Rescue of lethal hepatic failure by hepatized lymph nodes in mice

Abstract

Background & aims: Hepatocyte transplantation is a potential therapeutic approach for liver disease. However, most patients with chronic hepatic damage have cirrhosis and fibrosis, which limit the potential for cell-based therapy of the liver. The development of an ectopic liver as an additional site of hepatic function represents a new approach for patients with end-stage liver disease. We investigated the development and function of liver tissue in lymph nodes in mice with liver failure.

Methods: Hepatocytes were isolated from 8- to 12-week-old mice and transplanted by intraperitoneal injection into 8- to 12-week-old fumarylacetoacetate hydrolase mice (Fah(-/-)), a model of the human liver disease tyrosinemia type I. Survival was monitored and the locations and functions of the engrafted liver cells were determined.

Results: Lymph nodes of Fah(-/-) mice were colonized by transplanted hepatocytes; Fah(+) hepatocytes were detected adjacent to the CD45(+) lymphoid cells of the lymphatic system. Ten weeks after transplantation, these mice had substantial improvements in serum levels of transaminases, bilirubin, and amino acids. Homeostatic expansion of donor hepatocytes in lymph nodes rescued the mice from lethal hepatic failure.

Conclusions: Functional ectopic liver tissue in lymph nodes rescues mice from lethal hepatic disease; lymph nodes therefore might be used as sites for hepatocyte transplantation.

Figure 1

Fah^-/- mice are rescued from lethal hepatic failure by intraperitoneal injection of hepatocytes. (A) Body weight after splenic (SP) and intraperitoneal (IP) transplantation indicates hepatic regeneration. Body weight of the transplanted mice was monitored weekly after liver cell transplantation (time 0), in order to follow hepatic engraftment and rescue from tyrosinemia. Fah^-/- mice transplanted by either single SP or IP injections lost weight during the first 3 weeks. Weight loss is indicative of a decline in liver function. SP injected mice spontaneously regained weight. IP injected mice required two periods of selection prior to regaining weight for efficient survival, a protocol previously described for engrafting low levels of liver cells . (B) Spontaneous weight gain after a single period of selection was possible but with a lower survival rate. Fah^-/- mice were transplanted with 10⁶ wt liver cells followed by NTBC removal from their diet (blue and red line). n = number of mice analyzed. (C) Anatomic location of enlarged nodules 10 weeks after transplantation. Left panel: many enlarged nodules around the stomach region and on the mesenterium are observed in a mouse transplanted IP with wt hepatocytes (circles). Middle upper panel: native liver of the IP injected Fah^-/- mouse and a control wt liver. The native liver of the IP injected Fah^-/- mouse was atrophic with a couple of small regenerative nodules containing wt hepatocytes on its surface (arrows). Middle lower panel: isolated enlarged nodules from mouse in the left panel with diameters from 1 to 10 mm. Right upper and lower panels: mesenteric lymph nodes (mln) repopulated with GFP positive liver cells. Blood vessels (bv) and small intestine (si) are GFP negative.

Figure 1

Fah^-/- mice are rescued from lethal hepatic failure by intraperitoneal injection of hepatocytes. (A) Body weight after splenic (SP) and intraperitoneal (IP) transplantation indicates hepatic regeneration. Body weight of the transplanted mice was monitored weekly after liver cell transplantation (time 0), in order to follow hepatic engraftment and rescue from tyrosinemia. Fah^-/- mice transplanted by either single SP or IP injections lost weight during the first 3 weeks. Weight loss is indicative of a decline in liver function. SP injected mice spontaneously regained weight. IP injected mice required two periods of selection prior to regaining weight for efficient survival, a protocol previously described for engrafting low levels of liver cells . (B) Spontaneous weight gain after a single period of selection was possible but with a lower survival rate. Fah^-/- mice were transplanted with 10⁶ wt liver cells followed by NTBC removal from their diet (blue and red line). n = number of mice analyzed. (C) Anatomic location of enlarged nodules 10 weeks after transplantation. Left panel: many enlarged nodules around the stomach region and on the mesenterium are observed in a mouse transplanted IP with wt hepatocytes (circles). Middle upper panel: native liver of the IP injected Fah^-/- mouse and a control wt liver. The native liver of the IP injected Fah^-/- mouse was atrophic with a couple of small regenerative nodules containing wt hepatocytes on its surface (arrows). Middle lower panel: isolated enlarged nodules from mouse in the left panel with diameters from 1 to 10 mm. Right upper and lower panels: mesenteric lymph nodes (mln) repopulated with GFP positive liver cells. Blood vessels (bv) and small intestine (si) are GFP negative.

