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The differentiation of human multipotent adult progenitor cells into hepatocyte-like cells induced by coculture with human hepatocyte line L02.

Mu N, Liu HB, Meng QH, Du DW, Jiang Y, Hu HZ - Ann Surg Treat Res (2014)

Bottom Line: With the MACS method, (5-10) × 10(4)/mL hMAPCs could be separated from 1 × 10(6)/mL bone marrow mononuclear cells.The purity of CD45-/GlyA- cells separated from bone marrow adherent cells was more than 98%, as determined by flow cytometry.In the coculture without cell-to-cell contact, hMAPCs expressed high AFP on day 1, and then tapered daily to low expression on day 7; ALB expression reached its peak on day 5, and remained high on day 7; CK18 was initially expressed on day 5 and was higher on day 7; CK19 was negative in all assays.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatobiliary Surgery, Fuzhou General Hospital, Fuzhou, China.

ABSTRACT

Purpose: The aim of this study was to establish an in vitro method to purify human multipotent adult progenitor cells (hMAPCs) and assess their possible differentiation into hepatocytes by coculture with human hepatocyte line L02.

Methods: hMAPCs were isolated by magnetic activated cell sorting (MACS) depletion selection using CD45 and GlyA microbeads. After indirect or direct coculture of hMAPCs and human hepatocyte line L02, the expression of albumin (ALB), alpha-fetoprotein (AFP), cytokeratin (CK) 18, and CK19 by hMAPCs was detected by immunocytochemistry.

Results: With the MACS method, (5-10) × 10(4)/mL hMAPCs could be separated from 1 × 10(6)/mL bone marrow mononuclear cells. The purity of CD45-/GlyA- cells separated from bone marrow adherent cells was more than 98%, as determined by flow cytometry. In the coculture without cell-to-cell contact, hMAPCs expressed high AFP on day 1, and then tapered daily to low expression on day 7; ALB expression reached its peak on day 5, and remained high on day 7; CK18 was initially expressed on day 5 and was higher on day 7; CK19 was negative in all assays. In the coculture with cell-to-cell contact, ALB and CK18 were expressed by most cells while AFP appeared in only a few on day 5.

Conclusion: hMAPCs were induced to differentiate into mature hepatocyte-like cells by coculture with a hepatocyte cell line, either with or without cell-to-cell contact, but the former seemed more effective.

No MeSH data available.


Immunofluorescence staining of direct cocultures by laser scanning confocal microscope (LSCM). (A) On day 5, direct coculture, ALB immunofluorescence LSCM (×400). (B) On day 5, direct coculture, CK18 immunofluorescence LSCM (×400). (C) On day 5, direct coculture, AFP immunofluorescence LSCM (×400). (D) Separately cultured L02 hepatocytes, ALB immunofluorescence LSCM (positive control, ×400). (E) Separately cultured human multipotent adult progenitor cell (hMAPC), ALB immunofluorescence LSCM (negative control, ×400). CFSE and SABC-Cy3 double staining indirectly, Positive cells: yellow fluorescence. ALB, albumin; AFP, alpha-fetoprotein; CFSE, carboxyfluorescein diacetate succinimidyl ester; SABC, strept avidinbiotin complex; CK18, cytokeratin 18.
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Figure 4: Immunofluorescence staining of direct cocultures by laser scanning confocal microscope (LSCM). (A) On day 5, direct coculture, ALB immunofluorescence LSCM (×400). (B) On day 5, direct coculture, CK18 immunofluorescence LSCM (×400). (C) On day 5, direct coculture, AFP immunofluorescence LSCM (×400). (D) Separately cultured L02 hepatocytes, ALB immunofluorescence LSCM (positive control, ×400). (E) Separately cultured human multipotent adult progenitor cell (hMAPC), ALB immunofluorescence LSCM (negative control, ×400). CFSE and SABC-Cy3 double staining indirectly, Positive cells: yellow fluorescence. ALB, albumin; AFP, alpha-fetoprotein; CFSE, carboxyfluorescein diacetate succinimidyl ester; SABC, strept avidinbiotin complex; CK18, cytokeratin 18.

Mentions: In the direct coculture of hMAPCs and human hepatocyte line L02 (with cell-to-cell contact), on day 5, the expression of ALB, AFP, and CK18 on hMAPCs was determined by LSCM (as qualitative observation). The yellow immunofluorescence means expression of ALB and CK18 appeared on many cells, while AFP appeared on only a few (Fig. 4A-C).


