**2. Embryonic liver development**

During embryonic development, the liver arises from the definitive endoderm (for reviews on liver development, see (7-9)). The definitive endoderm is an embryonic layer, whereas visceral endoderm is a non-embryonic derived layer, also called extra-embryonic endoderm. The definitive endoderm is one of the three germ layers, which include also ectoderm and mesoderm. The definitive endoderm is initially located beneath the ectoderm and mesoderm. In the mouse, the definitive endoderm layer forms a liver bud between E8.5 and E9.5. This layer will also form the pancreas, lung, stomach, intestine, and thyroid. The cardiac mesoderm and septum transversum mesenchyme release signals, such as fibroblast growth factors (FGF) and bone morphogenetic proteins (BMP), which are necessary to induce liver specification. The septum transversum mesenchyme has been implicated to give rise to stellate cells (also called Ito cells), which are fat and vitamin A-storing and extracellular matrix producing liver cells (10); cells positive for the Lim-homeobox gene (Lhx2) migrate from the septum transversum into the forming liver bud and become desmin and Lhx2 positive stellate cells. Cells in the developing liver bud are termed hepatoblasts and express alpha-fetoprotein (AFP). Hepatoblasts have been described as bipotential progenitors, developing into mature hepatocytes as well as bile duct epithelial cells (cholangiocytes), based on findings from *ex vivo* and *in vitro* studies (11-15). Suppression of transcription factor CCAAT-enhancer-binding protein alpha (CEBPα) has been suggested to induce their specification towards biliary differentiation (16, 17).

#### **3. Human hepatic progenitors in fetal and adult livers**

Different hepatic progenitors in human livers have been described. Based on early findings in developmental biology, hepatic stem cells were originally defined as AFP positive hepatoblasts. More recent research, however, reveals that hepatic stem cells are AFP negative and are the precursors to hepatoblasts (12, 18). Furthermore, stem cells of assumed mesendodermal origin capable of multilineage differentiation towards liver- and mesenchymal lineages have been discovered (19). An overview about human hepatic progenitors that have been isolated and characterized is given in **Table 2**.

Stem cell Cell, which is capable to differentiate into multiple lineages and is

Hepatoblast Hepatic parenchymal cell of the fetal liver. Defined by its

Oval cell Small cells with oval-shaped nuclei that emerge in livers, which have been treated with certain toxins. Table 1. Common terminology relevant to liver progenitor biology. Further details can be

During embryonic development, the liver arises from the definitive endoderm (for reviews on liver development, see (7-9)). The definitive endoderm is an embryonic layer, whereas visceral endoderm is a non-embryonic derived layer, also called extra-embryonic endoderm. The definitive endoderm is one of the three germ layers, which include also ectoderm and mesoderm. The definitive endoderm is initially located beneath the ectoderm and mesoderm. In the mouse, the definitive endoderm layer forms a liver bud between E8.5 and E9.5. This layer will also form the pancreas, lung, stomach, intestine, and thyroid. The cardiac mesoderm and septum transversum mesenchyme release signals, such as fibroblast growth factors (FGF) and bone morphogenetic proteins (BMP), which are necessary to induce liver specification. The septum transversum mesenchyme has been implicated to give rise to stellate cells (also called Ito cells), which are fat and vitamin A-storing and extracellular matrix producing liver cells (10); cells positive for the Lim-homeobox gene (Lhx2) migrate from the septum transversum into the forming liver bud and become desmin and Lhx2 positive stellate cells. Cells in the developing liver bud are termed hepatoblasts and express alpha-fetoprotein (AFP). Hepatoblasts have been described as bipotential progenitors, developing into mature hepatocytes as well as bile duct epithelial cells (cholangiocytes), based on findings from *ex vivo* and *in vitro* studies (11-15). Suppression of transcription factor CCAAT-enhancer-binding protein alpha (CEBPα) has been suggested to

Different hepatic progenitors in human livers have been described. Based on early findings in developmental biology, hepatic stem cells were originally defined as AFP positive hepatoblasts. More recent research, however, reveals that hepatic stem cells are AFP negative and are the precursors to hepatoblasts (12, 18). Furthermore, stem cells of assumed mesendodermal origin capable of multilineage differentiation towards liver- and mesenchymal lineages have been discovered (19). An overview about human hepatic

several mature hepatic functions and proteins. Hepatocyte Hepatic parenchymal cell of the adult liver. In non-pathological

expression of immature protein alpha-fetoprotein and absence of

conditions defined by its expression of mature functions and proteins, such as albumin and cytochrome P450 enzymes, and the

absence of immature proteins such as alpha-fetoprotein.

also able of self-renewal.

