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Proteomic profiles of mesenchymal stem cells induced by a liver differentiation protocol.

Leelawat K, Narong S, Chaijan S, Sa-Ngiamsuntorn K, Disthabanchong S, Wongkajornsilp A, Hongeng S - Int J Mol Sci (2010)

Bottom Line: We demonstrated that MSCs treated with the liver differentiation protocol expressed significantly more albumin, CK19 and CK20, than did undifferentiated cells.In addition the results of proteomic study demonstrated increases expression of FEM1B, PSMC2 and disulfide-isomerase A3 in MSCs treated with the liver differentiation protocol.These results from proteomic profiling will not only provide insight into the global responses of MSCs to hepatocyte differentiation, but will also lead to in-depth studies on the mechanisms of proteomic changes in MSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Rajavithi Hospital, Rajathevi, Bangkok, 10400, Thailand; E-Mails: kawin.leelawat@gmail.com (K.L.); sirilucknarong@hotmail.com (S.N.); bubu_b600@hotmail.com (S.C.).

ABSTRACT
The replacement of disease hepatocytes and the stimulation of endogenous or exogenous regeneration by human mesenchymal stem cells (MSCs) are promising candidates for liver-directed cell therapy. In this study, we isolated MSCs from adult bone marrow by plastic adhesion and induced differentiation with a liver differentiation protocol. Western blot analyses were used to assess the expression of liver-specific markers. Next, MSC-specific proteins were analyzed with two-dimensional (2D) gel electrophoresis and peptide mass fingerprinting matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS). To confirm the results from the proteomic study, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed. We demonstrated that MSCs treated with the liver differentiation protocol expressed significantly more albumin, CK19 and CK20, than did undifferentiated cells. In addition the results of proteomic study demonstrated increases expression of FEM1B, PSMC2 and disulfide-isomerase A3 in MSCs treated with the liver differentiation protocol. These results from proteomic profiling will not only provide insight into the global responses of MSCs to hepatocyte differentiation, but will also lead to in-depth studies on the mechanisms of proteomic changes in MSCs.

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(A) Detection of glycogen in the cytoplasm of MSCs treated with liver differentiation protocol was demonstrated by PAS staining. PAS positive substances stain pink in the cytoplasm of the cells; (B) The urea production was determined. Cells were treated with liver differentiation protocol or control vehicle (DMSO) for 21 days. Studies were done in triplicate and repeated twice. Data represent the mean ± SD of three independent experiments (significant as compared with control, * p < 0.001).
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f3-ijms-11-04905: (A) Detection of glycogen in the cytoplasm of MSCs treated with liver differentiation protocol was demonstrated by PAS staining. PAS positive substances stain pink in the cytoplasm of the cells; (B) The urea production was determined. Cells were treated with liver differentiation protocol or control vehicle (DMSO) for 21 days. Studies were done in triplicate and repeated twice. Data represent the mean ± SD of three independent experiments (significant as compared with control, * p < 0.001).

Mentions: As revealed by morphological studies, MSCs cultured in the liver differentiation media for four weeks adopted polygonal cell morphology. The nucleus and cytoplasm appeared granulated (Figure 1) and were still viable after 60 days. To verify whether these differentiated cells had the characteristic expression of hepatic phenotypic markers, protein from undifferentiated and differentiated cells was extracted. Western blot analyses demonstrated that differentiated MSCs expressed significantly more albumin, CK19 and CK20 than did undifferentiated cells (control) (Figure 2A). In addition, the expression of albumin was also found in differentiated MSCs after 60 days (Figure 2B). The presence of glycogen in the cytoplasm of differentiated MSCs was demonstrated by PAS staining (Figure 3A). Additionally, MSCs themselves did not produce urea. When MSCs were cultured for three weeks in the liver differentiation media, urea was secreted in the supernatant (Figure 3B).


Proteomic profiles of mesenchymal stem cells induced by a liver differentiation protocol.

Leelawat K, Narong S, Chaijan S, Sa-Ngiamsuntorn K, Disthabanchong S, Wongkajornsilp A, Hongeng S - Int J Mol Sci (2010)

(A) Detection of glycogen in the cytoplasm of MSCs treated with liver differentiation protocol was demonstrated by PAS staining. PAS positive substances stain pink in the cytoplasm of the cells; (B) The urea production was determined. Cells were treated with liver differentiation protocol or control vehicle (DMSO) for 21 days. Studies were done in triplicate and repeated twice. Data represent the mean ± SD of three independent experiments (significant as compared with control, * p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3100820&req=5

f3-ijms-11-04905: (A) Detection of glycogen in the cytoplasm of MSCs treated with liver differentiation protocol was demonstrated by PAS staining. PAS positive substances stain pink in the cytoplasm of the cells; (B) The urea production was determined. Cells were treated with liver differentiation protocol or control vehicle (DMSO) for 21 days. Studies were done in triplicate and repeated twice. Data represent the mean ± SD of three independent experiments (significant as compared with control, * p < 0.001).
Mentions: As revealed by morphological studies, MSCs cultured in the liver differentiation media for four weeks adopted polygonal cell morphology. The nucleus and cytoplasm appeared granulated (Figure 1) and were still viable after 60 days. To verify whether these differentiated cells had the characteristic expression of hepatic phenotypic markers, protein from undifferentiated and differentiated cells was extracted. Western blot analyses demonstrated that differentiated MSCs expressed significantly more albumin, CK19 and CK20 than did undifferentiated cells (control) (Figure 2A). In addition, the expression of albumin was also found in differentiated MSCs after 60 days (Figure 2B). The presence of glycogen in the cytoplasm of differentiated MSCs was demonstrated by PAS staining (Figure 3A). Additionally, MSCs themselves did not produce urea. When MSCs were cultured for three weeks in the liver differentiation media, urea was secreted in the supernatant (Figure 3B).

Bottom Line: We demonstrated that MSCs treated with the liver differentiation protocol expressed significantly more albumin, CK19 and CK20, than did undifferentiated cells.In addition the results of proteomic study demonstrated increases expression of FEM1B, PSMC2 and disulfide-isomerase A3 in MSCs treated with the liver differentiation protocol.These results from proteomic profiling will not only provide insight into the global responses of MSCs to hepatocyte differentiation, but will also lead to in-depth studies on the mechanisms of proteomic changes in MSCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Rajavithi Hospital, Rajathevi, Bangkok, 10400, Thailand; E-Mails: kawin.leelawat@gmail.com (K.L.); sirilucknarong@hotmail.com (S.N.); bubu_b600@hotmail.com (S.C.).

ABSTRACT
The replacement of disease hepatocytes and the stimulation of endogenous or exogenous regeneration by human mesenchymal stem cells (MSCs) are promising candidates for liver-directed cell therapy. In this study, we isolated MSCs from adult bone marrow by plastic adhesion and induced differentiation with a liver differentiation protocol. Western blot analyses were used to assess the expression of liver-specific markers. Next, MSC-specific proteins were analyzed with two-dimensional (2D) gel electrophoresis and peptide mass fingerprinting matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS). To confirm the results from the proteomic study, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed. We demonstrated that MSCs treated with the liver differentiation protocol expressed significantly more albumin, CK19 and CK20, than did undifferentiated cells. In addition the results of proteomic study demonstrated increases expression of FEM1B, PSMC2 and disulfide-isomerase A3 in MSCs treated with the liver differentiation protocol. These results from proteomic profiling will not only provide insight into the global responses of MSCs to hepatocyte differentiation, but will also lead to in-depth studies on the mechanisms of proteomic changes in MSCs.

Show MeSH
Related in: MedlinePlus