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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) Proteome map of protein extraction from MSC control and treatment. Identification of proteins were performed by 2D gel electrophoresis follow by MALDI-TOF-MS. Isoelectric focusing were performed with 50 μg total proteins from MSC using 3–10 pH-strips, 18 cm. SDS-PAGE were performed on 12.5% gels and stained with Coomassie blue G-250; (B) Total RNA was isolated from MSCs cells treated with or without liver differentiation protocol and liver specimens. Expression of FEM1B, PSMC2 and disulfide-isomerase A3 mRNA levels was evaluated using RT-PCR. The results, based on the ratio of these mRNA amplification to that of GAPDH, are presented as the fold increase relative to the mRNA levels from liver specimens. The results are expressed as the mean ± SD of three separate experiments (* p < 0.001, ** p = 0.01 versus the control MSC).
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f4-ijms-11-04905: (A) Proteome map of protein extraction from MSC control and treatment. Identification of proteins were performed by 2D gel electrophoresis follow by MALDI-TOF-MS. Isoelectric focusing were performed with 50 μg total proteins from MSC using 3–10 pH-strips, 18 cm. SDS-PAGE were performed on 12.5% gels and stained with Coomassie blue G-250; (B) Total RNA was isolated from MSCs cells treated with or without liver differentiation protocol and liver specimens. Expression of FEM1B, PSMC2 and disulfide-isomerase A3 mRNA levels was evaluated using RT-PCR. The results, based on the ratio of these mRNA amplification to that of GAPDH, are presented as the fold increase relative to the mRNA levels from liver specimens. The results are expressed as the mean ± SD of three separate experiments (* p < 0.001, ** p = 0.01 versus the control MSC).

Mentions: We used a proteomic approach to profile the protein expression in MSCs treated with the liver differentiation protocol. The proteins in the cell lysates were separated by two-dimensional electrophoresis followed by Coomassie staining. A representative two-dimensional gel image of protein lysates from MSCs with and without being cultured in the liver differentiation protocol is shown in Figure 4A. These protein spots, encompassing a wide range of molecular weights, pI values, and abundance were resolved and identified with high confidence (95%). We demonstrated that increases in vimentin, FEM1B, PSMC2 and disulfide-isomerase A3 expression were found in MSCs treated with the liver differentiation protocol.


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) Proteome map of protein extraction from MSC control and treatment. Identification of proteins were performed by 2D gel electrophoresis follow by MALDI-TOF-MS. Isoelectric focusing were performed with 50 μg total proteins from MSC using 3–10 pH-strips, 18 cm. SDS-PAGE were performed on 12.5% gels and stained with Coomassie blue G-250; (B) Total RNA was isolated from MSCs cells treated with or without liver differentiation protocol and liver specimens. Expression of FEM1B, PSMC2 and disulfide-isomerase A3 mRNA levels was evaluated using RT-PCR. The results, based on the ratio of these mRNA amplification to that of GAPDH, are presented as the fold increase relative to the mRNA levels from liver specimens. The results are expressed as the mean ± SD of three separate experiments (* p < 0.001, ** p = 0.01 versus the control MSC).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4-ijms-11-04905: (A) Proteome map of protein extraction from MSC control and treatment. Identification of proteins were performed by 2D gel electrophoresis follow by MALDI-TOF-MS. Isoelectric focusing were performed with 50 μg total proteins from MSC using 3–10 pH-strips, 18 cm. SDS-PAGE were performed on 12.5% gels and stained with Coomassie blue G-250; (B) Total RNA was isolated from MSCs cells treated with or without liver differentiation protocol and liver specimens. Expression of FEM1B, PSMC2 and disulfide-isomerase A3 mRNA levels was evaluated using RT-PCR. The results, based on the ratio of these mRNA amplification to that of GAPDH, are presented as the fold increase relative to the mRNA levels from liver specimens. The results are expressed as the mean ± SD of three separate experiments (* p < 0.001, ** p = 0.01 versus the control MSC).
Mentions: We used a proteomic approach to profile the protein expression in MSCs treated with the liver differentiation protocol. The proteins in the cell lysates were separated by two-dimensional electrophoresis followed by Coomassie staining. A representative two-dimensional gel image of protein lysates from MSCs with and without being cultured in the liver differentiation protocol is shown in Figure 4A. These protein spots, encompassing a wide range of molecular weights, pI values, and abundance were resolved and identified with high confidence (95%). We demonstrated that increases in vimentin, FEM1B, PSMC2 and disulfide-isomerase A3 expression were found in MSCs treated with the liver differentiation protocol.

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