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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: After 4th week of post-transplantation, the liver was perfused with 4% paraformaldehyde solution and stored in 10% buffered formalin solution. After dewaxing and dehydrating, the sections were incubated overnight with anti-mouse Alb (A) and CK18 (B) antibodies and the expression of these proteins was viewed by fluorescent microscopy using secondary antibody tagged with FITC. Both Alb and CK18 were highly expressed in MSC and MSC+HGF-CNP treated groups but not in control, HSC and HSC+HGF-CNP treated groups. It displayed the differentiation of MSC and augmentation of differentiation by HGF-CNP (Control magnification 100 ×).
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Figure 2: Immunofluoresent expression: After 4th week of post-transplantation, the liver was perfused with 4% paraformaldehyde solution and stored in 10% buffered formalin solution. After dewaxing and dehydrating, the sections were incubated overnight with anti-mouse Alb (A) and CK18 (B) antibodies and the expression of these proteins was viewed by fluorescent microscopy using secondary antibody tagged with FITC. Both Alb and CK18 were highly expressed in MSC and MSC+HGF-CNP treated groups but not in control, HSC and HSC+HGF-CNP treated groups. It displayed the differentiation of MSC and augmentation of differentiation by HGF-CNP (Control magnification 100 ×).

Mentions: To verify hepatic differentiation of transplanted stem cells and subsequent recovery of liver cirrhosis, the expression of liver markers such as Alb and CK18 was examined in control and treated groups by immunostaining. Alb and CK18 proteins increasingly expressed in the MSC/+HGF-CNP treated group compared to other groups (Figure 2A and 2B) despite continuous injection of CCl4 after post-transplantation. On the other hand, the expression of these markers was less in the control and HSC/+ HGF-CNP treated groups. This observation is in good correlation with the results of biochemical parameters. The results of both biochemical and immunohistochemical studies were further confirmed by the expression of mRNAs of Alb, CK18, AFP and CK19 proteins (Figure 3A). In semiquantitative analysis of gene expression, all of these genes expressed significantly (P < 0.05). HSC, even in the presence of HGF-CNP, did not show significant increase in the expression of these proteins and their levels were observed to be less than in MSC treated groups. Analysis of chimerism after cell transplantation is important for assessing the graft by the presence of donor cells. It is usually detected by the expression of specific gene by donor cells. Sry gene from donor male cells was detected by RT-PCR. Sry gene was expressed in all treatment groups; however, the control group did not show sry gene expression (Figure 3C).


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: After 4th week of post-transplantation, the liver was perfused with 4% paraformaldehyde solution and stored in 10% buffered formalin solution. After dewaxing and dehydrating, the sections were incubated overnight with anti-mouse Alb (A) and CK18 (B) antibodies and the expression of these proteins was viewed by fluorescent microscopy using secondary antibody tagged with FITC. Both Alb and CK18 were highly expressed in MSC and MSC+HGF-CNP treated groups but not in control, HSC and HSC+HGF-CNP treated groups. It displayed the differentiation of MSC and augmentation of differentiation by HGF-CNP (Control magnification 100 ×).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Immunofluoresent expression: After 4th week of post-transplantation, the liver was perfused with 4% paraformaldehyde solution and stored in 10% buffered formalin solution. After dewaxing and dehydrating, the sections were incubated overnight with anti-mouse Alb (A) and CK18 (B) antibodies and the expression of these proteins was viewed by fluorescent microscopy using secondary antibody tagged with FITC. Both Alb and CK18 were highly expressed in MSC and MSC+HGF-CNP treated groups but not in control, HSC and HSC+HGF-CNP treated groups. It displayed the differentiation of MSC and augmentation of differentiation by HGF-CNP (Control magnification 100 ×).
Mentions: To verify hepatic differentiation of transplanted stem cells and subsequent recovery of liver cirrhosis, the expression of liver markers such as Alb and CK18 was examined in control and treated groups by immunostaining. Alb and CK18 proteins increasingly expressed in the MSC/+HGF-CNP treated group compared to other groups (Figure 2A and 2B) despite continuous injection of CCl4 after post-transplantation. On the other hand, the expression of these markers was less in the control and HSC/+ HGF-CNP treated groups. This observation is in good correlation with the results of biochemical parameters. The results of both biochemical and immunohistochemical studies were further confirmed by the expression of mRNAs of Alb, CK18, AFP and CK19 proteins (Figure 3A). In semiquantitative analysis of gene expression, all of these genes expressed significantly (P < 0.05). HSC, even in the presence of HGF-CNP, did not show significant increase in the expression of these proteins and their levels were observed to be less than in MSC treated groups. Analysis of chimerism after cell transplantation is important for assessing the graft by the presence of donor cells. It is usually detected by the expression of specific gene by donor cells. Sry gene from donor male cells was detected by RT-PCR. Sry gene was expressed in all treatment groups; however, the control group did not show sry gene expression (Figure 3C).

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