Hepatic differentiation

Results from clinical studies suggested that bone marrow-derived stem cells can engraft into liver tissue and differentiate towards hepatocytes (Theise, 2000). In order to identify the potential cell type, purified HSC and expanded MSC were tested in vivo. Lagasse et al showed that HSC can differentiate into hepatocytes in vivo, but the underlying mechanism was identified as cellular fusion rather than differentiation (Lagasse, 2000; Wang, 2003b). Then, MSC derived from umbilical cord blood (Lee, 2004a), umbilical cord matrix (Campard, 2008), placenta (Chien, 2006), bone marrow (Aurich, 2007), and fat tissue (Banas, 2007), have been tested in vitro and in vivo.

Knowledge of transcription regulation of embryonic development of the liver and hepatocytes specification within embryonic and fetal liver tissue has been used to design hepatogenic differentiation protocols (Lemaigre, 2004).

First positive results were reported by Jiang et al and Schwartz et al. They applied FGF-4 and HGF to mouse and human MAPC cultured on Matrigel (extra-cellular matrix proteins) (Jiang, 2002; Schwartz, 2002). Differentiated MAPC showed expression of early and late hepatic transcription factors (HNF1alpha, HNF3beta/Foxa2, Gata-4), hepatic proteins (albumin, CK18), and metabolic functions (Cytochrome P450 activity, Urea and albumin production, glycogen production and storage). These protocols were applied to MSC with variable success. Kakinuma et al used UCB-derived MSC and applied FGF-1, FGF-2 (basic FGF), Leukemia inhibitory factor (Cano), HGF, Stem cell factor (SCF) and oncostatin M for 21 days. RT-PCR and immunohistochemistry analysis demonstrated expression of albumin, CK18 and CK19 (Kakinuma, 2003). In vitro, cell morphology of spindle-shaped MSC changed to

round-shaped and epitheloid cells. Undifferentiated MSC engrafted in the liver of SCID mice after partial hepatectomy and differentiated into hepatocytes. Cells survived for 55 weeks with persistent albumin secretion.

Later, Lee et al showed that clonal-derived human MSC isolated from UCB and from bone marrow aspirates were both able to differentiate into hepatocytes (Lee, 2004a;

Lee, 2004b). They applied a two step protocol after serum deprivation for 48 hours.

During the first step, hepatic induction was obtained by medium containing basic FGF, HGF and nicotinamide. After 7 days of induction, final differentiation was obtained using a medium containing oncostatin M and dexamethasone for up to 42 days. Both, UCB-derived and bone marrow derived cells significantly changed morphology during maturation, harboring a cuboid-like, and epitheloid morphology.

Differentiated cells expressed alpha-fetoprotein, albumin, and cytochrome P450, at RNA and protein levels. Urea production was induced and increased with differentiation time, similar to glycogen storage capacity.

Others described protocols using MSC derived from various tissues, such as fat tissue of patients undergoing liposuction (age 38-49 years) (Talens-Visconti, 2007) or abdominal surgery (age 36-55 years; 19-55 years) (Banas, 2007; Seo, 2005), from bone marrow of voluntary donors (Aurich, 2007) or from elderly patients (60-69 years) (Talens-Visconti, 2006), from mouse and rat bone marrow (Chen, 2007; Kang, 2005; Ke, 2008; Li, 2006; Shi, 2008; Wang, 2004b), from human placenta (Chien, 2006), from human umbilical cord matrix (Campard, 2008) or even from adult human liver tissue (Najimi, 2007). MSC were cultured on fibronectin (Chien, 2006; Seo, 2005), collagen (Banas, 2007; Campard, 2008), poly-L-Lysine (Chien, 2006), or extra-cellular matrix (Kang, 2005) coated dishes. In several studies, cells were pre-treated cells with serum deprivation for 24-48 hours (Campard, 2008; Lee, 2004a;

