Persistence of a chimerical phenotype after hepatocyte differentiation of human bone marrow mesenchymal stem cells

Abstract

Objectives: Recent studies have suggested the potential of mesenchymal stem cells (MSCs) to differentiate into a hepatocyte-like lineage. Here, we evaluate the efficacy of hepatocyte differentiation of MSCs by studying acquisition of hepatocyte-like features together with alteration of the native mesenchymal phenotype.

Material and methods: In vitro, we have investigated protein and mRNA level expression of hepatocyte and mesenchymal markers of mesenchymal-derived hepatocyte-like cells (MDHLCs) and we have evaluated their functionality using metabolic assays. In vivo, we investigated co-expression of hepatocyte (albumin, alpha-foetoprotein, cytokeratin 18) and mesenchymal (fibronectin, vimentin) markers after transplantation of MSCs or MDHLCs into severe combined immune deficiency mice.

Results: We observed that while in vitro these cells acquired some phenotypic and functional features of mature hepatocytes, they partially preserved their mesenchymal phenotype. After intrasplenic transplantation, engrafted MSCs with isolated expression of fibronectin and alpha-foetoprotein were observed. When these cells were injected into the liver, they expressed all analysed markers, confirming the chimaeric co-expression observed in vitro. Conversely, liver-engrafted MDHLCs conserved their hepatocyte-lineage markers but lost their chimaeric phenotype.

Conclusions: Hepatocyte differentiation of MSCs predominantly allows the acquisition of phenotypic hallmarks and provides chimaeric cells that maintain expression of initial lineage markers. However, advanced maturation to the hepatocyte-like phenotype could be obtained in vivo by conditioning MSCs prior to transplantation or by infusing cells into the liver micro-environment.

Figure 1

Characterization of MSCs. Example of MSCs morphology at passage 1, day 15 (a) that was altered at passage 8, day 99 (b). Expression of mesodermal antigens in MSCs at passage 2 as revealed by immunofluorescence [vimentin (c), fibronectin (d), ASMA (e), laminin (f), n = 3 each]. Staining of extracellular calcium matrix deposition by von Kossa (g, h) and alizarin red (j) colorations in MSCs after osseous differentiation. Accumulation of intracytoplasmic lipid‐rich droplets revealed by oil red O staining (l, m) after adipocyte differentiation of MSCs. Respective controls are provided with undifferentiated MSCs (i, k, n). Example of representative immunophenotype of MSCs at passage 2 (o). Analyzed epitopes are indicated in the histograms. Bars indicate the fluorescence level of corresponding isotype. Pictures magnifications are: ×100 (b, g), ×200 (j), ×300 (c–f) and ×400 (a, i, k–n).

Figure 2

Description of the data collected for the elaboration of the complete hepatocyte differentiation protocol. (a) Description of the cytokines and factors tested in combinations (n = 30) following a two‐step procedure. Column A describes the factors invariably used in the protocols. Column B lists the factors that were added independently or in a combined manner (for step 1). (b) Description of the most representative hepatocyte differentiation protocols designed by combination of the cytokines/factors described in table A. Factors as dexa (dexamethasone), ascorbate (ascorbid acid 2‐phosphate), ITS and nicot (nicotinamide) were not considered in the description. (c) Pictures showing the results obtained after completion of the protocols described in table B (30 days procedures) in terms of morphology analysed by optic microscopy (OM), albumin content assayed by immunocytochemistry and glycogen storage depicted by periodic acid‐Schiff (PAS) staining. Morphology and PAS staining pictures were taken at magnification ×400. Albumin staining pictures were taken at magnification ×200. (d) Calculation, for each group of differentiation, of the amount of cells displaying a hepatocyte‐like morphology or a positive staining for albumin or glycogen. Whereas similar morphological changes were observed in each procedure, the highest albumin and glycogen‐positive cell content was noticed in protocol no. 7. Values are presented as mean ± SD. Total cell count for each experiment is provided between brackets. (e) Values of urea secretion for each differentiation protocol at 30 days (n = 6 for each experiment). Urea production of the protocol no. 7 was significantly higher than all of the other protocols. P values are indicated in the graph (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3

In vitro hepatocyte differentiation of human bone marrow‐MSCs. (a) Comparative aspect of MSCs, MDHLCs and HHs as observed in optic (OM) and electron (EM) microscopy. Pictures show that MDHLCs acquire round or polygonal‐shaped cells reduced in size and containing cytoplasmic granulations and central nucleus with prominent nucleolus. HHs comparative cell size was smaller. EM reveals that MDHLCs exhibited a higher content in cytoplasmic organites (endoplasmic reticulum, mitochondria) and a prominent nucleolus suggesting their enhanced metabolic activity. They produced intracytoplasmic glycogen vacuoles that are a hallmark of hepatocyte‐like functionality. Comparison with MSCs and HHs is provided. OM pictures were taken at magnification ×400 unless indicated and EM pictures magnification was ×4000. (b) MDHLCs immunostaining for hepatocyte markers showing positive expression of albumin, αFP, DPPIV and some epithelial markers (E‐cadherin, CX‐32) but not of HepPar‐1, CK8 and CK18. MSCs and HHs stainings were provided as negative and positive controls, respectively. Pictures were taken at magnification ×400. (c) Representative examples of albumin staining by flow cytometry in MDHLCs and MSCs. Bars indicate the fluorescence level of the corresponding isotype. αFP, α‐foetoprotein; CK18, cytokeratin 18; CX‐32, connexin‐32; DPPIV, dipeptidylpeptidase IV; HHs, human primary hepatocytes; MDHLCs, mesenchymal‐derived hepatocyte‐like cells; MSCs, mesenchymal stem cells.

