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Hepatocyte growth factor incorporated chitosan nanoparticles augment the differentiation of stem cell into hepatocytes for the recovery of liver cirrhosis in mice.

Pulavendran S, Rose C, Mandal AB - J Nanobiotechnology (2011)

Bottom Line: Immunostaining for alpha smooth muscle actin (αSMA) and type I collagen showed decreased expression in the MSC+HGF-CNP treatment.These results indicated that HGF-CNP enhanced the differentiation of stem cells into hepatocytes and supported the reversal of fibrolysis of extracellular matrix (ECM).Biodegradable biopolymeric nanoparticles were prepared with the pleotrophic protein molecule and it worked well for the differentiation of stem cells, especially mesenchymal phenotypic cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Central Leather Research Institute, Adyar, Chennai-600020, India.

ABSTRACT

Background: Short half-life and low levels of growth factors in the niche of injured microenvironment necessitates the exogenous and sustainable delivery of growth factors along with stem cells to augment the regeneration of injured tissues.

Methods: Here, recombinant human hepatocyte growth factor (HGF) was incorporated into chitosan nanoparticles (CNP) by ionic gelation method and studied for its morphological and physiological characteristics. Cirrhotic mice received either hematopoietic stem cells (HSC) or mesenchymal stemcells (MSC) with or without HGF incorporated chitosan nanoparticles (HGF-CNP) and saline as control. Biochemical, histological, immunostaining and gene expression assays were carried out using serum and liver tissue samples. One way analysis of variance was used for statics application

Results: Serum levels of selected liver protein and enzymes were significantly increased in the combination of MSC and HGF-CNP (MSC+HGF-CNP) treated group. Immunopositive staining for albumin (Alb) and cytokeratin 18 (CK18), and reverse transcription-polymerase chain reaction (RT-PCR) for Alb, alpha fetoprotein (AFP), CK18, cytokeratin 19 (CK19) ascertained that MSC-HGF-CNP treatment could be an effective combination to repopulate liver parenchymal cells in the liver cirrhosis. Zymogram and western blotting for matrix metalloproteinases 2 and 9 (MMP2 and MMP9) revealed that MMP2 actively involved in the fibrolysis of cirrhotic tissue. Immunostaining for alpha smooth muscle actin (αSMA) and type I collagen showed decreased expression in the MSC+HGF-CNP treatment. These results indicated that HGF-CNP enhanced the differentiation of stem cells into hepatocytes and supported the reversal of fibrolysis of extracellular matrix (ECM).

Conclusion: Bone marrow stem cells were isolated, characterized and transplanted in mice model. Biodegradable biopolymeric nanoparticles were prepared with the pleotrophic protein molecule and it worked well for the differentiation of stem cells, especially mesenchymal phenotypic cells. Transplantation of bone marrow MSC in combination with HGF-CNP could be an ideal approach for the treatment of liver cirrhosis.

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Immunofluoresent expression of ECM proteins: After 4th week, livers were harvested after perfusing with 4% paraformaldehyde solution and stored in 10% buffered formalin solution. For immunofluoresence assay, the specimens after dewaxing and dehydrating were blocked for nonspecific protein using BSA. Then, the sections were incubated overnight with anti-mouse α-SMA antibody (A): Control (A1), HSC (A2), MSC (A3), HSC+HGF-CNP (A4) and MSC+HGF-CNP (A5); and anti-mouse type I collagen antibody (B): Control (B1), HSC (B2), MSC (B3), HSC+HGF-CNP (B4) and MSC+HGF-CNP (B5). The expression of these proteins was viewed by fluorescent microscopy using secondary antibody tagged with FITC. Both α-SMA and type I collagen expressed high in control, HSC and HSC+HGF-CNP treated groups than MSC and MSC-HGF-CNP treated groups. MSC treatment suppressed the myofibroblasts and consequently made type I collagen disappeared (original magnification ×100).
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Figure 5: Immunofluoresent expression of ECM proteins: After 4th week, livers were harvested after perfusing with 4% paraformaldehyde solution and stored in 10% buffered formalin solution. For immunofluoresence assay, the specimens after dewaxing and dehydrating were blocked for nonspecific protein using BSA. Then, the sections were incubated overnight with anti-mouse α-SMA antibody (A): Control (A1), HSC (A2), MSC (A3), HSC+HGF-CNP (A4) and MSC+HGF-CNP (A5); and anti-mouse type I collagen antibody (B): Control (B1), HSC (B2), MSC (B3), HSC+HGF-CNP (B4) and MSC+HGF-CNP (B5). The expression of these proteins was viewed by fluorescent microscopy using secondary antibody tagged with FITC. Both α-SMA and type I collagen expressed high in control, HSC and HSC+HGF-CNP treated groups than MSC and MSC-HGF-CNP treated groups. MSC treatment suppressed the myofibroblasts and consequently made type I collagen disappeared (original magnification ×100).