Talens-Visconti, 2006). Media containing either 5-Aza (Aurich, 2007), or cocktails of growth factors, with basic FGF (Campard, 2008; Shi, 2008), FGF-1 (Banas, 2007), FGF-4 (Banas, 2007; Chien, 2006; Kang, 2005; Talens-Visconti, 2006), epidermal growth factor (EGF) (Aurich, 2007; Campard, 2008; Talens-Visconti, 2006) , HGF (Aurich, 2007; Banas, 2007; Campard, 2008; Chien, 2006; Kang, 2005; Li, 2006;

Seo, 2005; Talens-Visconti, 2006), Oncostatin M (Campard, 2008; Seo, 2005; Shi, 2008; Talens-Visconti, 2006) , and nicotinamide (Campard, 2008; Talens-Visconti, 2006), all at variable concentrations. Expression of albumin and other hepatic markers was detected at RNA and/or protein levels in all studies cited above, several

also demonstrating hepatocyte metabolic activities, such as urea production (Banas, 2007; Seo, 2005) or glycogen storage (Aurich, 2007; Banas, 2007; Campard, 2008).

However, few studies showed negative results using only growth factors (Lange, 2005; Lange, 2006; Snykers, 2007). Lange et al showed that rat bone marrow-derived MSC when cultured in medium containing HGF, SCF, FGF-4 and EGF did not differentiate into hepatocyte-like cells (Lange, 2005). When co-cultured with adult primary hepatocytes (Lange, 2005) or with primary fetal liver cells (Lange, 2006) in differentiation medium, MSC expressed hepatocyte specific genes. Snykers et al studied the importance of sequential application of growth factors: when all growth factors were applied at the same time, no differentiation occurred, but when growth factors were applied sequentially, using a time sequence similar to embryonic development, CK18 was expressed and glycogen stored by MSC (Snykers, 2007). In the same study, when trichostatin A, a histone deacetylase inhibitor, known to inhibit proliferation and induce differentiation in primary hepatocytes (Herold, 2002;

Vanhaecke, 2004), was added to culture and growth factors were applied sequentially, MSC acquired an epitheloid morphology. These cells then expressed HNF-3beta, alpha-fetoprotein, CK18, albumin, and demonstrated urea production and inducible cytochrome P450 activity (Snykers, 2007).

Serum from patients with cholestasis or medium conditioned with primary hepatocyte cultures induced expression of hepatic markers in MSC (Chen, 2007; Li, 2006). The responsible hepatogenic factors were not identified. Ke et al showed that hepatic cellular extracts could induce expression of hepatic markers in MSC (Ke, 2008). They also showed that mRNA of factors implicated in the Wnt signaling pathway were lower in differentiated MSC. Furthermore, supplementation of culture medium with dickkopf-1 (DKK-1), a Wnt-1 inhibitor, induced earlier expression of albumin in MSC (Ke, 2008). Thus, differentiation of MSC into albumin expressing cells is dependent on Wnt inhibition, as it is the case for early embryonic endodermal specification (McLin, 2007).

To investigate the potential of MSC as cell therapy for hepatocyte replacement, in vivo studies in animal models with liver diseases or injury are required. The major working hypothesis of transplanting MSC within a damaged liver is that local secretion of growth factors and injured environment stimulate MSC to differentiate into hepatocytes and thereby repopulate the injured organ. Kakinuma et al transplanted human UCB cells without selection for MSC into the portal vein of severe combined immunodeficient mice (SCID), after treatment of