**Term Description** 

**2. Embryonic liver development** 

induce their specification towards biliary differentiation (16, 17).

**3. Human hepatic progenitors in fetal and adult livers** 

progenitors that have been isolated and characterized is given in **Table 2**.

found also in (5, 6).


Hepatic Progenitors of the Liver and Extra-Hepatic Tissues 47

structures. Adipogenic differentiation could not be induced. As these cells were cultureselected, percentages of their *in vivo* occurrence were not established. Cells *in vitro* demonstrated exponential growth rates. When transplanted, human cells could be localized *in vivo* within the liver parenchyma of severe-combined immunodeficient (SCID) mice

Mesenchymal progenitors isolated from adult human livers were investigated for their potential to differentiate into hepatocytes (20, 21). Mesenchymal-like cells were obtained by selective culture (not sorting) of total liver cells. FACS analyses of cultured cells revealed a phenotype similar to mesenchymal stem cells with positive expression for CD90, CD73, CD29, CD44, CD13, and HLA-class I, but negative expression for CD105, CD133, CD117, CD45, CD34, and HLA-DR; cells were weakly positive for CD49e and CD49b, and only a minor fraction expressed CD49f. When cells were intrasplenically transplanted into uPA+/+- SCID mice, human albumin and AFP positive cells could be observed and human albumin secretion was detected. When transplanted into SCID mice with and without 70% hepatectomy, human albumin gene expression could be measured in mice livers that had undergone hepatectomy, and human albumin positive cells could be detected in mouse liver sections in both models. Potential fusion events were not analyzed. When cells were induced to hepatic lineages *in vitro* (21), hepatic functions were increased compared to non-

Hepatic stem cells can be isolated from fetal, neonatal, pediatric, and adult human livers with identical characteristics (12, 18), as described by Schmelzer *et al*. Cell surface and intracellular markers include CD326, CD133, CD56, E-cadherin, CD29, Patched (24), claudin 3 (18), CK19, and show weak positivity for albumin. Cells are negative for AFP, CD45, CD34, CD38, CD14, CD90, CD235a, VEGFr, vWF, CD31, CD146, desmin, ASMA, transferrin, connexins, PEPCK, DPP4, CYP450; CD117 is variably expressed. Sonic and Indian Hedgehog signaling pathway components are expressed (24). Stem cells could be selected by MACS sorting as well as under selective culture conditions, which included serum-free medium and culture on plastic. Under these culture conditions, hepatic stem cell colonies formed. These colonies (**Figure 1**) exhibit a typical epithelial morphology of densely packed, small cells with high nucleus-to-cytoplasm ratio. Stem cell colonies are positive for CD326

induced controls, but lower than those of freshly isolated adult liver cells.

(**Figure 2**), CD44h, CD56, and weakly express albumin, but are negative for AFP.

Cells were capable of self-renewal, as shown by clonogenic expansion for more than 150 population doublings. 0.5 – 2.5% of all liver cells from all ages were positive for CD326 expression. Hepatic stem cells have a small diameter of about 9 μm. *In vivo*, they are located in the ductal plates in fetal and neonatal livers and in the Canals of Hering in pediatric and adult livers. The Canal of Hering has been previously described as the reservoir of stem cells in postnatal livers (25, 26). Carpentier *et al*. recently studied lineage tracing by using a Cre recombinase Sox9 mouse model and confirmed that ductal plate cells give rise to cholangiocytes, periportal hepatocytes, and adult liver progenitor cells (27). Furuyama *et al*. (28) demonstrated that adult intestinal cells, hepatocytes and pancreatic acinar cells are physiologically supplied from Sox9-expressing progenitors using Cre-based lineage tracing in mice. In CCl4 mediated liver injury, Sox9-positive progenitors contributed to liver

treated with N-acetyl-p-aminophen.