Figure 1

Fah^-/- mice are rescued from lethal hepatic failure by intraperitoneal injection of hepatocytes. (A) Body weight after splenic (SP) and intraperitoneal (IP) transplantation indicates hepatic regeneration. Body weight of the transplanted mice was monitored weekly after liver cell transplantation (time 0), in order to follow hepatic engraftment and rescue from tyrosinemia. Fah^-/- mice transplanted by either single SP or IP injections lost weight during the first 3 weeks. Weight loss is indicative of a decline in liver function. SP injected mice spontaneously regained weight. IP injected mice required two periods of selection prior to regaining weight for efficient survival, a protocol previously described for engrafting low levels of liver cells . (B) Spontaneous weight gain after a single period of selection was possible but with a lower survival rate. Fah^-/- mice were transplanted with 10⁶ wt liver cells followed by NTBC removal from their diet (blue and red line). n = number of mice analyzed. (C) Anatomic location of enlarged nodules 10 weeks after transplantation. Left panel: many enlarged nodules around the stomach region and on the mesenterium are observed in a mouse transplanted IP with wt hepatocytes (circles). Middle upper panel: native liver of the IP injected Fah^-/- mouse and a control wt liver. The native liver of the IP injected Fah^-/- mouse was atrophic with a couple of small regenerative nodules containing wt hepatocytes on its surface (arrows). Middle lower panel: isolated enlarged nodules from mouse in the left panel with diameters from 1 to 10 mm. Right upper and lower panels: mesenteric lymph nodes (mln) repopulated with GFP positive liver cells. Blood vessels (bv) and small intestine (si) are GFP negative.

Figure 2

Immunohistochemistry of lymph nodes from the gastric and common hepatic arteries. (A) 2 and 3 days after IP injection of hepatocytes in Fah^-/- mice. On day 2, some wt Fah⁺CK18⁺ hepatocytes could be detected in lymphatic system near lymphocytes. On day 3, clusters of CK18⁺ hepatocytes were seen in association with CD45⁺ hematopoietic cells. Meca79 (CD62L ligand), a marker of high endothelial venules found in lymph nodes, is co-localized with CK18⁺ donor hepatocytes (B) 2 and 3 weeks after IP injection, Fah⁺ hepatocytes (green) have colonized the lymph nodes and have a high index of proliferation, as demonstrated by the high ratio of BrdU incorporation (red nuclei). 8 weeks after IP injection, few liver cells are proliferating in lymph nodes. (C) Immunofluorescence analysis of hematopoietic markers in hepatized lymph nodes 10 weeks after IP injection of hepatocytes in Fah^-/- mice. Both sections of hepatized lymph nodes and control (wt) lymph nodes were stained with hematopoietic markers. Each staining has two panels, the upper panel represents the lymph nodes engrafted with hepatocytes (hepatized LN) and lower panel is normal wt mouse lymph node (Control LN). Stainings were performed on serial sections. Bar: 100μm.