2-acetylaminofluorene and 30% hepatectomy (Kakinuma, 2003). They observed engraftment of UCB-derived cells with a functional phenotype and human albumin within sera of mice, up to 55 weeks after transplantation (Kakinuma, 2003). HSC engraftment into liver after transplantation was already demonstrated at that time (Lagasse, 2000). Seo et al induced liver damage with carbon tetrachloride (CCl4) and injected differentiated and undifferentiated MSC into the tail vein of SCID mice (Seo, 2005). Analysis of liver tissue at 10 days after transplantation demonstrated that undifferentiated MSC engrafted as efficiently as pre-differentiated MSC within injured livers. Despite the high number of injected cells, engraftment was low as quantification of human cells within mouse recipient liver parenchyma showed only 0.32% of human undifferentiated cells at 3 days and 0.13% of human albumin-expressing cells at 10 days (Seo, 2005). Aurich et al transplanted EGF-HGF- pre-differentiated human bone marrow-derived MSC into the spleen of immunodeficient mice, which were previously subjected to partial hepatectomy and to inhibition of endogenous liver regeneration (Aurich, 2007). Cells engrafted predominantly in the periportal regions. Glycogen storage was detected, suggesting functional activity of engrafted cells. Cell fusion between transplanted MSC and endogenous hepatocytes was not detected (Aurich, 2007). Systemic injection of pre-differentiated adipose-tissue-derived MSC selected for CD105 (Banas, 2007), or bone marrow-derived MSC (Oyagi, 2006) into immunodeficient mice after CCl4 induced liver injury, induced increased albumin secretion after transplantation, compared to transplantation of undifferentiated cells. Oyagi et al measured higher albumin concentration in serum after transplantation of predifferentiated MSC, suggesting either a direct or a trophic effect of MSC on injured liver tissue.

MSC from various sources, such as umbilical cord matrix (Campard, 2008), human bone marrow (Lysy, 2008), human liver (Najimi, 2007), as well as human skin fibroblasts (Lysy, 2007), were transplanted into immuno-deficient mice after partial hepatectomy, or retrorsine–allylalcohol treatment. Engraftment and differentiation into hepatocytes, with expression of alpha-fetoprotein, albumin, and/or CK18, were detected, independently of administration route. Therefore, MSC and even human skin fibroblasts can engraft and differentiate towards hepatocyte-like cells (Campard, 2008; Lysy, 2008; Lysy, 2007). It appears, however, that despite high numbers of injected cells (500’000 to 1 million), few cells could be detected, questioning the possible clinical use and function in liver failure (Campard, 2008; Lysy, 2008; Lysy, 2007; Najimi, 2007).

Popp et al compared efficiency of liver engraftment between rat MSC and rat hepatocytes after intraportal or intra-parenchymal injection (Popp, 2007). They used injection of CCl4 or allylalcohol, and retrorsine treatment, to induce liver injury and inhibit endogenous, regeneration respectively Rat hepatocytes engrafted and even proliferated within livers after transplantation. However, rat MSC did not repopulate liver parenchyma and could not be recovered at 2 days, 2 weeks or 2 months after transplantation, despite demonstration of their transitory presence immediately after transplantation. These negative results in a syngeneic model raised some doubts about in vivo differentiation potential of undifferentiated MSC.

Di Bonzo et al transplanted human bone marrow-derived MSC into the tail vein of mice suffering from either acute or chronic liver injury (di Bonzo, 2008). They observed engraftment especially in the chronic liver injury model, but with few cells differentiating into hepatocytes. Importantly, they demonstrated differentiation of MSC into myofibroblasts, showing a pro-fibrogenic potential of MSC within liver parenchyma. Hence, in the presence of liver injury, MSC from the bone marrow may favor scar formation, instead of contributing to organ recovery (di Bonzo, 2008).

However, fibrogenic potential of MSC transplanted into liver parenchyma remains controversial. In rats suffering from CCl4- or dimethylnitrosamine-induced liver fibrosis, MSC transplantation reduced fibrosis and decreased collagen deposist, by mechanisms which remain unknown (Zhao, 2005a).

In summary, MSC and even human skin-derived fibroblasts may differentiate into hepatocytes both in vitro and in vivo, but quantification was performed only in few studies. In contrast, well-conducted, controlled experiments questioned this hepatogenic differentiation potential, both in vitro and in vivo, making it difficult to draw a definitive conclusion. Finally, numbers of differentiated cells after transplantation seem low in most studies, implying further improvement of cell-isolation and differentiation protocols, in order to apply this stem cell technology to clinical settings of liver failure.