**3.2 Hepatic stem cells in the human liver** 


Abbreviations: AFP: alpha-fetoprotein; CCl4: Carbon tetrachloride; CD: cluster of differentiation; CK: cytokeratin; GGT: γ-glutamyl transpeptidase; HLA: human leukocyte antigen; NOD: non-obese diabetic; SCID: severe-combined immunodeficient; MACS: magnetic activated cells sorting; PDT: population doubling time; uPA: urokinase-type plasminogen activator.

Table 2. Progenitors with hepatic potential isolated from human livers. Details are given in the respective sections.

#### **3.1 Human liver multipotent progenitors**

Dan *et al*. isolated liver stem cells co-expressing endodermal and mesenchymal phenotypes from human fetal liver by culture selection on feeder cells (19). These cells could differentiate not only into hepatocytes and bile duct cells, but also into fat, bone, cartilage, and endothelial cells. Because of their multilineage differentiation potential, these cells were termed human fetal liver multipotent progenitor cells (hFLMPC). The *in vivo* percentage of this progenitor was not given, as these cells were isolated by culture selection. Cell surface and intracellular markers included: CD34, CD90, CD117, CD326 (also called epithelial cell adhesion molecule (EpCAM)), c-met, SSEA4, CK18, CK19, CD44h, and vimentin. Cells were negative for albumin, CD133, CD45, and AFP. They could be cultured monoclonal and longterm for up to 100 population doublings. Cells had population doubling times of 46h. Early and late passages demonstrated identical morphology, differentiation potential, and telomere length. Cultured cells formed typical clusters with cells having a high nuclear to cytoplasm ratio. The morphology of these clusters resembled hepatic stem cells colonies described by Schmelzer *et al.* (12). When transplanted into immunotolerant Rag2- /- γ- /- mice (using a modified retrorsine/carbon tetra-chloride model), human-specific albumin in mouse serum and human-specific albumin in sections of the liver could be detected. Liver sections of transplanted mice demonstrated clusters of human hepatocytes. A repopulation of 0.8–1.7% was estimated. The multipotential differentiation potential and resemblance to hepatic stem cell colonies suggests that hFLMPC represent mesendodermal precursors of hepatic stem cells.

Herrera *et al*. isolated a similar population from human adult livers (22) using culture selection. These cells also expressed hepatic and mesenchymal markers. Cell surface and intracellular markers included albumin, AFP, CD29, CD73, CD44, CD90, vimentin and nestin; however, there was a negative expression of CD34, CD45, CD117, CD133, and CK19, and a weakly positive expression of CK8 and CK18. The cells were different from those described by Dan *et al*., as albumin and AFP expression could be observed and hematopoietic markers CD34 and CD117 were absent. *In vitro*, progenitors differentiated not only into hepatocytes, but also into osteogenic, endothelial, and islet-like, insulin-producing

**Isolation method** 

MACS, culture **Phenotype** *In vivo*

Positive: AFP. Variable: CD326

**model for repopulation** 

*In vitro*  **characteristics**

NOD/SCID Can arise from hHpSC colonies in culture

/- γ-/- mice

**Term used by authors** 

Abbreviations: AFP: alpha-fetoprotein; CCl4: Carbon tetrachloride; CD: cluster of differentiation; CK: cytokeratin; GGT: γ-glutamyl transpeptidase; HLA: human leukocyte antigen; NOD: non-obese diabetic; SCID: severe-combined immunodeficient; MACS: magnetic activated cells sorting;

Table 2. Progenitors with hepatic potential isolated from human livers. Details are given in

Dan *et al*. isolated liver stem cells co-expressing endodermal and mesenchymal phenotypes from human fetal liver by culture selection on feeder cells (19). These cells could differentiate not only into hepatocytes and bile duct cells, but also into fat, bone, cartilage, and endothelial cells. Because of their multilineage differentiation potential, these cells were termed human fetal liver multipotent progenitor cells (hFLMPC). The *in vivo* percentage of this progenitor was not given, as these cells were isolated by culture selection. Cell surface and intracellular markers included: CD34, CD90, CD117, CD326 (also called epithelial cell adhesion molecule (EpCAM)), c-met, SSEA4, CK18, CK19, CD44h, and vimentin. Cells were negative for albumin, CD133, CD45, and AFP. They could be cultured monoclonal and longterm for up to 100 population doublings. Cells had population doubling times of 46h. Early and late passages demonstrated identical morphology, differentiation potential, and telomere length. Cultured cells formed typical clusters with cells having a high nuclear to cytoplasm ratio. The morphology of these clusters resembled hepatic stem cells colonies

described by Schmelzer *et al.* (12). When transplanted into immunotolerant Rag2-