Figure 3

Hepatized lymph nodes 10 weeks after IP transplantation. (A) Sections were immunostained with an anti-Fah antibody (brown, HRP staining) then counterstained with hematoxylin. Fah⁺ hepatocytes and several islands of small hematopoietic cells were present but no biliary structures were observed. Bar: 100μm. (B) Immunofluorescence of lymph nodes engrafted with hepatocytes (hepatized LN), control liver and control lymph nodes. Frozen sections were stained with hepatocyte marker CK18 and the endothelial marker CD31. CD26, dipeptidyl-peptidase-IV, was used as a hepatocyte maker and E-Cadherin as an epithelial maker. Most cells in the hepatized LN were CK18⁺ hepatocytes, with expression patterns similar to those of control liver. These cells were also albumin positive (brown cells, insert in left upper panel) with CK18 and CD26 co-localized (insert in CD26 staining panel). Hepatocyte and epithelial markers were negative in normal lymph node. In the hepatized LN, CD31⁺ endothelial cells corresponding to vessels and were similar in size and morphology to those found in normal (control) liver. In contrast, CD31⁺ cells indicative of high endothlelial vessels (HEV) found in normal (control) lymph nodes differ in morphology. Bar: 100μm. (C) The ratio ± s.d. of the weight of liver and enlarged nodules to body weight. The ratio of hepatic tissues to body weight was determined in Fah^-/- mice transplanted IP and both liver (atrophic) and enlarged nodules (hypertrophic) were collected and compared to normal wt liver. n = number of mice analyzed. (D) Transmission electron microscopy of the hepatized lymph nodes. Ultrastructure of a hepatized lymph node (upper panels) and control liver (lower panels). Left upper panel: hepatocytes present in lymph nodes have large prominent nuclei (N), bile canaliculi (BC), mitochondria (M), peroxisomes (P) and rough endoplasmic reticulum (RER). Bar: 2μm. Center upper panel: higher magnification of the bile canaliculus, containing microvilli (MV) with tight junctions (arrowheads) and adherent junctions (AJ). A lipid vacuole is seen within the canaliculus. Bar: 500nm. Right upper panel: Vessels in hepatized lymph node consisted of non-fenestrated sinusoidal endothelial cells (SECs). Bar: 1μm. Left lower panel: hepatocytes in control liver showing fenestrations (arrows) in SECs. Bar: 2μm. Center lower panel: higher magnification of bile canaliculus showing tight junctions (arrowheads), lipid vacuoles and Space of Disse (SD). Bar: 500nm. Right lower panel: Organization of hepatic plates in control livers with bile canaliculi at the apical surface and fenestrated sinusoids (S) at the basolateral surface. Bar: 2μm. (E) Immunofluorescence analysis with non-hematopoietic liver cells markers in hepatized lymph nodes, normal liver and normal lymph node (LN). Staining was performed with the hepatocyte marker, CK18 and the non-parenchymal cell markers F4/80, Desmin, and GFAP, CK19 and ER-TR7. F4/80⁺ (Kupffer cells), CK19 (biliary cells), and ER-TR7 (reticular fibroblasts) were negative in hepatized lymph nodes. Bar: 100μm.

Figure 3

Hepatized lymph nodes 10 weeks after IP transplantation. (A) Sections were immunostained with an anti-Fah antibody (brown, HRP staining) then counterstained with hematoxylin. Fah⁺ hepatocytes and several islands of small hematopoietic cells were present but no biliary structures were observed. Bar: 100μm. (B) Immunofluorescence of lymph nodes engrafted with hepatocytes (hepatized LN), control liver and control lymph nodes. Frozen sections were stained with hepatocyte marker CK18 and the endothelial marker CD31. CD26, dipeptidyl-peptidase-IV, was used as a hepatocyte maker and E-Cadherin as an epithelial maker. Most cells in the hepatized LN were CK18⁺ hepatocytes, with expression patterns similar to those of control liver. These cells were also albumin positive (brown cells, insert in left upper panel) with CK18 and CD26 co-localized (insert in CD26 staining panel). Hepatocyte and epithelial markers were negative in normal lymph node. In the hepatized LN, CD31⁺ endothelial cells corresponding to vessels and were similar in size and morphology to those found in normal (control) liver. In contrast, CD31⁺ cells indicative of high endothlelial vessels (HEV) found in normal (control) lymph nodes differ in morphology. Bar: 100μm. (C) The ratio ± s.d. of the weight of liver and enlarged nodules to body weight. The ratio of hepatic tissues to body weight was determined in Fah^-/- mice transplanted IP and both liver (atrophic) and enlarged nodules (hypertrophic) were collected and compared to normal wt liver. n = number of mice analyzed. (D) Transmission electron microscopy of the hepatized lymph nodes. Ultrastructure of a hepatized lymph node (upper panels) and control liver (lower panels). Left upper panel: hepatocytes present in lymph nodes have large prominent nuclei (N), bile canaliculi (BC), mitochondria (M), peroxisomes (P) and rough endoplasmic reticulum (RER). Bar: 2μm. Center upper panel: higher magnification of the bile canaliculus, containing microvilli (MV) with tight junctions (arrowheads) and adherent junctions (AJ). A lipid vacuole is seen within the canaliculus. Bar: 500nm. Right upper panel: Vessels in hepatized lymph node consisted of non-fenestrated sinusoidal endothelial cells (SECs). Bar: 1μm. Left lower panel: hepatocytes in control liver showing fenestrations (arrows) in SECs. Bar: 2μm. Center lower panel: higher magnification of bile canaliculus showing tight junctions (arrowheads), lipid vacuoles and Space of Disse (SD). Bar: 500nm. Right lower panel: Organization of hepatic plates in control livers with bile canaliculi at the apical surface and fenestrated sinusoids (S) at the basolateral surface. Bar: 2μm. (E) Immunofluorescence analysis with non-hematopoietic liver cells markers in hepatized lymph nodes, normal liver and normal lymph node (LN). Staining was performed with the hepatocyte marker, CK18 and the non-parenchymal cell markers F4/80, Desmin, and GFAP, CK19 and ER-TR7. F4/80⁺ (Kupffer cells), CK19 (biliary cells), and ER-TR7 (reticular fibroblasts) were negative in hepatized lymph nodes. Bar: 100μm.