Mentions: Inflammatory cascade activates the quiescent hepatic stellate cells into myofibroblasts which was confirmed by the immunostaining of α-SMA (an indicator of activated myofibroblasts). Secretion of type I collagen by myofibroblasts that leads to fibrosis was analyzed by immunofluorescence. The results of imunofluorescence study for distribution of α-SMA positive cells around the sinusoid portion (Figure 5A) confirmed the histological observation (Figure 4D) in respect of the presence of large number of nonparenchymal cells with consistant morphology of activated myofibroblast-like cells clustered around the fibrotic septae at the end of fourth week in control, HSC or HSC+HGF-CNP treated groups. Increased expression of α-SMA in the periportal region of liver of control, or HSC+HGF-CNP treated groups and the reduced expression of this protein in MSC and MSC+HGF-CNP groups were also noticed. Decreased expression of type I collagen, due to reduced myofibroblast activity, in MSC or MSC+HGF-CNP compared to control and other treated groups is shown in Figure 5B.


Hepatocyte growth factor incorporated chitosan nanoparticles augment the differentiation of stem cell into hepatocytes for the recovery of liver cirrhosis in mice.

Pulavendran S, Rose C, Mandal AB - J Nanobiotechnology (2011)

Immunofluoresent expression of ECM proteins: After 4th week, livers were harvested after perfusing with 4% paraformaldehyde solution and stored in 10% buffered formalin solution. For immunofluoresence assay, the specimens after dewaxing and dehydrating were blocked for nonspecific protein using BSA. Then, the sections were incubated overnight with anti-mouse α-SMA antibody (A): Control (A1), HSC (A2), MSC (A3), HSC+HGF-CNP (A4) and MSC+HGF-CNP (A5); and anti-mouse type I collagen antibody (B): Control (B1), HSC (B2), MSC (B3), HSC+HGF-CNP (B4) and MSC+HGF-CNP (B5). The expression of these proteins was viewed by fluorescent microscopy using secondary antibody tagged with FITC. Both α-SMA and type I collagen expressed high in control, HSC and HSC+HGF-CNP treated groups than MSC and MSC-HGF-CNP treated groups. MSC treatment suppressed the myofibroblasts and consequently made type I collagen disappeared (original magnification ×100).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3108285&req=5