(using a modified retrorsine/carbon tetra-chloride model), human-specific albumin in mouse serum and human-specific albumin in sections of the liver could be detected. Liver sections of transplanted mice demonstrated clusters of human hepatocytes. A repopulation of 0.8–1.7% was estimated. The multipotential differentiation potential and resemblance to hepatic stem cell colonies suggests that hFLMPC represent mesendodermal precursors of

Herrera *et al*. isolated a similar population from human adult livers (22) using culture selection. These cells also expressed hepatic and mesenchymal markers. Cell surface and intracellular markers included albumin, AFP, CD29, CD73, CD44, CD90, vimentin and nestin; however, there was a negative expression of CD34, CD45, CD117, CD133, and CK19, and a weakly positive expression of CK8 and CK18. The cells were different from those described by Dan *et al*., as albumin and AFP expression could be observed and hematopoietic markers CD34 and CD117 were absent. *In vitro*, progenitors differentiated not only into hepatocytes, but also into osteogenic, endothelial, and islet-like, insulin-producing

Human hepatoblasts

PDT: population doubling time; uPA: urokinase-type plasminogen activator.

**Publication Develop-**

the respective sections.

hepatic stem cells.

**3.1 Human liver multipotent progenitors** 

Schmelzer *et al*. 2006, 2007 (12, 18) **mental stage of liver tissue** 

Fetal (16-20 weeks of gestation)

**Presumable lineage** 

structures. Adipogenic differentiation could not be induced. As these cells were cultureselected, percentages of their *in vivo* occurrence were not established. Cells *in vitro* demonstrated exponential growth rates. When transplanted, human cells could be localized *in vivo* within the liver parenchyma of severe-combined immunodeficient (SCID) mice treated with N-acetyl-p-aminophen.

Mesenchymal progenitors isolated from adult human livers were investigated for their potential to differentiate into hepatocytes (20, 21). Mesenchymal-like cells were obtained by selective culture (not sorting) of total liver cells. FACS analyses of cultured cells revealed a phenotype similar to mesenchymal stem cells with positive expression for CD90, CD73, CD29, CD44, CD13, and HLA-class I, but negative expression for CD105, CD133, CD117, CD45, CD34, and HLA-DR; cells were weakly positive for CD49e and CD49b, and only a minor fraction expressed CD49f. When cells were intrasplenically transplanted into uPA+/+- SCID mice, human albumin and AFP positive cells could be observed and human albumin secretion was detected. When transplanted into SCID mice with and without 70% hepatectomy, human albumin gene expression could be measured in mice livers that had undergone hepatectomy, and human albumin positive cells could be detected in mouse liver sections in both models. Potential fusion events were not analyzed. When cells were induced to hepatic lineages *in vitro* (21), hepatic functions were increased compared to noninduced controls, but lower than those of freshly isolated adult liver cells.

#### **3.2 Hepatic stem cells in the human liver**

Hepatic stem cells can be isolated from fetal, neonatal, pediatric, and adult human livers with identical characteristics (12, 18), as described by Schmelzer *et al*. Cell surface and intracellular markers include CD326, CD133, CD56, E-cadherin, CD29, Patched (24), claudin 3 (18), CK19, and show weak positivity for albumin. Cells are negative for AFP, CD45, CD34, CD38, CD14, CD90, CD235a, VEGFr, vWF, CD31, CD146, desmin, ASMA, transferrin, connexins, PEPCK, DPP4, CYP450; CD117 is variably expressed. Sonic and Indian Hedgehog signaling pathway components are expressed (24). Stem cells could be selected by MACS sorting as well as under selective culture conditions, which included serum-free medium and culture on plastic. Under these culture conditions, hepatic stem cell colonies formed. These colonies (**Figure 1**) exhibit a typical epithelial morphology of densely packed, small cells with high nucleus-to-cytoplasm ratio. Stem cell colonies are positive for CD326 (**Figure 2**), CD44h, CD56, and weakly express albumin, but are negative for AFP.