Figure 3

Hepatized lymph nodes 10 weeks after IP transplantation. (A) Sections were immunostained with an anti-Fah antibody (brown, HRP staining) then counterstained with hematoxylin. Fah⁺ hepatocytes and several islands of small hematopoietic cells were present but no biliary structures were observed. Bar: 100μm. (B) Immunofluorescence of lymph nodes engrafted with hepatocytes (hepatized LN), control liver and control lymph nodes. Frozen sections were stained with hepatocyte marker CK18 and the endothelial marker CD31. CD26, dipeptidyl-peptidase-IV, was used as a hepatocyte maker and E-Cadherin as an epithelial maker. Most cells in the hepatized LN were CK18⁺ hepatocytes, with expression patterns similar to those of control liver. These cells were also albumin positive (brown cells, insert in left upper panel) with CK18 and CD26 co-localized (insert in CD26 staining panel). Hepatocyte and epithelial markers were negative in normal lymph node. In the hepatized LN, CD31⁺ endothelial cells corresponding to vessels and were similar in size and morphology to those found in normal (control) liver. In contrast, CD31⁺ cells indicative of high endothlelial vessels (HEV) found in normal (control) lymph nodes differ in morphology. Bar: 100μm. (C) The ratio ± s.d. of the weight of liver and enlarged nodules to body weight. The ratio of hepatic tissues to body weight was determined in Fah^-/- mice transplanted IP and both liver (atrophic) and enlarged nodules (hypertrophic) were collected and compared to normal wt liver. n = number of mice analyzed. (D) Transmission electron microscopy of the hepatized lymph nodes. Ultrastructure of a hepatized lymph node (upper panels) and control liver (lower panels). Left upper panel: hepatocytes present in lymph nodes have large prominent nuclei (N), bile canaliculi (BC), mitochondria (M), peroxisomes (P) and rough endoplasmic reticulum (RER). Bar: 2μm. Center upper panel: higher magnification of the bile canaliculus, containing microvilli (MV) with tight junctions (arrowheads) and adherent junctions (AJ). A lipid vacuole is seen within the canaliculus. Bar: 500nm. Right upper panel: Vessels in hepatized lymph node consisted of non-fenestrated sinusoidal endothelial cells (SECs). Bar: 1μm. Left lower panel: hepatocytes in control liver showing fenestrations (arrows) in SECs. Bar: 2μm. Center lower panel: higher magnification of bile canaliculus showing tight junctions (arrowheads), lipid vacuoles and Space of Disse (SD). Bar: 500nm. Right lower panel: Organization of hepatic plates in control livers with bile canaliculi at the apical surface and fenestrated sinusoids (S) at the basolateral surface. Bar: 2μm. (E) Immunofluorescence analysis with non-hematopoietic liver cells markers in hepatized lymph nodes, normal liver and normal lymph node (LN). Staining was performed with the hepatocyte marker, CK18 and the non-parenchymal cell markers F4/80, Desmin, and GFAP, CK19 and ER-TR7. F4/80⁺ (Kupffer cells), CK19 (biliary cells), and ER-TR7 (reticular fibroblasts) were negative in hepatized lymph nodes. Bar: 100μm.