Figure 4

Analysis of hepatocyte and mesodermal markers expression by RT‐PCR in MDHLCs, MSCs (both on passage 1 to 3) and freshly isolated human liver cell suspension (LCS). The figure shows concomitant immature (αFP, CK8) and mature (albumin, α1AT, G6Pase, PEPCK, TAT, TDO) hepatocyte gene expression in MDHLCs while other markers are absent (CK18, CYP2A3, CYP2B6, HNF4). Both MSCs and MDHLCs expressed HGF receptor c‐met. Controls without reverse transcriptase (Cont) are shown for each analysed primer. Abbreviations of oligonucleotides are listed in Table 1.

Figure 5

Characterization of the chimerical phenotype of MDHLCs. (a) Immunofluorescence assay showing co‐staining of MDHLCs, HHs and MSCs for albumin/fibronectin (1), ASMA/αFP (2), DPPIV/fibronectin (3), E‐cadherin/fibronectin (4), CX‐32/fibronectin (5) and vimentin/albumin (6). Pictures were taken at magnification ×300. (b) RT‐PCR analysis revealing the persistence of vimentin, ASMA and fibronectin expression in MDHLCs population. (c) Flow cytometry assay showing a representative example of albumin, CD73 and CD90 co‐staining on MDHLCs. (d) Comparative flow cytometric phenotype of MSCs (passage 2 to 4), MDHLCs (passage 1 to 3), and human hepatocytes. MDHLCs and hepatocytes are specified respectively by (‐d) and (‐h) suffixes. P values are indicated below graph (*P < 0.05, **P < 0.01, ***P < 0.001). αFP, α‐foetoprotein; ASMA, α‐smooth muscle actin; CX‐32, connexin‐32; DPPIV, dipeptidylpeptidase IV; HHs, human primary hepatocytes; MDHLCs, mesenchymal‐derived hepatocyte‐like cells; MSCs, mesenchymal stem cells.

Figure 6

Functional characterization of MDHLCs. (a) Periodic acid‐Schiff (PAS) staining showing cytoplasmic glycogen deposition in MDHLCs as compared to HHs and MSCs. (b) G6Pase assay (n = 5) showing lack of enzyme activity in MDHLCs revealed by absence of brownish lead sulphide cytoplasmic precipitate. Positive and negative controls were performed respectively on HHs and MSCs. Similar results were obtained with overnight 5 mm G6Pate incubation. Pictures were taken at magnification ×400. (c) Colorimetric assay showing significant urea production in MDHLCs (n = 4) as compared to MSCs (n = 4). By comparison, no difference could be observed between the urea production of MDHLCs and mouse hepatocytes. (d) Gluconeogenesis assay showing the decrease of glucose neoformation in mouse hepatocytes (plain bars) after exposure of growing concentrations of pentachlorophenol. No glucose production was observed in MDHLCs (shaded bars) or in MSCs (empty bars) (n = 3 each). HHs, human primary hepatocytes; MDHLCs, mesenchymal‐derived hepatocyte‐like cells; MSCs, mesenchymal stem cells.

Figure 7

In vivo characterization of MSCs and MDHLCs after transplantation into SCID mice. (a) Analysis of in vivo differentiation potential of MSCs and MDHLCs. Pictures show immunostaining for human mesodermal (fibronectin, vimentin) and hepatocyte (albumin, αFP) antigens. In groups A and B, the engrafted cells formed clusters presenting staining for fibronectin or αFP separately. In group C, cell clusters co‐expressed mesodermal and hepatocyte markers as assessed by serial sections. In groups D and E, where MDHLCs were injected respectively into the spleen or the liver, the engrafted cell clusters expressed only hepatocyte markers. Stains were performed on human and mouse livers respectively as positive and negative controls. (b) Analysis of CK18 expression in groups C and E mice. In group C, the engrafted cells displayed co‐expression of fibronectin, albumin and CK18 whereas in group E, isolated staining for CK18 was observed in single or clustering cells. Control stains for CK18 were performed on human and mouse livers. Pictures were taken at magnification ×400. αFP, α‐foetoprotein.

Figure 8

Control immunohistochemistry. Pictures show staining assays performed without primary antibodies using ARK kit (ARK) or HRP‐conjugated anti‐rabbit antibody (HRP Ab). For monoclonal antibodies, stains with corresponding isotypes were performed (IgG1 for anti‐albumin and CK18 antibodies, IgG2a for anti‐vimentin antibody). No cross‐reactivity was observed in any of these conditions. Pictures were taken at magnification ×400.

Figure 9

In vivo tracking of human cells. Pictures show that engrafted human cells were co‐stained at the nuclear level with Alu probe by in situ hybridization technique and at the cytoplasmic level with antihuman fibronectin antibody by immunohistochemical assay on serial sections. Human and mouse livers are provided respectively as positive and negative controls. Arrows indicate human positive cells. Pictures were taken at magnification ×400.