Cells were capable of self-renewal, as shown by clonogenic expansion for more than 150 population doublings. 0.5 – 2.5% of all liver cells from all ages were positive for CD326 expression. Hepatic stem cells have a small diameter of about 9 μm. *In vivo*, they are located in the ductal plates in fetal and neonatal livers and in the Canals of Hering in pediatric and adult livers. The Canal of Hering has been previously described as the reservoir of stem cells in postnatal livers (25, 26). Carpentier *et al*. recently studied lineage tracing by using a Cre recombinase Sox9 mouse model and confirmed that ductal plate cells give rise to cholangiocytes, periportal hepatocytes, and adult liver progenitor cells (27). Furuyama *et al*. (28) demonstrated that adult intestinal cells, hepatocytes and pancreatic acinar cells are physiologically supplied from Sox9-expressing progenitors using Cre-based lineage tracing in mice. In CCl4 mediated liver injury, Sox9-positive progenitors contributed to liver

Hepatic Progenitors of the Liver and Extra-Hepatic Tissues 49

Human hepatoblasts do not express the mesenchymal or hematopoietic markers CD90, vimentin, and CD34 (34). In mice, hepatoblasts express the surface marker Dlk-1 (35-37), which was subsequently demonstrated to be expressed by human fetal hepatoblasts as well (34). Mouse fetal liver cells sorted for Dlk-1 can be cultured long-term; transplantation of Dlk-1 positive cells into the spleen gives rise to hepatocytes in the liver. Several signaling pathways and transcription factors contribute towards differentiation into either cell type. In mice, Notch signaling controls differentiation towards biliary epithelium by upregulation of HNF1β but downregulation of HNF1α, HNF4, and C/EBPα (38), and, in turn, suppression of C/EBPα expression in periportal hepatoblasts is suggested to induce biliary epithelial

Fig. 2. Human hepatic stem cell colonies established as described in (12) are positive for CD326 (**A**). Fluorescence microscopy for the transmembrane glycoprotein CD326 (also named epithelial cell adhesion molecule (EpCAM)) in (**A**), and corresponding nuclei stained

Various surface markers have been applied to identify hepatic stem or progenitor

Germain *et al*. described the bipotential capacity of fetal rat liver cells to differentiate into hepatic and biliary cells *in vitro* (39), as did Kubota *et al*. using clonal cultures (40). Small hepatocytes were detected in non-parenchymal fractions of adult rat liver cells (41-44). These small hepatocytes produced colonies that expressed hepatic and biliary markers. A similar type of colony could be obtained when adult liver cell clusters were placed into culture (45). Suzuki *et al*. sorted progenitor populations from fetal mice and rat livers with a

into albumin and glycogen positive cells when transplanted into retrorsine-treated adult rats that had undergone two-third partial hepatectomy. Cells negative for c-met or positive for CD45 could not repopulate recipient livers. These progenitors could be also cultured clonally. Feng *et al*. (51) demonstrated that these cells could also undergo pancreatic differentiation in culture as well as *in vivo* when transplanted into alloxan-induced diabetic

, CD45-, and TER119- (46-50). Sorted cells developed

**4. Murine and rat hepatic progenitors in the fetal and adult liver** 

differentiation by increasing HNF6 and HNF1β expression (17).

with 4',6-diamidino-2-phenylindole in (**B**).

phenotype of c-met+, CD49f+, CD117-

populations in rodents.

regeneration. Hepatic stem cells have been shown to differentiate into biliary and hepatocytic lineages *in vivo* and *in vitro* (12). Freshly isolated cells or stem cells expanded in culture developed into mature liver tissue expressing human-specific proteins when transplanted into NOD/SCID mice, and lost their expression of stem cell marker CD326, CD133, and CK19. Whether those cells also possess multilineage differentiation potential beyond endodermal fates, i.e. mesodermal or ectodermal, has not yet been investigated. Khan *et al*. transplanted human fetal liver derived CD326+ sorted progenitors into patients with liver fibrosis (29). Patients demonstrated improvements in clinical and biochemical parameters and a decrease in mean MELD (model for end-stage liver disease) score at sixmonth follow-up.

Fig. 1. Human hepatic stem cell colony in culture, established from fetal liver cell suspensions as described in (12); phase contrast microscopy (**A**), and fluorescence microscopy (**B**) of proliferating cells with positive nuclei for incorporated thymidine analog bromodeoxyuridine (**B-I**) and corresponding total nuclei stained with 4',6-diamidino-2 phenylindole (**B-II**).