Figure 3

Hepatized lymph nodes 10 weeks after IP transplantation. (A) Sections were immunostained with an anti-Fah antibody (brown, HRP staining) then counterstained with hematoxylin. Fah⁺ hepatocytes and several islands of small hematopoietic cells were present but no biliary structures were observed. Bar: 100μm. (B) Immunofluorescence of lymph nodes engrafted with hepatocytes (hepatized LN), control liver and control lymph nodes. Frozen sections were stained with hepatocyte marker CK18 and the endothelial marker CD31. CD26, dipeptidyl-peptidase-IV, was used as a hepatocyte maker and E-Cadherin as an epithelial maker. Most cells in the hepatized LN were CK18⁺ hepatocytes, with expression patterns similar to those of control liver. These cells were also albumin positive (brown cells, insert in left upper panel) with CK18 and CD26 co-localized (insert in CD26 staining panel). Hepatocyte and epithelial markers were negative in normal lymph node. In the hepatized LN, CD31⁺ endothelial cells corresponding to vessels and were similar in size and morphology to those found in normal (control) liver. In contrast, CD31⁺ cells indicative of high endothlelial vessels (HEV) found in normal (control) lymph nodes differ in morphology. Bar: 100μm. (C) The ratio ± s.d. of the weight of liver and enlarged nodules to body weight. The ratio of hepatic tissues to body weight was determined in Fah^-/- mice transplanted IP and both liver (atrophic) and enlarged nodules (hypertrophic) were collected and compared to normal wt liver. n = number of mice analyzed. (D) Transmission electron microscopy of the hepatized lymph nodes. Ultrastructure of a hepatized lymph node (upper panels) and control liver (lower panels). Left upper panel: hepatocytes present in lymph nodes have large prominent nuclei (N), bile canaliculi (BC), mitochondria (M), peroxisomes (P) and rough endoplasmic reticulum (RER). Bar: 2μm. Center upper panel: higher magnification of the bile canaliculus, containing microvilli (MV) with tight junctions (arrowheads) and adherent junctions (AJ). A lipid vacuole is seen within the canaliculus. Bar: 500nm. Right upper panel: Vessels in hepatized lymph node consisted of non-fenestrated sinusoidal endothelial cells (SECs). Bar: 1μm. Left lower panel: hepatocytes in control liver showing fenestrations (arrows) in SECs. Bar: 2μm. Center lower panel: higher magnification of bile canaliculus showing tight junctions (arrowheads), lipid vacuoles and Space of Disse (SD). Bar: 500nm. Right lower panel: Organization of hepatic plates in control livers with bile canaliculi at the apical surface and fenestrated sinusoids (S) at the basolateral surface. Bar: 2μm. (E) Immunofluorescence analysis with non-hematopoietic liver cells markers in hepatized lymph nodes, normal liver and normal lymph node (LN). Staining was performed with the hepatocyte marker, CK18 and the non-parenchymal cell markers F4/80, Desmin, and GFAP, CK19 and ER-TR7. F4/80⁺ (Kupffer cells), CK19 (biliary cells), and ER-TR7 (reticular fibroblasts) were negative in hepatized lymph nodes. Bar: 100μm.

Figure 3

Hepatized lymph nodes 10 weeks after IP transplantation. (A) Sections were immunostained with an anti-Fah antibody (brown, HRP staining) then counterstained with hematoxylin. Fah⁺ hepatocytes and several islands of small hematopoietic cells were present but no biliary structures were observed. Bar: 100μm. (B) Immunofluorescence of lymph nodes engrafted with hepatocytes (hepatized LN), control liver and control lymph nodes. Frozen sections were stained with hepatocyte marker CK18 and the endothelial marker CD31. CD26, dipeptidyl-peptidase-IV, was used as a hepatocyte maker and E-Cadherin as an epithelial maker. Most cells in the hepatized LN were CK18⁺ hepatocytes, with expression patterns similar to those of control liver. These cells were also albumin positive (brown cells, insert in left upper panel) with CK18 and CD26 co-localized (insert in CD26 staining panel). Hepatocyte and epithelial markers were negative in normal lymph node. In the hepatized LN, CD31⁺ endothelial cells corresponding to vessels and were similar in size and morphology to those found in normal (control) liver. In contrast, CD31⁺ cells indicative of high endothlelial vessels (HEV) found in normal (control) lymph nodes differ in morphology. Bar: 100μm. (C) The ratio ± s.d. of the weight of liver and enlarged nodules to body weight. The ratio of hepatic tissues to body weight was determined in Fah^-/- mice transplanted IP and both liver (atrophic) and enlarged nodules (hypertrophic) were collected and compared to normal wt liver. n = number of mice analyzed. (D) Transmission electron microscopy of the hepatized lymph nodes. Ultrastructure of a hepatized lymph node (upper panels) and control liver (lower panels). Left upper panel: hepatocytes present in lymph nodes have large prominent nuclei (N), bile canaliculi (BC), mitochondria (M), peroxisomes (P) and rough endoplasmic reticulum (RER). Bar: 2μm. Center upper panel: higher magnification of the bile canaliculus, containing microvilli (MV) with tight junctions (arrowheads) and adherent junctions (AJ). A lipid vacuole is seen within the canaliculus. Bar: 500nm. Right upper panel: Vessels in hepatized lymph node consisted of non-fenestrated sinusoidal endothelial cells (SECs). Bar: 1μm. Left lower panel: hepatocytes in control liver showing fenestrations (arrows) in SECs. Bar: 2μm. Center lower panel: higher magnification of bile canaliculus showing tight junctions (arrowheads), lipid vacuoles and Space of Disse (SD). Bar: 500nm. Right lower panel: Organization of hepatic plates in control livers with bile canaliculi at the apical surface and fenestrated sinusoids (S) at the basolateral surface. Bar: 2μm. (E) Immunofluorescence analysis with non-hematopoietic liver cells markers in hepatized lymph nodes, normal liver and normal lymph node (LN). Staining was performed with the hepatocyte marker, CK18 and the non-parenchymal cell markers F4/80, Desmin, and GFAP, CK19 and ER-TR7. F4/80⁺ (Kupffer cells), CK19 (biliary cells), and ER-TR7 (reticular fibroblasts) were negative in hepatized lymph nodes. Bar: 100μm.

