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

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.

Keywords: Cell differentiation; Culture; Hepatocytes; Human multipotent adult progenitor cells; Stem cells.

Fig. 1

Expression of CD29, CD44, CD34, and human leukocyte antigen (HLA)-DR on human multipotent adult progenitor cells (hMAPCs). (A) The purity of CD45- GlyA- cells separated from bone marrow adherent cells was more than 98%, as determined by flow cytometry. Expression of CD29 (B), CD44 (C), CD34 (D), and HLA-DR (E) on hMAPCs. Panel F is for negative control. FITC, fluorescein isothiocyanate.

Fig. 2

Morphology observations during human multipotent adult progenitor cell (hMAPC) coculture (A) hMAPC growing morphology (6 generation, ×100). (B) hMAPC morphology on day 7, indirect coculture (×100). (C) Direct coculture morphology on day 5 (×100). (D) L-02 morphology (×100).

Fig. 3

Immunocytochemistry staining of indirect cocultures. (A) On day 1, indirect coculture, AFP cells (ABC method, DAB developing, ×200), Positive: light brown staining. (B) On day 5, indirect coculture, ALB cells (S-P method AEC developing, ×200), Positive: reddish brown staining. (C) On day 7, indirect coculture, cytokeratin 18 cells (ABC method, DAB developing, ×200), Positive: light brown staining. (D) L02 hepotocytes ALB cells (S-P method, AEC developing, positive control, ×200), Positive: red stain. (E) Undifferentiated human multipotent adult progenitor cells AFP immunocytochemistry (ABC method, DAB developing, no light brown staining, negative control, ×200). ABC, avidin-biotin complex; DAB, diaminobezidin; S-P, streptavidin-perosidase; AEC, 3-amino-9-ethylcarbazole.

Fig. 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.