#### **3.3 Hepatoblasts in human liver**

Hepatoblasts are the main parenchymal cell type of the fetal liver and are defined by their expression of AFP. AFP positive cells are rare in normal adult livers, except in livers with severe injury or disease (30-32) (for review, see (33)). Hepatoblasts can give rise to hepatocytes and cholangiocytes, and are therefore also named bipotential progenitors (15). AFP-negative hepatic stem cells are the precursors to hepatoblasts that can mature into AFPpositive hepatoblasts (12). Human fetal hepatoblasts could be cultured long-term and clonally, and contributed to liver parenchyma when transplanted into SCID mice (23). Hepatoblasts express biliary and hepatocyte markers such as CK19, CK14, gamma glutamyl transpeptidase, glucose-6-phosphatase, glycogen, albumin, AFP, E-cadherin (34), α-1 microglobulin, HepPar1, glutamate dehydrogenase, and dipeptidyl peptidase IV (15, 18).

regeneration. Hepatic stem cells have been shown to differentiate into biliary and hepatocytic lineages *in vivo* and *in vitro* (12). Freshly isolated cells or stem cells expanded in culture developed into mature liver tissue expressing human-specific proteins when transplanted into NOD/SCID mice, and lost their expression of stem cell marker CD326, CD133, and CK19. Whether those cells also possess multilineage differentiation potential beyond endodermal fates, i.e. mesodermal or ectodermal, has not yet been investigated. Khan *et al*. transplanted human fetal liver derived CD326+ sorted progenitors into patients with liver fibrosis (29). Patients demonstrated improvements in clinical and biochemical parameters and a decrease in mean MELD (model for end-stage liver disease) score at six-

Fig. 1. Human hepatic stem cell colony in culture, established from fetal liver cell suspensions as described in (12); phase contrast microscopy (**A**), and fluorescence

microscopy (**B**) of proliferating cells with positive nuclei for incorporated thymidine analog bromodeoxyuridine (**B-I**) and corresponding total nuclei stained with 4',6-diamidino-2-

Hepatoblasts are the main parenchymal cell type of the fetal liver and are defined by their expression of AFP. AFP positive cells are rare in normal adult livers, except in livers with severe injury or disease (30-32) (for review, see (33)). Hepatoblasts can give rise to hepatocytes and cholangiocytes, and are therefore also named bipotential progenitors (15). AFP-negative hepatic stem cells are the precursors to hepatoblasts that can mature into AFPpositive hepatoblasts (12). Human fetal hepatoblasts could be cultured long-term and clonally, and contributed to liver parenchyma when transplanted into SCID mice (23). Hepatoblasts express biliary and hepatocyte markers such as CK19, CK14, gamma glutamyl transpeptidase, glucose-6-phosphatase, glycogen, albumin, AFP, E-cadherin (34), α-1 microglobulin, HepPar1, glutamate dehydrogenase, and dipeptidyl peptidase IV (15, 18).

month follow-up.

phenylindole (**B-II**).

**3.3 Hepatoblasts in human liver** 

Human hepatoblasts do not express the mesenchymal or hematopoietic markers CD90, vimentin, and CD34 (34). In mice, hepatoblasts express the surface marker Dlk-1 (35-37), which was subsequently demonstrated to be expressed by human fetal hepatoblasts as well (34). Mouse fetal liver cells sorted for Dlk-1 can be cultured long-term; transplantation of Dlk-1 positive cells into the spleen gives rise to hepatocytes in the liver. Several signaling pathways and transcription factors contribute towards differentiation into either cell type. In mice, Notch signaling controls differentiation towards biliary epithelium by upregulation of HNF1β but downregulation of HNF1α, HNF4, and C/EBPα (38), and, in turn, suppression of C/EBPα expression in periportal hepatoblasts is suggested to induce biliary epithelial differentiation by increasing HNF6 and HNF1β expression (17).

Fig. 2. Human hepatic stem cell colonies established as described in (12) are positive for CD326 (**A**). Fluorescence microscopy for the transmembrane glycoprotein CD326 (also named epithelial cell adhesion molecule (EpCAM)) in (**A**), and corresponding nuclei stained with 4',6-diamidino-2-phenylindole in (**B**).