Figure 4

Biochemical liver functions are restored by hepatized lymph nodes. (A) Biochemical measurement of liver function in blood. Tyrosinemic (Fah^-/-) mice were rescued by intraperitoneal injection (IP) or by splenic injection (SP) of wild-type (wt) liver cells. Ten weeks after transplantation, mean biochemical measurements of various liver functions ± s.d. were compared between littermate wt controls, Fah^-/- mice under NTBC and untreated Fah^-/- mice (NTBC withheld for 5 weeks and experiencing hepatic failure). All animals were between 3 and 6 months old. The number of mice (serum) analyzed is indicated in parentheses. ALT stands for alanine aminotransferase. (B) Plasma concentration of Albumin, Fibrinogen and HGF after IP injection of wt hepatocytes in Fah^-/- mice at 1, 3, 4, 6 and 10 weeks. Controls correspond to normal wild type mice. Plasma samples were tested by ELISA. Each open circle represents the value from one mouse. t-tests were run using Prism to determine significant differences between particular groups. HGF increased at 6 weeks after the second and final selection (off NTBC) reflecting the massive expansion of hepatocytes in lymph nodes necessary for the Fah^-/- survival. Bars indicate mean values. For HGF * p_{control & 6wk} =0.0079 by Mann Whitney test. (C) Serum concentration of blood urea nitrogen (BUN), total cholesterol and triglycerides in Fah^-/- mice over 10 weeks after IP injection and rescue of the animals, and compared wild type mice with p values. (D) Glycogen storage in hepatized lymph nodes. Glycogen storage was determined by Periodic Acid Schiff (PAS) staining. Bar: 100μm. The black and white electron microscopy panel identified glyocogen rosettes (arrowheads) in hepatocytes. Bar: 500nm. (E) Fah enzyme assay. A standard curve to measure enzyme activity was established using wt liver (100% activity), Fah^-/- liver (0% activity) and wt/Fah^-/- mixes to achieve 15%, 25% and 80% enzyme activity. Fah enzyme activities in engrafted lymph nodes (LN) ranged from 80% to almost 100% of wt liver levels. In contrast, Fah enzyme activity in native livers of Fah^-/- mice rescued by hepatized LN had Fah activity ranging from 25% of wt liver activity to 0% (mean Fah activity of 15% of wt liver levels). n = number of mice analyzed.

Figure 4

Biochemical liver functions are restored by hepatized lymph nodes. (A) Biochemical measurement of liver function in blood. Tyrosinemic (Fah^-/-) mice were rescued by intraperitoneal injection (IP) or by splenic injection (SP) of wild-type (wt) liver cells. Ten weeks after transplantation, mean biochemical measurements of various liver functions ± s.d. were compared between littermate wt controls, Fah^-/- mice under NTBC and untreated Fah^-/- mice (NTBC withheld for 5 weeks and experiencing hepatic failure). All animals were between 3 and 6 months old. The number of mice (serum) analyzed is indicated in parentheses. ALT stands for alanine aminotransferase. (B) Plasma concentration of Albumin, Fibrinogen and HGF after IP injection of wt hepatocytes in Fah^-/- mice at 1, 3, 4, 6 and 10 weeks. Controls correspond to normal wild type mice. Plasma samples were tested by ELISA. Each open circle represents the value from one mouse. t-tests were run using Prism to determine significant differences between particular groups. HGF increased at 6 weeks after the second and final selection (off NTBC) reflecting the massive expansion of hepatocytes in lymph nodes necessary for the Fah^-/- survival. Bars indicate mean values. For HGF * p_{control & 6wk} =0.0079 by Mann Whitney test. (C) Serum concentration of blood urea nitrogen (BUN), total cholesterol and triglycerides in Fah^-/- mice over 10 weeks after IP injection and rescue of the animals, and compared wild type mice with p values. (D) Glycogen storage in hepatized lymph nodes. Glycogen storage was determined by Periodic Acid Schiff (PAS) staining. Bar: 100μm. The black and white electron microscopy panel identified glyocogen rosettes (arrowheads) in hepatocytes. Bar: 500nm. (E) Fah enzyme assay. A standard curve to measure enzyme activity was established using wt liver (100% activity), Fah^-/- liver (0% activity) and wt/Fah^-/- mixes to achieve 15%, 25% and 80% enzyme activity. Fah enzyme activities in engrafted lymph nodes (LN) ranged from 80% to almost 100% of wt liver levels. In contrast, Fah enzyme activity in native livers of Fah^-/- mice rescued by hepatized LN had Fah activity ranging from 25% of wt liver activity to 0% (mean Fah activity of 15% of wt liver levels). n = number of mice analyzed.