The differentiation of human multipotent adult progenitor cells into hepatocyte-like cells induced by coculture with human hepatocyte line L02.

Mu N, Liu HB, Meng QH, Du DW, Jiang Y, Hu HZ - Ann Surg Treat Res (2014)

Immunofluorescence staining of direct cocultures by laser scanning confocal microscope (LSCM). (A) On day 5, direct coculture, ALB immunofluorescence LSCM (×400). (B) On day 5, direct coculture, CK18 immunofluorescence LSCM (×400). (C) On day 5, direct coculture, AFP immunofluorescence LSCM (×400). (D) Separately cultured L02 hepatocytes, ALB immunofluorescence LSCM (positive control, ×400). (E) Separately cultured human multipotent adult progenitor cell (hMAPC), ALB immunofluorescence LSCM (negative control, ×400). CFSE and SABC-Cy3 double staining indirectly, Positive cells: yellow fluorescence. ALB, albumin; AFP, alpha-fetoprotein; CFSE, carboxyfluorescein diacetate succinimidyl ester; SABC, strept avidinbiotin complex; CK18, cytokeratin 18.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4279986&req=5

Figure 4: Immunofluorescence staining of direct cocultures by laser scanning confocal microscope (LSCM). (A) On day 5, direct coculture, ALB immunofluorescence LSCM (×400). (B) On day 5, direct coculture, CK18 immunofluorescence LSCM (×400). (C) On day 5, direct coculture, AFP immunofluorescence LSCM (×400). (D) Separately cultured L02 hepatocytes, ALB immunofluorescence LSCM (positive control, ×400). (E) Separately cultured human multipotent adult progenitor cell (hMAPC), ALB immunofluorescence LSCM (negative control, ×400). CFSE and SABC-Cy3 double staining indirectly, Positive cells: yellow fluorescence. ALB, albumin; AFP, alpha-fetoprotein; CFSE, carboxyfluorescein diacetate succinimidyl ester; SABC, strept avidinbiotin complex; CK18, cytokeratin 18.
Mentions: In the direct coculture of hMAPCs and human hepatocyte line L02 (with cell-to-cell contact), on day 5, the expression of ALB, AFP, and CK18 on hMAPCs was determined by LSCM (as qualitative observation). The yellow immunofluorescence means expression of ALB and CK18 appeared on many cells, while AFP appeared on only a few (Fig. 4A-C).

Bottom Line: With the MACS method, (5-10) × 10(4)/mL hMAPCs could be separated from 1 × 10(6)/mL bone marrow mononuclear cells.The purity of CD45-/GlyA- cells separated from bone marrow adherent cells was more than 98%, as determined by flow cytometry.In the coculture without cell-to-cell contact, hMAPCs expressed high AFP on day 1, and then tapered daily to low expression on day 7; ALB expression reached its peak on day 5, and remained high on day 7; CK18 was initially expressed on day 5 and was higher on day 7; CK19 was negative in all assays.

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatobiliary Surgery, Fuzhou General Hospital, Fuzhou, China.

ABSTRACT

Purpose: The aim of this study was to establish an in vitro method to purify human multipotent adult progenitor cells (hMAPCs) and assess their possible differentiation into hepatocytes by coculture with human hepatocyte line L02.

Methods: hMAPCs were isolated by magnetic activated cell sorting (MACS) depletion selection using CD45 and GlyA microbeads. After indirect or direct coculture of hMAPCs and human hepatocyte line L02, the expression of albumin (ALB), alpha-fetoprotein (AFP), cytokeratin (CK) 18, and CK19 by hMAPCs was detected by immunocytochemistry.

Results: With the MACS method, (5-10) × 10(4)/mL hMAPCs could be separated from 1 × 10(6)/mL bone marrow mononuclear cells. The purity of CD45-/GlyA- cells separated from bone marrow adherent cells was more than 98%, as determined by flow cytometry. In the coculture without cell-to-cell contact, hMAPCs expressed high AFP on day 1, and then tapered daily to low expression on day 7; ALB expression reached its peak on day 5, and remained high on day 7; CK18 was initially expressed on day 5 and was higher on day 7; CK19 was negative in all assays. In the coculture with cell-to-cell contact, ALB and CK18 were expressed by most cells while AFP appeared in only a few on day 5.

Conclusion: hMAPCs were induced to differentiate into mature hepatocyte-like cells by coculture with a hepatocyte cell line, either with or without cell-to-cell contact, but the former seemed more effective.

No MeSH data available.