Figure 5: Immunofluoresent expression of ECM proteins: After 4th week, livers were harvested after perfusing with 4% paraformaldehyde solution and stored in 10% buffered formalin solution. For immunofluoresence assay, the specimens after dewaxing and dehydrating were blocked for nonspecific protein using BSA. Then, the sections were incubated overnight with anti-mouse α-SMA antibody (A): Control (A1), HSC (A2), MSC (A3), HSC+HGF-CNP (A4) and MSC+HGF-CNP (A5); and anti-mouse type I collagen antibody (B): Control (B1), HSC (B2), MSC (B3), HSC+HGF-CNP (B4) and MSC+HGF-CNP (B5). The expression of these proteins was viewed by fluorescent microscopy using secondary antibody tagged with FITC. Both α-SMA and type I collagen expressed high in control, HSC and HSC+HGF-CNP treated groups than MSC and MSC-HGF-CNP treated groups. MSC treatment suppressed the myofibroblasts and consequently made type I collagen disappeared (original magnification ×100).
Mentions: Inflammatory cascade activates the quiescent hepatic stellate cells into myofibroblasts which was confirmed by the immunostaining of α-SMA (an indicator of activated myofibroblasts). Secretion of type I collagen by myofibroblasts that leads to fibrosis was analyzed by immunofluorescence. The results of imunofluorescence study for distribution of α-SMA positive cells around the sinusoid portion (Figure 5A) confirmed the histological observation (Figure 4D) in respect of the presence of large number of nonparenchymal cells with consistant morphology of activated myofibroblast-like cells clustered around the fibrotic septae at the end of fourth week in control, HSC or HSC+HGF-CNP treated groups. Increased expression of α-SMA in the periportal region of liver of control, or HSC+HGF-CNP treated groups and the reduced expression of this protein in MSC and MSC+HGF-CNP groups were also noticed. Decreased expression of type I collagen, due to reduced myofibroblast activity, in MSC or MSC+HGF-CNP compared to control and other treated groups is shown in Figure 5B.

Bottom Line: Immunostaining for alpha smooth muscle actin (αSMA) and type I collagen showed decreased expression in the MSC+HGF-CNP treatment.These results indicated that HGF-CNP enhanced the differentiation of stem cells into hepatocytes and supported the reversal of fibrolysis of extracellular matrix (ECM).Biodegradable biopolymeric nanoparticles were prepared with the pleotrophic protein molecule and it worked well for the differentiation of stem cells, especially mesenchymal phenotypic cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biotechnology, Central Leather Research Institute, Adyar, Chennai-600020, India.

ABSTRACT

Background: Short half-life and low levels of growth factors in the niche of injured microenvironment necessitates the exogenous and sustainable delivery of growth factors along with stem cells to augment the regeneration of injured tissues.

Methods: Here, recombinant human hepatocyte growth factor (HGF) was incorporated into chitosan nanoparticles (CNP) by ionic gelation method and studied for its morphological and physiological characteristics. Cirrhotic mice received either hematopoietic stem cells (HSC) or mesenchymal stemcells (MSC) with or without HGF incorporated chitosan nanoparticles (HGF-CNP) and saline as control. Biochemical, histological, immunostaining and gene expression assays were carried out using serum and liver tissue samples. One way analysis of variance was used for statics application

Results: Serum levels of selected liver protein and enzymes were significantly increased in the combination of MSC and HGF-CNP (MSC+HGF-CNP) treated group. Immunopositive staining for albumin (Alb) and cytokeratin 18 (CK18), and reverse transcription-polymerase chain reaction (RT-PCR) for Alb, alpha fetoprotein (AFP), CK18, cytokeratin 19 (CK19) ascertained that MSC-HGF-CNP treatment could be an effective combination to repopulate liver parenchymal cells in the liver cirrhosis. Zymogram and western blotting for matrix metalloproteinases 2 and 9 (MMP2 and MMP9) revealed that MMP2 actively involved in the fibrolysis of cirrhotic tissue. Immunostaining for alpha smooth muscle actin (αSMA) and type I collagen showed decreased expression in the MSC+HGF-CNP treatment. These results indicated that HGF-CNP enhanced the differentiation of stem cells into hepatocytes and supported the reversal of fibrolysis of extracellular matrix (ECM).

Conclusion: Bone marrow stem cells were isolated, characterized and transplanted in mice model. Biodegradable biopolymeric nanoparticles were prepared with the pleotrophic protein molecule and it worked well for the differentiation of stem cells, especially mesenchymal phenotypic cells. Transplantation of bone marrow MSC in combination with HGF-CNP could be an ideal approach for the treatment of liver cirrhosis.

Show MeSH
Related in: MedlinePlus