Figure 4

Biochemical liver functions are restored by hepatized lymph nodes. (A) Biochemical measurement of liver function in blood. Tyrosinemic (Fah^-/-) mice were rescued by intraperitoneal injection (IP) or by splenic injection (SP) of wild-type (wt) liver cells. Ten weeks after transplantation, mean biochemical measurements of various liver functions ± s.d. were compared between littermate wt controls, Fah^-/- mice under NTBC and untreated Fah^-/- mice (NTBC withheld for 5 weeks and experiencing hepatic failure). All animals were between 3 and 6 months old. The number of mice (serum) analyzed is indicated in parentheses. ALT stands for alanine aminotransferase. (B) Plasma concentration of Albumin, Fibrinogen and HGF after IP injection of wt hepatocytes in Fah^-/- mice at 1, 3, 4, 6 and 10 weeks. Controls correspond to normal wild type mice. Plasma samples were tested by ELISA. Each open circle represents the value from one mouse. t-tests were run using Prism to determine significant differences between particular groups. HGF increased at 6 weeks after the second and final selection (off NTBC) reflecting the massive expansion of hepatocytes in lymph nodes necessary for the Fah^-/- survival. Bars indicate mean values. For HGF * p_{control & 6wk} =0.0079 by Mann Whitney test. (C) Serum concentration of blood urea nitrogen (BUN), total cholesterol and triglycerides in Fah^-/- mice over 10 weeks after IP injection and rescue of the animals, and compared wild type mice with p values. (D) Glycogen storage in hepatized lymph nodes. Glycogen storage was determined by Periodic Acid Schiff (PAS) staining. Bar: 100μm. The black and white electron microscopy panel identified glyocogen rosettes (arrowheads) in hepatocytes. Bar: 500nm. (E) Fah enzyme assay. A standard curve to measure enzyme activity was established using wt liver (100% activity), Fah^-/- liver (0% activity) and wt/Fah^-/- mixes to achieve 15%, 25% and 80% enzyme activity. Fah enzyme activities in engrafted lymph nodes (LN) ranged from 80% to almost 100% of wt liver levels. In contrast, Fah enzyme activity in native livers of Fah^-/- mice rescued by hepatized LN had Fah activity ranging from 25% of wt liver activity to 0% (mean Fah activity of 15% of wt liver levels). n = number of mice analyzed.

Figure 4

Biochemical liver functions are restored by hepatized lymph nodes. (A) Biochemical measurement of liver function in blood. Tyrosinemic (Fah^-/-) mice were rescued by intraperitoneal injection (IP) or by splenic injection (SP) of wild-type (wt) liver cells. Ten weeks after transplantation, mean biochemical measurements of various liver functions ± s.d. were compared between littermate wt controls, Fah^-/- mice under NTBC and untreated Fah^-/- mice (NTBC withheld for 5 weeks and experiencing hepatic failure). All animals were between 3 and 6 months old. The number of mice (serum) analyzed is indicated in parentheses. ALT stands for alanine aminotransferase. (B) Plasma concentration of Albumin, Fibrinogen and HGF after IP injection of wt hepatocytes in Fah^-/- mice at 1, 3, 4, 6 and 10 weeks. Controls correspond to normal wild type mice. Plasma samples were tested by ELISA. Each open circle represents the value from one mouse. t-tests were run using Prism to determine significant differences between particular groups. HGF increased at 6 weeks after the second and final selection (off NTBC) reflecting the massive expansion of hepatocytes in lymph nodes necessary for the Fah^-/- survival. Bars indicate mean values. For HGF * p_{control & 6wk} =0.0079 by Mann Whitney test. (C) Serum concentration of blood urea nitrogen (BUN), total cholesterol and triglycerides in Fah^-/- mice over 10 weeks after IP injection and rescue of the animals, and compared wild type mice with p values. (D) Glycogen storage in hepatized lymph nodes. Glycogen storage was determined by Periodic Acid Schiff (PAS) staining. Bar: 100μm. The black and white electron microscopy panel identified glyocogen rosettes (arrowheads) in hepatocytes. Bar: 500nm. (E) Fah enzyme assay. A standard curve to measure enzyme activity was established using wt liver (100% activity), Fah^-/- liver (0% activity) and wt/Fah^-/- mixes to achieve 15%, 25% and 80% enzyme activity. Fah enzyme activities in engrafted lymph nodes (LN) ranged from 80% to almost 100% of wt liver levels. In contrast, Fah enzyme activity in native livers of Fah^-/- mice rescued by hepatized LN had Fah activity ranging from 25% of wt liver activity to 0% (mean Fah activity of 15% of wt liver levels). n = number of mice analyzed.

