors declare that they have no competing interests. Acknowledgements We would like to thank the support of research grants from the General Program of National Natural Science Foundation of China (Grant No.81470898). Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.174. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.174.
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Please cite this article as: W. Wang et al., Myofibroblasts control the proliferation of fetal hepatoblasts and their differentiated cholangiocytes during the hepatoblast-to-cholangiocyte transition, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/ j.bbrc.2019.11.174
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