Figure 4

Biochemical liver functions are restored by hepatized lymph nodes. (A) Biochemical measurement of liver function in blood. Tyrosinemic (Fah^-/-) mice were rescued by intraperitoneal injection (IP) or by splenic injection (SP) of wild-type (wt) liver cells. Ten weeks after transplantation, mean biochemical measurements of various liver functions ± s.d. were compared between littermate wt controls, Fah^-/- mice under NTBC and untreated Fah^-/- mice (NTBC withheld for 5 weeks and experiencing hepatic failure). All animals were between 3 and 6 months old. The number of mice (serum) analyzed is indicated in parentheses. ALT stands for alanine aminotransferase. (B) Plasma concentration of Albumin, Fibrinogen and HGF after IP injection of wt hepatocytes in Fah^-/- mice at 1, 3, 4, 6 and 10 weeks. Controls correspond to normal wild type mice. Plasma samples were tested by ELISA. Each open circle represents the value from one mouse. t-tests were run using Prism to determine significant differences between particular groups. HGF increased at 6 weeks after the second and final selection (off NTBC) reflecting the massive expansion of hepatocytes in lymph nodes necessary for the Fah^-/- survival. Bars indicate mean values. For HGF * p_{control & 6wk} =0.0079 by Mann Whitney test. (C) Serum concentration of blood urea nitrogen (BUN), total cholesterol and triglycerides in Fah^-/- mice over 10 weeks after IP injection and rescue of the animals, and compared wild type mice with p values. (D) Glycogen storage in hepatized lymph nodes. Glycogen storage was determined by Periodic Acid Schiff (PAS) staining. Bar: 100μm. The black and white electron microscopy panel identified glyocogen rosettes (arrowheads) in hepatocytes. Bar: 500nm. (E) Fah enzyme assay. A standard curve to measure enzyme activity was established using wt liver (100% activity), Fah^-/- liver (0% activity) and wt/Fah^-/- mixes to achieve 15%, 25% and 80% enzyme activity. Fah enzyme activities in engrafted lymph nodes (LN) ranged from 80% to almost 100% of wt liver levels. In contrast, Fah enzyme activity in native livers of Fah^-/- mice rescued by hepatized LN had Fah activity ranging from 25% of wt liver activity to 0% (mean Fah activity of 15% of wt liver levels). n = number of mice analyzed.

Figure 5

Expansion of hepatized lymph nodes after hepatectomy. (A) Anatomic location of hepatized lymph nodes in IP injected Fah^-/- mice after hepatectomy. Enlarged nodules were found around the stomach region and on the mesenterium (yellow circles). (B) Native liver and extra-hepatic nodules after hepatectomy from the mouse on the left panel. The native tyrosinemic liver of the IP injected Fah^-/- mouse was atrophic and the enlarged nodules had a diameter from 3 to 15 mm. (C) The ratio ± sem of the weight of native liver and hepatized lymph nodes to body weight. The ratio of hepatic tissues to body weight was determined between transplanted Fah^-/- mice with or without hepatectomy. The hepatized lymph nodes show a significant increase in their weight after hepatectomy (P=0.0052). n = number of mice analyzed.

Figure 6

Serial transplantation of lymph node derived hepatocytes. Hepatocytes isolated from hepatized lymph nodes were serially transplanted into Fah^-/- mice by splenic (SP) injection. (A) Body weight of Fah^-/- mice after splenic transplantation. The body weight lost and spontaneous gain after lymph nodes derived hepatocytes transplantation is very similar to the change observed when liver derived hepatocytes are transplanted. Two selections were necessary due to the low number of hepatocytes transplanted. (B) Fah⁺ hepatocytes were observed only in the repopulated liver of Fah^-/- mice 8 weeks after transplantation. Counterstaining was done with eosin. Bar: 100μm.